Essential Oil from Sweet Potato Vines, a Potential New Natural

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Essential oil from sweet potato vines, a potential new natural preservative, antioxidant on sweet potato tuber: Assessment of the activity and the constitute Bo Yuan, Lingwei Xue, Qiu-yue Zhang, Wan-wan Kong, Jun Peng, Men Kou, and Jihong Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03175 • Publication Date (Web): 14 Sep 2016 Downloaded from http://pubs.acs.org on September 17, 2016

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Journal of Agricultural and Food Chemistry

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Essential oil from sweet potato vines, a potential new natural preservative,

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antioxidant on sweet potato tuber: Assessment of the activity and the constitute

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Bo Yuan, §, † Ling-wei Xue, §, † Qiu-yue Zhang, § Wan-wan Kong, £, Jun Peng, § Men

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Kou, §,ƫ Ji-hong Jiang§*

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§

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School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116, China.

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ƫ

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Xuzhou, Jiangsu 221131, China.

Xuzhou Sweetpotato Research Institute, Chinese Academy of Agricultural Science,

£

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The Key Laboratory of Biotechnology for Medicinal Plants of Jiangsu Province &

Department environmental monitoring and protection, Peixian, Xuzhou, Jiangsu

221600, China.

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12

*

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83403515; Email address:[email protected]);

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†

Corresponding author: Ji-hong Jiang, (Tel: +86 (0)516 83403515, Fax: +86 (0)516

Bo Yuan and Ling-wei Xue contributed equally to this study.

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ABSTRACT

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The pathogenic fungi and oxidation are the major factors to cause the

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deterioration of sweet potatoes and also causes the loss of quality and makes

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consumption unsafe. In the present study, the in vitro results demonstrate that the

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essential oil from sweet potatoes vines exhibits a significantly enhanced activity

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compared to the control. Furthermore, the essential oil can actively inhibit the growth

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of some common microgram inducing pathogenic bacteria and fungi (inhibition rates

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above 50% at low concentrations). A total of 31 constituents were identified using the

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GC-MS and confirmed that linalool and p-hydroxybenzoic acid are the major active

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ingredients. The experiment involving actual tubers showed the essential oil could

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keep the quality of and against the fungus disease. It suggests that it could be used in

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the food industry to increase the shelf life of stored produce (tubers) to ensure food

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safety without the use of additives or preservatives.

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Keywords: Essential oil; Sweet potato vines; Potential activity; Active constituent

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INTRODUCTION

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Sweet potato (Ipomoea batatas L.) is a typical dicotyledonous plant of the

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Convolvuceae family that is believed to have originated in tropical America.1 It is

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widely grown as a food crop in developing countries (such as China, India) and is

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considered to be one of the important economic crops in these countries, after rice,

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wheat, maize, and cassava.2-4 The main commercial producers of sweet potato are

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China, Indonesia, Vietnam, Japan, India, Tanzania, and Uganda.5 As well as being a

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major source of food, animal feed, and industrial raw materials, sweet potato has even

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been employed in clinical therapy in China. Many nutritionists believe that sweet

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potatoes promote health because of their high vitamin and trace element content,

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whilst being low in fat and cholesterol.6 However, sweet potato is also relatively

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easily invaded by fungi. Meanwhile, the infected tubers could produce fungal toxin

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which could threaten the health, even life of human and livestock.

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Pathogenic fungi can attack the sweet potato’s roots during industrial storage. In

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particular, black rot disease is a serious disease of sweet potato caused by the

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pathogenic fungus, Ceratosistis fimbriata and causes significant economic losses

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during storage.7 Microbiological control is a common method of controlling the

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growth of C. fimbriata.8 However, the efficacy of antifungal activity is often impaired

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by environmental factors such as rainfall, temperature, and so on. Pathogenic fungi

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thus remain significant destroyers of food during storage.

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Traditional methods (physical and chemical) are generally applied to deal with

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the problems associated with food storage, 9 however, there are also some drawbacks

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for those. It believes that those methods could threat the quality and safety of food,

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especially the synthetic chemicals. In modern agriculture, the synthetic pesticide has

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been widely and successfully used for the crop protection and plant disease control.

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There are many environmental contamination problems which caused by synthetic

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pesticide all over the word.10, 11 Therefore, more and more substitutes or alternative

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methods that are actively being researched, e.g. microbial sourced antifungal agents 12

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and insecticides.13

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Plants are important industrial sources of bioactive ingredients many of which

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can be used in various areas of interest, e.g. pharmacy, agriculture, cosmetics, and so

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on.14 Botanical pesticides consist of chemicals from plants that have evolved in crops

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and may be used for their defense against pathogenic fungi. Some authorities on the

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subject believe that plant-derived pesticides are friendly to the environment, animals,

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and humans because of low toxicity and residue. And the natural compounds consider

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as a good agent to control postharvest rots that would be as effective as synthetic

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fungicides.

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Essential oils are known for their antimicrobial and antioxidant properties and

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are already widely used in the food protection and other areas. Essential oils extracted

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from plants are volatile and usually consist of a complex mixture of ingredients, most

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of which are widespread in plants and have a variety of beneficial properties such as

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antioxidant,15, 16 insecticidal,17 antitumor,18 antifungal,19 and antimicrobial activity.20

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Most studies have been focused on essential oils from aromatic plants and have been

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applied to crops and antifungal food agents. However, these oils are not always


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obtained from the protected plants themselves.

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Research has shown that vines contain many bioactive chemical ingredients

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(alkaloids, flavonoids, steroids, etc.). In particular, extracts from sweet potato vines

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show good antimicrobial and antifungal activity against Escherichia coli, Salmonella

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typhi, Staphylococcus aureus, Penicillium spp., and Pseudomonas aeroginosa to name

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just a few.21 In some African countries, the vines or leaves of the sweet potato, which

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contain several nutrients, are commonly eaten like the tubers themselves. Therefore,

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not only can the sweet potato be harvested for its fleshy tubers but also for its vines as

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well. However, most vines are discarded after harvest or used as material for biogas

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fermentation or animals feed in China.22 Industrial and rational use of sweet potato

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vines (and leaves) may follow subject to further study.

