Accepted Manuscript

UHPLC-QqQ-MS/MS identification, quantification of

polyphenols from Passiflora subpeltata fruit pulp and
determination of nutritional, antioxidant, α-amylase and α-
glucosidase key enzymes inhibition properties

Saravanan Shanmugam, Isla Alcântara Gomes, Marina Denadai,

Bruno dos Santos Lima, Adriano Antunes de Souza Araújo,
Narendra Narain, Maria Terezinha Santos Leite Neta, Mairim
Russo Serafini, Lucindo José Quintans-Júnior, Parimelazhagan
Thangaraj

PII: S0963-9969(18)30277-1
DOI: doi:10.1016/j.foodres.2018.04.006
Reference: FRIN 7524
To appear in: Food Research International
Received date: 27 November 2017
Revised date: 17 March 2018
Accepted date: 3 April 2018

Please cite this article as: Saravanan Shanmugam, Isla Alcântara Gomes, Marina Denadai,
Bruno dos Santos Lima, Adriano Antunes de Souza Araújo, Narendra Narain, Maria
Terezinha Santos Leite Neta, Mairim Russo Serafini, Lucindo José Quintans-Júnior,
Parimelazhagan Thangaraj , UHPLC-QqQ-MS/MS identification, quantification of
polyphenols from Passiflora subpeltata fruit pulp and determination of nutritional,
antioxidant, α-amylase and α-glucosidase key enzymes inhibition properties. The address
for the corresponding author was captured as affiliation for all authors. Please check if
appropriate. Frin(2018), doi:10.1016/j.foodres.2018.04.006

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 ACCEPTED MANUSCRIPT

UHPLC-QqQ-MS/MS identification, quantification of polyphenols from Passiflora

subpeltata fruit pulp and determination of nutritional, antioxidant, α -amylase and α-

glucosidase key enzymes inhibition properties.

Saravanan Shanmugam1* , Isla Alcântara Gomes1 , Marina Denadai2 , Bruno dos Santos Lima1 ,

Adriano Antunes de Souza Araújo1* , Narendra Narain2 , Maria Terezinha Santos Leite Neta2 ,

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Mairim Russo Serafini1 , Lucindo José Quintans-Júnior1 , Parimelazhagan Thangaraj3

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1

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Department of Pharmacy/Federal University of Sergipe, Av. Marechal Rondon, Jardim Rosa

Elze, CEP: 4910 0-000 São Cristóvão – Sergipe, Brazil.

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2
Laboratory of Flavor and Chromatographic Analysis/Federal University of Sergipe, Av.

Marechal Rondon, Jardim Rosa Elze, CEP: 4910 0-000 São Cristóvão – Sergipe, Brazil.
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3
Department of Botany/Bharathiar University, Coimbatore – 641 046, Tamilnadu, India.
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* Corresponding authors address:

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Department of Pharmacy, Federal University of Sergipe, CEP 49100-000, Sao Cristovao, Brazil,

Email address: saranflora04@gmail.com, Tel.: +55 79 991024838 and

adriasa2001@yahoo.com.br, Tel.: +55 79 991924545

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Abstract

The diabetic key enzymes inhibition, nutritional, antioxidant activity and bioactive

compounds identification of Passiflora subpeltata fruit pulp were investigated. Fifteen

polyphenolic compounds including protocatechuic acid, ferulic acid, vanillic acid,

epicatechin, p-coumaric acid, cinnamic acid, eriodictyol and quercetin-3-glucoside were

identified in the pulp of this species by using UHPLC-QqQ-MS/MS analysis. The total

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carbohydrates and crude protein contents in fruit pulp were 2.62 mg glucose equivalent/g

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sample fruit pulp and 8.80 mg BSA equivalent/g sample fruit pulp, respectively. The fresh

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fruit pulp of P. subpeltata contained high total phenolic (724.76 mg GAE/g sample) content

and it revealed very high DPPH• (IC50 of 5.667 µg/mL) and ABTS+• (6794.96 µM trolox
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equivalent/g sample) scavenging activities. In the key enzymes assays useful for diabetic

inhibition the fresh fruit pulp characterized maximum inhibition of α-amylase and α-
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glucosidase IC50 of 18.69 and 32.63 µg/mL, respectively. Thus, these results lead to conclude

that this fruit specie could be very useful source in nutraceutical products preparations for
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Type 2 diabetic suffering humans.