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In this work, we employ sweet potato vines and leaves (waste materials left over

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from sweet potato harvesting) as a resource for plant-sourced antifungal agents for

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use in storing tubers. We also investigate their effects on a homemade quorum sensing

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system and their antioxidant potential. The major objectives of the present study are

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to estimate the antioxidant, antibacterial, antifungal, and refreshing activities of the

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extracted essential oil, and to identify its chemical composition via gas

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chromatography – mass spectrometry (Q-Exactive GC Orbitrap).

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MATERIALS AND METHODS

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Sample preparation. Sweet potato tubers (4 kg, cultivar is Xushu-18) were

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obtained from the Xuzhou Sweet Potato Research Institute at November, 2015

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(Xuzhou, Jiangsu, China) and used as a representative sample (it is the vines and



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leaves collected from the tubers, which form the actual samples used). The tubers

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were washed with water and cut into slices of equal thickness/size. 10 of identical

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sweet potato slices with same weight were choose and put them into a 10 L glass pots.

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Essential oil extraction. Essential oil was extracted from the fresh leaves and

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vines of the sweet potatoes following the method described by Sriramavaratharajan

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method.23 The samples (50g) were then subjected to hydro-distillation in a Clevenger

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apparatus for about 4 h (a sample: water ratio of 1: 5 w/v was employed). The

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essential oil was collected and dried over a circular, rigid glass tube filled with

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anhydrous Nitrogen gas and count the oil yield was 0.54% (w/w). Then stored the

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essential oil at 4 oC for further study.

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In vivo activity analysis. (1) Antimicrobial activity. The antimicrobial activity

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of the essential oil was evaluated using the method reported in Ud-Daula.16 Five

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different microorganisms were used to carry out the evaluation: Bacillus subtilis

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ATCC 6633, B. cereus Frankland ATCC 14579, Staphylococcus aureus NCTC 7447,

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Escherichia coli CICC 10389, and Pseudomonas aeruginosa CICC 10419.

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Ceratocystis fimbriata ATCC 24096 and Fusarium oxysporum ATCC 52429 were

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used to evaluate the antifungal activity.

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Stock bacterial cultures were prepared on Nutri-Bertani plates and then incubated

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at 37 oC for 24 h. Bacterial suspensions were added to Luria–Bertani broth and the

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broth kept in a constant temperature incubator at 37 oC for 24 h. Disc diffusion assays

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were used to determine the antimicrobial activity. Sterile filter paper discs (6 mm in

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diameter) were placed into solutions of the essential oil (100µg/mL) of different


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concentrations and impregnated until saturated (10 min). The paper discs were then

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transferred over to the Nutri-Bertani plates. To create a positive control, 100 µg/mL of

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ampicillin, miconazole, and amphotericin were used. Sterilized distilled water (10 µL)

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was used as the control. All inoculated plates were incubated in an incubator at a

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constant temperature of 37 oC for 24 h. After incubation, antimicrobial activity was

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evaluated by measuring the diameters of the inhibition zones (including the disc).

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Antifungal activity (with respect to C. fimbriata ATCC 24096 and F. oxysporum

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ATCC 52429) was determined using the same method but using a potato dextrose

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agar medium. Also, the temperature was kept at 28 oC for 100 h using an isoperibol.

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The same positive and negative controls were used as for the antimicrobial activity

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determination.

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(2) Determination of the minimum inhibitory concentration (MIC). The MIC

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value was determined using the microdilution method described by Ud-Daula et al.

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The essential oil was diluted two-fold with ethyl acetate to adjust the concentration

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range to 0.80–1000 µg/mL. A mixture formed from filter-sterilized essential oil

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(10 µL) and liquid broth growth medium (170 µL) was then added to the cells of a

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96-cell microtiter plate. Then, a standardized suspension of the organism (20 µL) was

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added to each cell. The 96-cell microtiter plates were kept at a constant temperature of

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37 oC in an incubator for 24 h for the bacteria (28 oC for 100 h for the fungi).

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Ampicillin and amphotericin B were used as antibacterial and antifungal control

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compounds at ranges of 0.5–1000 and 0.1–1000 µg/mL, respectively. After the

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incubation period, the cells were checked for microorganism growth. The lowest



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concentration for which there are no visible signs of growth corresponds to the MIC

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value.

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(3) Evaluation of the antioxidant activity of the essential oil. Various assays

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were used to evaluate the antioxidant activity of the essential oil: 2,

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2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays, β-carotene/linoleic

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acid assays, ferric thiocyanate (FTC) assays, and NO radical scavenging assays.

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The DPPH scavenging assays were evaluated as described by the Blois’s

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method24. It carried out by mixing essential oil samples of different concentrations (10,

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20, 30, 40, 50, and 100 mg/mL) with α-tocopherol and adding them to methanolic

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solutions containing DPPH radicals (0.1 mM) to yield a final volume of 1 mL. The

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mixtures were shaken vigorously and placed in the dark for 30 min at 25 oC. The

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absorbance values of the solutions at 517 nm were then determined using a UV-vis

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spectrophotometer (Evolution 300, Thermo Fisher Co., USA). The percentage

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scavenging ability was the calculated using the following formula:

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% DPPH radical scavenging = (Ablank – Asample)/Ablank × 100.

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For the β-carotene/linoleic acid assays, the method was described as the Safaei

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method with some modifi-cations.25 The β-carotene was dissolved in 0.2 mL of

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chloroform. Then, a solution of linoleic acid (20 mg) in Tween 40 (in 200 mg) was

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prepared and the β-carotene solution added to it. The chloroform was then evaporated

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under vacuum (40 oC for 5 min). Ultra-pure water (50 mL) was added to the emulsion

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which was then vigorously shaken. Meanwhile, different concentrations of essential

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oil were mixed with α-tocopherol. These mixtures were then added to the prepared


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β-carotene/linoleic acid emulsions. The mixtures were incubated at 50 oC for 3 h and

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then their absorbance values determined at 470 nm. The results were finally

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calculated using the expression:

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% inhibition of β-carotene bleaching = (Asample,3h – Ablank,3h)/(Ablank,0h –Ablank,3h) ×100.

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The method used to carry out the FTC assays was adapted from the method used

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by Dong et al. method.26 Different amounts of essential oil were dissolved in 5 mL of

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ethanol. These were then mixed with linoleic acid (2% v/v) in 4 mL of ethanol, 0.05

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mol phosphate buffer, distilled water (4 mL), respectively, and the pH adjusted to 7.0.

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Then, the mixtures were added to 75% ethanol (v/v) and 30% (w/v) ammonium

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thiocyanate. Then, exactly 0.1 mL of 20 mM ferrous chloride in hydrochloric acid

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(3.5%) was added and the mixture allowed to react. In the final step, the absorbance

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of the resulting red solution was measured at 500 nm every 24 h until the

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time-controlled absorbance value reached its maximum value. The inhibition of

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linoleic acid peroxidation was calculated using the following formula:

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% inhibition = 100 – [(Asample /Acontrol) × 100].

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Note that all the absorbance values increased in the samples and controls. All

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tests were run in triplicate and averaged.

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To determine NO radical-scavenging activity, different concentrations of the

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essential oil and α-tocopherol were mixed with 0.1 mL of 100 mM sodium

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nitroprusside and phosphate buffer solution to yield a final volume of 1 mL. This was

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then incubated at 25 oC for 3 h and added to Griess reagent (0.05 mL to 0.05 mL),



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kept at constant temperature for 10 min, and then its absorbance at 540 nm

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determined. The NO radical scavenging activity was calculated according to:

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% NO radical scavenging = (Ablank – Asample)/Ablank ×100.

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Assays to determine the reducing power activity (RPA) and ferric reducing

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antioxidant power (FRAP) were carried out using 96-well plates. The steps used to

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perform these assays were all identical to those described by Ud-Daula.16

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Inhibition of mycotoxin production. Tuber preservation experiments were

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carried out using the apparatus depicted Figure 1. A sample of the essential oil was

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placed in an airtight, brown-glass pot fitted with an air-input tube. The pot was placed

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on a heater and the temperature maintained at 70 oC. The arrangement allowed the

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essential oil in the brown pot to be volatilized into a glass tuber storage pot via a

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conduit. The glass storage pot had previously had slices of sweet potato tubers and C.

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fimbriata placed inside it and was then evacuated using a vacuum pump. A cribellum

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in the glass pot separated the tubers from the C. fimbriata, which had been cultured in

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an open Petri dish. The system contains three (calibrated) micro gas monometers,

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which were used to determine the amount of escaping gas and the actual amount

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consumed. The instrument was located in a warm room (20–25 oC). At the end of the

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experiment, the tubers were collected, cut up, and extracted twice with

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dichloromethane. The dichloromethane was removed from the combined organic

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phases using a rotary evaporator. The dry residue was then weighed.

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GC-MS detection condition. Chromatography was used to purify the crude

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extract (using 50–100 µm silica gel and a hexane–ethyl acetate gradient). The toxicant


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fraction was eluted with 90% hexane: ethyl acetate, which was further cleaned using

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SPE-C18 chromatography (using a hexane–acetate gradient) to yield a toxicant mix.

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The structures of the isolated toxicants were then determined using gas

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chromatography – mass spectrometry (using a Thermo Q-Exactive GC Orbitrap), and

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the toxins identified via comparison with spectroscopic data in the literature (NIST).

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The Q-Exactive GC Orbitrap device consists of a Thermo Fisher Trace 1300 gas

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chromatograph coupled to an ISQ EI mass spectrometer (Thermo Fisher Technology,

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CA). It was equipped with a BD-5MS column (30 m × 0.25 mm i.d. × 0.25 µm film

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thickness, Thermo Fisher Technology, CA). Helium was used as the carrier gas and

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the flow rate was set at 1.5 mL/min. The temperature programming was refer to the

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method of Wamalwa.27 The oven temperature was held at 80 oC for 5 min and then

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programmed to increase in 20 oC per minute steps to 280 oC and then held this

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temperature for 10 min. The target peaks were identified using X-calibur software

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(Thermo Fisher Technology, CA). The mass spectra were compared with NIST, Wiley,

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and SADITER library data. The masses of the ion peaks were analyzed to determine

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the contents of the toxicant. Additionally, the diameters of the C. fimbriata samples

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were measured and compared with the control.

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Chemical composition of the essential oil and identification of the active

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ingredients. The chemical composition of the essential oil was determined using the

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Q-Exactive GC Orbitrap fitted with a TR-5MS capillary column (30 m × 0.25 mm,

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0.25 µm film thickness). Helium was used as the carrier gas (delivered at 1 mL/min).

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The injector temperature was set to 250 oC and splitless injection was used. The



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original temperature of the oven was 80 oC for 5 min, increasing to 200 oC at a rate of

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5 oC/min, and then this temperature was held for 10 min. To continue, the temperature

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was raised to 250oC at a constant rate of 5 oC per minute. The EI and transline

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temperatures were set to 280 and 250 oC, respectively.

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Solid-phase microextraction (SPME, Supelco Co., Bellefonte, USA) coupled

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with the Q-Exactive GC Orbitrap device was used to identify the active ingredients of

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the essential oil. When the reading on micro gas monometer 3 had changed and

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finally remained constant, the SPME handle was inserted into the glass pot to extract

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some of the chemical ingredients in the glass pot at that time. After 30 min, the SPME

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handle was removed and injected into the injection port. By pushing the lever, the

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extraction head (quartz fibre) was pushed out of the needle tubing. Then, the

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adsorbate entered into the oven as a result of thermal desorption and was analyzed by

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the programmed temperature changes described above. By comparing the results with

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standards, the essential oil composition and available ingredients could then be

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evaluated.

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Test of actual tubers. 60 kg of fresh sweet potato (Xushu-18) were collected

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and ensure all of them were same variety, same harvest time, and keep the same size

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or weight. Six groups were designed and each of which include 10 kg of tubers. There

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were three experiment groups (difference contraction of essential oil treatment),

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positive control (blank control) and negative control (topsin) groups. For the

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experiment groups, the essential oil was dressing with the tubers. Difference ratios

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(1:15, 1:30, 1:45, 1:60) of essential oil and tubers were set. Group 5 accepted no


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special treatment (positive control, CK1), group 6 was deal with topsin (1:60,

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negative control, CK2). The negative control was used the topsin to spraying

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sanitization of the cellars and the positive groups was deal with sterile water. Six

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groups into six potato cellars and each one capacity about 5±m3. The temperature in

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the sweet potato cellars were keep 15±5oC. Count the incidence of sweet potato

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black/soft rot at short-time intervals. Extraction the tubes and determine the water

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content, α- amylase activity, starch content, soluble protein content, and soluble sugar

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content. The experiment time was continuing about 150 days. All of the determine

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method were all reference to the detection method of EN food standards and GB food

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standards (China).