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Keywords: Passiflora subpeltata; UHPLC-QqQ-MS/MS; DPPH; α-amylase; α-glucosidase;

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phenolic compounds.
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1. Introduction

Owing to their natural pleasant sweet-acid taste and an intense flavor, the fruits of

Passiflora popularly known as passion fruit, are very much appreciated in the world. In food

industries, the fruit juice is the main source of nutritional and therapeutic properties mainly

due to the presence of many phytochemical constituents such as phenolic and flavonoid

compounds. Although hundreds of Passiflora species can be found worldwide, only a few

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species are identified as edible. Furthermore, these fruits are reported to contribute for some

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health beneficial properties such as being antioxidant, anti-inflammatory, antipyretic,

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analgesic, sedative and hypotensive activities (Dembitsky et al., 2011; Saravanan, Arunachalam, &

Parimelazhagan, 2014; Saravanan & Parimelazhagan, 2014; Shanmugam et al., 2017; Shanmugam,
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Murugaiyan, et al., 2016; Shanmugam, Thangaraj, et al., 2016). Fruits of Passiflora are also known

to exert neuroactive function, demonstrating antianxiety and anticonvulsant effects (Porto-Figueira,

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Freitas, Cruz, Figueira, & Câmara, 2015). Moreover, the fruits have rich in minerals (calcium and

phosphorus) and vitamins, especially A, C, thiamine, riboflavin and niacin. It is also a good source
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of carotenoids, anthocyanins and alkaloids (Jiménez et al., 2011). All these important features of

Passiflora species are explored in the folklore for traditional medicinal system to cure various
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ailments.
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Diabetes mellitus (DM) is a chronic metabolic disorder, which is characterized by

hyperglycemia. This happens mainly due to two reasons - either the pancreas does not
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produce enough insulin or the body cannot effectively use the insulin it produces (WHO,

2016). World Health Organization (WHO) estimated that DM will be the 7 th leading cause of

death in 2030 (Mathers & Loncar, 2006). Type Two Diabetes Mellitus (T2DM) consists

especially of diminished insulin secretion and activity which results in eventual pancreatic β-

cells failure (Vilcacundo, Martínez-Villaluenga, & Hernández-Ledesma, 2017). Main

stratagems adopted to treat T2DM involve inhibition of carbohydrate-digesting enzymes such

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as α-amylase and α-glucosidase in the small intestine, which are associated with delaying of

intestinal glucose absorption and lowering of postprandial blood glucose levels (Balan et al.,

2017). An exocyclic enzyme, α-glucosidase hydrolyses the oligosaccharides which are

converted to monosaccharides and these are later absorbed into the blood from the intestine.

Blood carries these monosaccharides to the individual cells where they are converted to

energy by the cell machinery with the help of insulin. Another enzyme, α-amylase hydrolyzes

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glycosidic bonds in starch to glucose, maltose, malt triose and dextrin (Ngoh, Lim, & Gan,

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2016). Thus, inhibition of these starch digestive enzymes or glucose transporters can suppress

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postprandial hyperglycemia by reducing the rate of glucose release and absorption in the

small intestine (Hanhineva et al., 2010).

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Modern system of medicine shows that phenolic and flavonoid compounds can

inactivate α-amylase, α-glucosidase and lipase through non-specific binding to enzymes

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(Zhang et al., 2015). Passiflora species such as P. edulis (flavicarpa), P. alata, P. incarnata,

P. mollissima, P. tripartita, P. ligularis and P. quadrangularis are widely grown and have
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many biologically active secondary metabolites including phenolic and flavonoid compounds.