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RESULTS AND DISCUSSION

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Antimicrobial activity. The antimicrobial activity results for the essential oil

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from sweet potato vines and leaves are shown Table 1. The table clearly shows that

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the essential oil has better antimicrobial activity against bacterial and fungal growth

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than known antimicrobial substances. The essential oil shows activity against bacteria

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and fungi with inhibition zones ranging from 2.9 to 87.5 mm. E. coli appears to be the

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microorganism most sensitive to the essential oil and B. subtilis the least. The

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essential oil inhibited the growth of E. coli with inhibition zones ranging from 5.5 to

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87.7 mm, depending on concentration.

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Different concentrations of essential oil exhibit different degrees of activity

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against all the microorganisms tested, even P. aeruginose, a highly drug-resistant

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microorganism. Some researchers have pointed out that because of its high level of



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intrinsic resistance, P. aeruginose is resistant to all known antimicrobials and

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antibiotics — even synthetic drugs do not work on it.16, 28 However, the essential oil

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extracted from sweet potato vines inhibits the growth of this strain. Therefore, search

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and verification of the active ingredient(s) in the essential oil from the sweet potato

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vines would provide a novel antimicrobial can be found for P. aeruginose.

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From the results, the essential oil appears to have a better activity against fungi

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than bacteria. On the other hand, the essential oil is equally active against both

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gram-positive and gram-negative bacteria. As gram-negative bacteria have

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lipopolysaccharide outer membranes that generally restrict the diffusion of

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hydrophobic compounds, these bacteria are less susceptible to antibacterial agents. In

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several recent studies, various essential plant oils have been found to have good

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activity against the growth of bacteria regardless of their gram-negative/gram-positive

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nature.29-32 Comparing the results in these papers with the current antimicrobial

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results, it appears that sweet potato vine oil could be a more effective oil to use than

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those oils. Given these sorts of results, the essential oil from sweet potato vines could

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be used against most of the bacteria encountered in the food industry and so it could

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be a better preservative for storage of food products.

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As for fungal growth, both C. fimbriata and F. oxysporum were inhibited by the

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essential oil and the former was more strongly affected than the latter. The inhibition

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zones on the C. fimbriata plates (12.7–80.5 mm) were smaller than that of the positive

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control, flutriafol. At the equal concentration of 100 µg/mL, the inhibition zone of

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essential oil was also smaller than the control. As is well known, flutriafol is an


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economically important agricultural chemical that is able to control several diseases

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affecting a wide range of crops. However, its high mobility potential in the soil and

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groundwater means that flutriafol is a problematic pesticide. Indeed, flutriafol is a

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potentially toxic chemical pesticide, which may disrupt fertility in women and affect

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the endocrine system.33

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The measured minimum inhibitory concentrations are shown in Table 2. As can

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be seen, the MICs of the essential oil are 176.5, 16.7, 12.6, 10.8, 142.6, 78.4, and

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96.5 µg/mL for the microorganisms, B. subtilis, B. cereus, S. aureus, E. coli, P.

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aeruginosa, C. fimbriata, and F. oxysporum, respectively. Compared with the control

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antibiotics, the MICs of the essential oil against B. subtilis, B. cereus, S. aureus, C.

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fimbriata, and F. oxyspoum are close to those of the best antibiotics. The essential oil

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showed strong activity against E. coli (10.8 µg/mL), along with erythromycin, topsin,

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tetracycline, and was better than gentamicin and flutriafol. The essential oil also

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showed good activity against C. fimbriata and F. oxysporum, two fungi commonly

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encountered during the storage of sweet potatoes (78.4 and 96.5 µg/mL).

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Unfortunately, these MIC values are larger than those of flutriafol (56.7 and

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69.4 µg/mL). However, compared with the toxicity of flutriafol, the essential oil

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seems to have very low toxicity.

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There are several studies reporting the antimicrobial activity of essential oils.

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Ud-Daula found that essential oil from different parts of Etlingera fimbriobracteata

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has good antimicrobial activity against gram-positive bacteria (B. subtilis, B.

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spizizenii, and S. aureus, with MIC values ranging from 19.5 to 156 µg/mL).16 It



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appears that the MIC values of the oil from sweet potato vines are much lower than

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those of the essential oil from E. fimbriobracteata. E. coli is a very important germ in

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the food industry and in medicine. Interestingly, most of the essential oils from the

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plants reported in the literature have their best inhibitory activity against E. coli. A

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report recently reported that the essential oil extracted from Alpinia guilinensis has

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strong antibacterial activity against pathogenic foodborne bacteria (S. aureus, B.

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subtilis, E. coli, P. aeruginosa).34 However, it seems that the MIC values of the oil

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from sweet potato vines are much lower than those of the oil extracted from A.

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guilinensis. There are various other reports similar to the ones mentioned here;

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however, none of them consider inhibition of the pathogens important to storage, viz.

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C. fimbriata and F. oxysporum. Not only that, the antimicrobial activity of sweet

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potato vine oil is strong and has a broad spectrum of applicability. Hence, the

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essential oil from sweet potato vines is more suitable as a preservative for storage

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purposes in the food industry.

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Antioxidant activity. Antioxidant activity is an important property to investigate

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when evaluating the bioactivity of an essential oil. The different components in the

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essential oil are chemical compounds with different functional groups, polarities, and

347

chemical behavior. For the present study, we used six different assays to evaluate the

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antioxidant properties of the sweet potato vine oil.

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The radical scavenging potential of the essential oil determined via DPPH assay

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is illustrated in Figure 2A. The figure clearly shows that the oil’s radical scavenging

351

capacity increases as the concentration of the oil increases. At the same time, the


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effect of the oil is clearly time-dependent: the longer the oil is allowed to interact, the

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better the radical scavenging effect. After 50 min of interaction, the oil’s IC50 capacity

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(34.3 mg/mL) is, in fact, better than that of vitamin C (36.2 mg/mL).

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Vitamin C (Vc) and butylated hydroxytoluene (BHT) were used as references to

356

gauge NO radical scavenging ability. Essential oils that possess NO scavenging

357

activity inhibit nitrite formation by competing with oxygen as it tries to react with NO.

358

From Figure 2B, it can be seen that the NO radical scavenging capacity of all three

359

substances tested increase with concentration. The sweet potato oil clearly has a better

360

IC50 value (75.5 mg/mL) than both Vc (81.2 mg/mL) and BHT (116.5 mg/mL).

361

Although vitamin C has a better activity than the essential oil when the concentration

362

is 60 mg/mL, the essential oil is superior when the concentration reaches the

363

maximum-used dosage of 100 mg/mL (equal to 66.2%).

364

In keeping with the DPPH results, the essential oil also exhibited the most potent

365

antioxidant activity in the β-carotene/linoleic acid assay (Figure 2C). Its IC50 value is

366

8.35 mg/mL. The other method of antioxidant assay (FTC) also showed

367

concentration-dependence, and the oil’s IC50 value (75.80 mg/mL) was better than

368

those of both the reference substances (Vc: 127.8 mg/mL and BHT: 110.8 mg/mL).

369

The DPPH radical scavenging activity values exhibited by the essential oil and Vc are

370

almost equal (IC50 values of 34.3 and 36.2 mg/mL, respectively). However, the

371

antioxidant activity of the essential oil exceeds that of Vc in the NO radical

372

scavenging, β-carotene/linoleic acid, and FTC assays.

373

The two reduction assays (RPA and FRAP) measure the ability of the oil to



374

reduce Fe3+ to Fe2+. The results of the RPA assays (Figure 3B) indicate that the

375

essential oil has markedly lower reducing potentials than Vc and BHT. Based on their

376

EC50 values, the tested substances can be ranked (strongest to weakest) in the

377

following order: BHR (33.9 mg/mL) > Vc (41.4 mg/mL) > essential oil (83.9 mg/mL).

378

In contrast, in the FRAP assay, the reducing ability of the essential oil was better than

379

that of the two controls. The essential oil produced an EC50 value of 7.42 mg/mL,

380

significantly lower than that of Vc and BHT (12.2 and 9.54 mg/mL, respectively).

381

To the best of our knowledge, the antioxidant activity of the essential oil from

382

sweet potato vines has not been reported before in the literature. Some researchers

383

have suggested that the essential oil from the plant could have good activity with

384

respect to DPPH radical scavenging, reducing power, and so on. Other researchers

385

have also observed similar results. For example, the essential oils from the Etlingera

386

fimbriobracteata and Flos Lonicerae showed good antioxidant or anti-microbial

387

activity.16,

388

ingredients. Thus, an analysis of the chemical composition in our case is very

389

important.

35

All of those study, the activity were all dependent on the chemical

390

Composition of the essential oil, activity evaluation, and quantitative

391

toxicant calculations. GC–MS was used to analyze the chemical composition of the

392

essential oil because of its high sensitivity. The results of the analysis were compared

393

with several databases (e.g. Wiley and NIST) to obtain the final assignments. Over 30

394

ingredients were identified in the essential oil, as listed in Table 3. The most abundant

395

ingredients detected in the essential oil are linalool (49.65%), followed by β-pinene


Page 18 of 44

Page 19 of 44


396

(12.54%), p-hydroxybenzoic acid (12.51%), α-pinene (5.09%), ledol (2.65%), and

397

sitosterol (2.05%). Oxygenated acyclic monoterpenes form the dominant ingredient

398

type in the oil (49.65%), followed by bicyclic monoterpenes (21.02%), and acyclic

399

monoterpenes (13.16%).

400

In another study of essential oil from sweet potato leaves, there are about 84 and

401

45 components were tentatively identified in two cultivars of sweet potato by

402

GC-MS.35 Compared with the results, the components of our study are quite different.

403

There are only four compounds were existed in the sweet potato vines essential oil

404

including benzaldehyde, linalool, α-terpineol, and caryophyllene oxide. Although

405

some ingredients are present in both results, the amounts are not the same. Difference

406

cultivars of sweet potato and part have different quantities and concertation of

407

ingredients.

408

We extracted the sweet potato tuber during the activity evaluation in vitro and

409

analyzed the change in ingredients using the Q-Exactive GC Orbitrap (concentrating

410

on those components related to the toxins produced by C. fimbriata). The toxicant

411

ingredients were successfully and quantitatively identified using GC-MS/MS by

412

comparing the data with the databases. The full scan showed up four peaks of interest.

413

These were identified as being due to four furanoterpenoids: ipomeanine (IPN),

414

4-ipomeanol (4-IPO), ipomeamarone (IPAO), and dehydroipomeamarone (D-IPAO).

415

To clarify the results of the comparison, MS/MS scans were carried out on the parent

416

ions. The molecular ion peak appeared at 248 m/z and the target ion was at 207 m/z.

417

Meanwhile, the fragment ion at 207 m/z was also the peak with the largest abundance.



418

Because of its structure, there is a possibility that a McLafferty rearrangement could

419

have occurred during the pyrolytic process. The mass spectral fragmentation

420

pathways typical of furans verified the results of the comparison as 4-IPO. The other

421

three MS/MS spectra were consistent with the Q-Exactive GC Orbitrap analysis.

422

We further investigated the change in the toxin level during storage over a

423

50-day period (Figure 4). The 4-IPO concentration ranged between 0.08 and

424

10.55 mg/kg in the sample treated with essential oil from the sweet potato.

425

Comparison with the control group shows that the oil clearly inhibits toxin production

426

in the sweet potato. As is well known, the toxin (i.e. IPO) is produced by C. fimbriata

427

when the fungus infects the sweet potato tuber. As the essential oil inhibits toxin

428

production, it might mean that the oil inhibits the C. fimbriata from infecting the

429

sweet potato tuber because of the toxin is the products when the C. fimbriata

430

infection.

431

Table 4 shows how the composition of the essential oil changed after the

432

experiment compared to before (derived using MSPE technology). The table clearly

433

shows that many of the ingredients undergo a change. The biggest changes occur for

434

linalool and p-hydroxybenzoic acid. About 50% was expended during the experiment

435

and other microconstituents (50% inhibition) against B. subtilis, B. cereus, S. aureus, E. coli,

441

C. fimbriata, and F. oxysporum. The concentrations of the two compounds employed

442

ranged from 50 to 300 µg/mL and growth inhibition increased with increasing

443

concentration of the linalool and p-hydroxybenzoic acid for all the strains tested.

444

Using 300 µg/mL of linalool, growth was inhibited by 77.10%, 57.39%, 74.54%,

445

76.60%, 73.37%, and 73.58% for B. subtilis, B. cereus, S. aureus, E. coli, C. fimbriata,

446

and F. oxysporum, respectively. The corresponding figures for p-hydroxybenzoic acid

447

are 80.22%, 62.23%, 63.6%, 72.42%, 79.31%, and 78.63%. Growth inhibition was in

448

the range 20–40% when the lowest concentration was used. Linalool seemed to have

449

better activity compared to p-hydroxybenzoic acid. However, it was possible to

450

observe a reduction in growth, even at the lowest concentration used. As expected,

451

linalool and p-hydroxybenzoic are strong candidates for the antimicrobial ingredients

452

in the essential oil from sweet potato vines.

453

Linalool is a monoterpene that has been reported to be a major component of

454

essential oil from various aromatic species. As a biogenic unsaturated terpene alcohol,

455

it is also used in food as a flavor and aroma enhancer, especially for fruits and

456

vegetables.36, 37 It is the most flavorsome component in orange juice and other fruit,

457

such as tomato, grape, and mango.38 Linalool has been found to exhibit strong activity

458

against periodontopathic microorganisms with MIC and MBC values ranging from

459

0.1 to 1.6 mg/mL and 0.1 to 0.8 mg/mL, respectively.39 In another study, linalool was

460

found to be a good antifungal agent for Campylobacter jejuni and C. coli.40

461

The other active ingredient, p-hydroxybenzoic acid, has previously been shown



462

to have antimicrobial activity against some microbial strains.41 In the present study,

463

p-hydroxybenzoic acid has shown good antibacterial and antifungal activity. In the

464

food industry, p-hydroxybenzoic acid is well established as an effective preservative.

465

Overall, the two ingredients identified here seem to be good preservatives for sweet

466

potato tubers based on their good inhibitory activity towards fungal growth during

467

tuber storage. In other situations, linalool and/or p-hydroxybenzoic acid, may also be

468

equally effective. This suggests that they could be of general use in the food industry

469

to enhance the shelf life of food products and increase their safety without requiring

470

chemical additives or preservatives.

471

Assessment of the actual tubers storage. The results of assessment of actual

472

tubers showed that the CK1 sweet potato became rot at the 60th day, then large-scale

473

of rotten rate was appear and more than half were rotten at the 150th day (Figure 6A).

474

The essential oil treatment could delay this phenomenon to 90th day later and the

475

higher ratio of essential oil and tubers, effect is better. The essential oil could keep a

476

quite low rotten rote than CK1. The best refreshment was to reach 120 days and the

477

topsin could help the sweet potato refreshment only 80 days under the same ratio

478

(1:60). So in the actual storage, the essential oil could keep the sweet potato from

479

decay in a long time.

480

For the water content, the essential oil also could hele the sweet potato tubers

481

keep more water. From the result (Figure 6B) exhibition, the treatment groups showed

482

higher water content than the control in each stage. And a negative relation of the

483

essential oil ratio and water content. Lower ratio could help sweet potato to hold more


Page 22 of 44

Page 23 of 44


484

water. The essential oil and topsin have same moisturizing function, merit attention,

485

topsin is still some toxicity for human and livestock even then low toxicity.

486

The α-amylase in the sweet potato could help the starch transform to the

487

reducing sugar and improve the taste. However, higher activity of α-amylase, lower

488

starch in the storage tubers. So modest activity of α-amylase in the sweet potato is an

489

important factor for the storage of sweet potato. In the experiment, the α-amylase

490

exerts an advanced growth and then decline. From the Figure 6C, the group which

491

deal with essential oil showed lower activity than CK. A positive correlation between

492

the ratio and the activity. Compared with the CK1, there were decline about 4.7% at

493

the 150th day and other three groups were 6.33%, 9.57%, and 15.35%, respectively.

494

The starch is the important composition of the sweet potato so high content of starch

495

in the sweet potato reflects the quality is good or bad. In the storage, the starch in the

496

tubers reveal decline in the first stage and keep gentle in the last stage. There was also

497

a positive correlation between essential oil and starch content (Figure 6D). The starch

498

content had increased 3.36%, 2.96%, 2.76%, and 2.06%, respectively under

499

difference ratio of essential oil. For the soluble protein and sugar, the essential oil was

500

also showed positive correlation between essential oil and them (Figure 6E and 6F).

501

From above results, we could believe that the essential oil of sweet potato vine could

502

help the sweet potato against the fungal infestation and keep the most nutrient

503

substance in the storage tubers.

504

In the present study, essential oil was obtained from sweet potato vines and

505

evaluated for activity with regard to storage of sweet potato tubers. The essential oil



506

showed good antifungal, antibacterial, and antioxidant activity in vivo and in vitro. It

507

was able to inhibit the growth of the sweet potato tuber fungus C. fimbriata and its

508

ability to produce mycotoxin. The in vivo antimicrobial effects of the essential oil

509

were better than those of common antibacterial agents. Terpenoids form the majority

510

of the ingredients in the essential oil, as shown via Q-Exactive GC Orbitrap analysis.

511

Active verification tests showed that linalool and p-hydroxybenzoic acid are the main

512

active ingredients (>50%).

513

Our results demonstrate that the essential oils from sweet potato vines (and, in

514

particular, its major active ingredients, linalool and p-hydroxybenzoic acid) show

515

good antioxidant and antimicrobial potential. All of the

516

essential oil extracted from (normally discarded) sweet potato vines could be used as

517

a good natural preservative for industrial food storage, especially sweet potatoes (and

518

it might be better than existing preservatives). It could be decrease the amount of

519

chemical ingredients used in the industrial storage and improve environmental

520

protection. It certainly seems prudent to conduct appropriate industrial warehouse

521

tests in subsequent studies.

522

ABBREVIATIONS USED

results suggest that the

523

ATCC: American type culture collection; NCTC: National Counterterrorism

524

Center; CICC: China Center of Industrial Culture Collection; MIC: Minimum

525

Inhibitory Concentration; DPPH: 2, 2-diphenyl-1-picrylhydrazyl; FTC: ferric

526

thiocyanate; RPA: Reducing Power Activity; GC-MS: Gas Chromatography-Mass

527

Spectrometer. NIST: National Institute of Standards and Technology.


Page 24 of 44

Page 25 of 44


528

ACKNOWLEDGMENT

529

National Science Foundation of China (31370646, 31672148), the Program of

530

Natural Science Foundation of the Jiangsu Higher Education Institutions of China

531

(15KJD180004), Jiangsu Province Science and Technology Support Project

532

(BK20150232), Grants from Natural Science Foundation by Jiangsu Normal

533

University (15XLR025, 15XLR026) and Student Innovation Training Program of

534

China (201510320103X).

535



536

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activities of essential oils from leaves, aerial stems, basal stems, and rhizomes of

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17. Buentello-Wong, S.; Galán-Wong, L.; Arévalo-Niño, K.; Almaguer-Cantú, V.;

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effect from Curcuma kwangsiensis. Afr. J. Pharm. Pharmaco. 2012, 6, 1348-1351.

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Vanloot, P.; Abbad, A.; Boudyach, E. H.; Aoumar, A. A. B. Chemical characterization

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and antifungal activities of four Thymus species essential oils against postharvest

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20. Yousefzadi, M.; Riahi-Madvar, A.; Hadian, J.; Rezaee, F.; Rafiee, R.; Biniaz, M.

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anti-microbial activity. J. Immunotoxicol. 2014, 11, 50-55.


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21. Mbaeyi-Nwa, I. E.; Emejulu, V. N. Evaluation of Phytochemical Composition

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and Antimicrobial Activity of Sweet Potato ( Ipomoea batatas ) Leaf. Pak. J. Nutr.

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22. Zhan, J.; Hui, L.; Qi, S.; Zhou, Q.; Zhao, Y. Potential utilization of waste

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sweetpotato vines hydrolysate as a new source for single cell oils production by

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Trichosporon fermentans. Bioresource Technol. 2012, 135, 622-629.

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23. Sriramavaratharajan, V.; Stephan, J.; Sudha, V.; Murugan, R. Leaf essential oil of

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Cinnamomum agasthyamalayanum from the Western Ghats, India—A new source of

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24. Blois, M. S. Antioxidant Determinations by the Use of a Stable Free Radical.

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25. Safaei-Ghomi, J.; Ebrahimabadi, A. H.; Djafari-Bidgoli, Z.; Batooli, H. GC/MS

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analysis and in vitro antioxidant activity of essential oil and methanol extracts of

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Thymus caramanicus Jalas and its main constituent carvacrol. Food Chem. 2009, 115,

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26. Dong-Jiann, H.; Hsien-Jung, C.; Wen-Chi, H.; Chun-Der, L.; Yaw-Huei, L. Active

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recombinant thioredoxin h protein with antioxidant activities from sweet potato

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4720-4.

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27. Wamalwa, L. N.; Cheseto, X.; Ouna, E.; Kaplan, F.; Maniania, N. K.; Machuka,

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Sweetpotato (Ipomoea batatas L. Lam) Storage Roots upon Infection by Rhizopus



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chitosan films containing Eucalyptus globulus essential oil. LWT - Food Sci.Techno.

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30. Dhouioui, M.; Boulila, A.; Chaabane, H.; Zina, M. S.; Casabianca, H. Seasonal

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31. Ma, Q.; Davidson, P. M.; Zhong, Q. Antimicrobial properties of microemulsions

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formulated with essential oils, soybean oil, and Tween 80. Int. J. Food Microbiol.

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32. Radaelli, M.; da Silva, B. P.; Weidlich, L.; Hoehne, L.; Flach, A.; da Costa, L. A.

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M. A.; Ethur, E. M. Antimicrobial activities of six essential oils commonly used as

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condiments in Brazil against Clostridium perfringens. Braz. J. Microbiol. 2016, 47,

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33. Santana, M. B.; Rodrigues, K. J. A.; Durán, R.; Alfonso, M.; Vidal, L.; Campos,

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microdialysis in freely moving rats. Ecotox. Environ. Safe. 2009, 72, 1565-1571.

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34. Chen, Z.; He, B.; Zhou, J.; He, D.; Deng, J.; Zeng, R. Chemical compositions and

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antibacterial activities of essential oils extracted from Alpinia guilinensis against

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selected foodborne pathogens. Ind. Crop. Prod. 2016, 83, 607-613.

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35. Mei, W.; Xiong, Y.; Zeng, M.; Li, H.; Zhang, T.; Liang, Y. GC–MS Combined

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with Chemometrics for Analysis of the Components of the Essential Oils of Sweet

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Potato Leaves. Chromatographia 2010, 71, 891-897.

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36. Chen, X.; Hopke, P. K. A chamber study of secondary organic aerosol formation

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by linalool ozonolysis. Atmos. Environ. 2009, 43, 3935-3940.

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37. Bäcktorp, C.; Wass, J. R. T. J.; Panas, I.; Sköld, M.; Börje, A.; Nyman, G.

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39. Park, S.-N.; Lim, Y. K.; Freire, M. O.; Cho, E.; Jin, D.; Kook, J.-K. Antimicrobial

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669

FIGURE CAPTIONS

670

Figure 1. Schematic diagram of the apparatus used in the tuber-preservation

671

experiments.

672

Figure 2. Antioxidant activity of the essential oil from sweet potato vines as a

673

function of concentration: (A) DPPH radical scavenging activity, (B) NO radical

674

scavenging activity, (C) inhibition of β-carotene/linoleic acid bleaching, and (D) FTC

675

activity.

676

Figure 3. In vitro antioxidant analysis of the essential oil: (A) the layout used in the

677

96-well-based assay for different concentrations of essential oil and positive and

678

negative controls, (B) reducing power activity (RPA) results, and (D) FRAP assay

679

results.

680

Figure 4. The effect of the essential oil on mycotoxin production from C. fimbriata.

681

Values are given as mean±standard deviation (n = 10). Bars labeled with different

682

letters indicate significant differences at p < 0.05.

683

Figure 5. Effect of different concentrations of p-hydroxybenzoic acid (A) and linalool

684

(B) on antimicrobial activity as determined using the disc diffusion method. The

685

results are expressed as percentage of growth inhibition. Bars labeled with different

686

letters indicate significant differences at p < 0.05.

687

Figure 6. The change of physiology and biochemistry in the tubers under difference

688

ratio of essential oil. (A) Rotten rate; (B) Water content; (C) The α-amylase activity of

689

the sweet potato tubers; (D) Starch content in the sweet potato tubers; (E) Soluble

690

protein in the tubers; (F) Soluble sugar in the tubers.



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Table 1 Diameters of the inhibition zones produced by different concentrations of the essential oil from sweet potato vines and controls (mm).

Microorganism

Essential oil concentration (µg/mL, in DMSO)

Control (200 µg/mL) 1500

1000

500

200

100

Bacillus subtilis

25.2±0.2 (erythromycin)

67.5±0.5

52.2±0.2

37.6±0.7

25.7±0.5

11.5±0.6

Bacillus cereus

24.2±0.2 (erythromycin)

80.6±0.6

56.5±0.5

33.1±0.7

21.6±0.1

10.5±0.5

Staphylococcus aureus

28.5±0.8 (penicillin G)

75.5±0.2

54.7±0.5

30.5±0.9

28.7±0.4

7.2±0.5

Escherichia coli

24.6±0.5 (tetracycline)

87.7±0.5

50.7±0.2

36.5±0.1

20.5±0.6

5.5±0.2

Pseudomonas aeruginosa

20.7±0.7 (gentamicin)

72.5±0.7

56.7±0.5

30.6±0.4

17.5±0.3

2.9±0.5

Ceratocystis fimbriata

20.7±0.5 (flutriafol)

80.5±0.2

53.6±0.7

36.5±0.5

28.7±0.1

20.5±0.3

Fusarium oxysporum

21.1±0.2 (flutriafol)

70.2±0.8

68.7±0.6

35.7±0.8

26.4±0.2

21.7±0.5

34


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Table 2 Minimum inhibitory concentrations of the essential oil from sweet potato vines for certain test microorganisms. Minimum inhibitory concentration (µg/mL) Microorganism Essential oil

Erythromycin

Topsin

Bacillus subtilis

176.5

17.2

19.7

24.6

22.7

/

Bacillus cereus

16.7

15.6

20.4

18.7

17.6

/

Staphylococcus aureus

12.6

2.1

12.6

11.8

15.7

/

Escherichia coli

10.8

9.2

7.1

6.5

11.7

/

Pseudomonas aeruginosa

142.6

27.4

65.9

/

/

/

Ceratocystis fimbriata

78.4

/

27.4

/

/

56.7

Fusarium oxysporum

96.5

/

/

/

/

69.4

35


Tetracycline

Gentamicin

Flutriafol


Page 36 of 44

Table 3 Percentage composition and relative retention indices (RI) of the compounds identified in the essential oil from sweet potato vines. Typea

RI

Compound

Molecular Formula

Composition (%) 0.65 49.65 0.92 5.09 0.27 0.66 12.54 0.68 0.48 0.99 0.55 0.32 0.33 0.17 2.65 0.65 0.88 0.76 12.51 1.54 0.27 0.32

927 Benzaldehyde C7 H6 O AS 1021 Linalool C10H18O OAM 1027 α-Terpineol C10H16 BM 837 α-Pinene C10H16 BM 1033 Sabinene C10H16 BM 1145 Camphene C10H16 BM 1181 β-Pinene C10H16 BM 1196 (Z)-β-Ocimene C10H16 AM 1243 (E)-β-Ocimene C10H16 AM 1249 δ-3-Carene C10H16 BM 1257 Carvone C10H14O BM 1263 α-Terpinene C10H16 MM 1279 Terpinen-4-ol C10H18O OMM 1255 γ-Elemene C15H24 AM 1368 ledol C15H26O DP 1375 Fenchyl alcohol C10H18O OBM 1426 Spathulenol C15H24O DP 1432 Nonanal C9H18O AH 1162 p-hydroxybenzoic acid C7H6O3 AS 1498 Myrtenol C10H16O OBM 1501 Borneol C10H18O OBM 1506 Caryophyllene oxide C15H24O OBS 1516 Borneol C10H18O 0.58 OBM 1527 Pentacosane C25H52 0.65 AK 1545 Pentylfuran C9H14O 0.32 HC 1565 Hexadecanoic acid C16H32O2 0.44 FA 0.28 1578 Myristic acid C14H28O2 FA 1592 Octadecanol C18H38O 0.36 SA 1625 Lanosterol C30H50O 0.75 STO 1633 6-Ketocholestanol C27H46O2 0.21 STO 1647 Octacosanol C28H58O 0.33 SA 1670 Sitosterol C29H50O 2.05 STO a AH – aldehyde, AK – alkane, AM – acyclic monoterpene, AS – aromatic series, BM – bicyclic monoterpene, DP – diterpene, FA – fatty acid, HC – heterocycle, MM – monocyclic monoterpene, OAM – oxygenated acyclic monoterpene, OMM – oxygenated monocyclic monoterpene, OBM – oxygenated bicyclic monoterpene, OBS – oxygenated bicyclic sesquiterpene, SA – saturated alcohol, STO – sterol. 36 ACS Paragon Plus Environment

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Table 4 Ingredient changes, before and after the experiment. No

Compositiona (%)

Compound

Before After Benzaldehyde 0.51 NDb 1 Linalool 24.5 10.25 2 α-Terpineol 0.54 ND 3 α-Pinene 2.55 2.05 4 Sabinene ND ND 5 Camphene 0.21 ND 6 β-Pinene 6.21 5.98 7 (Z)-β-Ocimene 0.05 ND 8 (E)-β-Ocimene ND ND 9 δ-3-Carene 0.25 ND 10 Carvone 0.35 ND 11 α-Terpinene ND ND 12 Terpinen-4-ol ND ND 13 γ-Elemene ND ND 14 ledol 1.02 ND 15 Fenchyl alcohol 0.02 ND 16 Spathulenol 0.21 ND 17 Nonanal 0.15 ND 18 p-hydroxybenzoic acid 2.12 0.98 19 Myrtenol 1.05 0.95 20 Borneol ND ND 21 Caryophyllene oxide ND ND 22 Borneol 0.25 ND 23 Pentacosane 0.35 ND 24 Pentylfuran ND ND 25 Hexadecanoic acid ND ND 26 Myristic acid ND ND 27 Octadecanol ND ND 28 Lanosterol 0.42 ND 29 6-Ketocholestanol ND ND 30 Octacosanol ND ND 31 a Proportion in the gaseous state in the glass pot based on the area of the peak (total ion chromatogram). b ‘ND’ means ‘not detected’ by the Q-Exactive GC Orbitrap.



Figure 1.


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Figure 2.



Figure 3.


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Figure 4.



Figure 5.


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Figure 6.



Table of Contents Graphic (TOC):


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Essential Oil from Sweet Potato Vines, a Potential New Natural

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