C-glycosyl derivatives apigenin and luteolin, vitexin, isovitexin, orientin, schaftoside, 2´´-O-
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rhamnoside and luteolin-7-O-(2-rhamnosylglucoside), quercetin 3-β-D-glucoside, isoscoparin

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-2´´-O-glucoside are some novel functional compounds of Passiflora species (Gazola et al.,

2015; Saravanan & Parimelazhagan, 2014; Shuayprom, Sanguansermsri, Sanguansermsri,

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Fraser, & Wongkattiya, 2016).

Passiflora subpeltata (Synonym P. calcarata) is one of the species of Passiflora,

widely distributed in all over the world. The plant bears attractive flowers and their fruits and

leaves are edible. The tribal peoples of Kurumba, Paniya and Kattunaikka living across the

Western Ghats of India consume this plant as a heathy diet and traditionally, leaves of this

plant are used to treat pain and inflammation related health disorders (Ratheesh Narayanan &
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Anil Kumar, 2007). Earlier publications showed that this plant contains phenolic and

flavonoid compounds like gallic acid, apigenin, catechin and cumarin derivatives, cyanogenic

glycosides and these were identified and quantified in this plant leaves (Olafsdottir,

Thorgeirsdottir, & Jaroszewski, 1997; Saravanan et al., 2014). Our previous report revealed

that the acetone extract of leaves of P. subpeltata exhibit antioxidant, analgesic, antipyretic, anti-

inflammatory and hepatoprotective properties (Saravanan et al., 2014; Shanmugam, et al., 2016).

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However to the best of our knowledge, there is no detailed work as yet reported on the functional

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properties and phytochemical constituents of the pulp of P. subpeltata fruits. Thus, the objective

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of the present study was to identify and quantify the bioactive functional compounds present in

the pulp of this plant species by using Ultra High Performance Liquid Chromatography with
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Triple Quadrupole type Mass Spectrometer (UHPLC-QqQ–MS/MS) and to determine its

nutritional profile, antioxidant capacity and inhibition of α-amylase, α-glucosidase enzymes for
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their in vitro antidiabetic property.

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2. Materials and methods

2.1. Chemicals
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Water used for the mobile phase was purified through a Milli-Q system from Millipore
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(São Paulo, Brazil). Artepillin C, cinnamic acid, ferulic acid, daidzein, epicatechin,

eriodictyol, gallic acid, luteolin, naringenin, p-coumaric acid, protocatechuic acid, quercetin-3-
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glucoside, chrologenic acid, vanillin and vanillic acid, 2, 2-diphenyl-1-picryl-hydrazyl (DPPH),

2,2´azinobis (3-ethylbenzothiozoline-6- sulfonic acid) disodium salt (ABTS), α-amylase (porcine

pancreas) and α-glucosidase (rat intestine) were purchased from Sigma-Aldrich (Saint Louis, MO,

USA). Methanol and acetonitrile were of HPLC grade, purchased from Merck (São Paulo, Brazil).

2.2. Collection of plant materials

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The fresh plant fruits of P. subpeltata were collected from the altitude of 2600 mts

from the places of Ooty, Coonor, Mynala and Thottapetta during the months of November to

May 2016. The collected plant material was identified and their authenticity was confirmed by

comparing the voucher specimen at the herbarium of Botanical Survey of India, Southern circle,

Coimbatore, Tamilnadu (No. BSI/SRC/5/23/2013-14/Tech/532). The plant specimen was deposited

in the herbarium (Voucher specimen No: BU/HB/006167) Department of Botany, Bharathiar

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University, Coimbatore. Freshly collected fruits were cleaned to remove adhering dust and then

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dried under shade. The dried sample were powdered and used for further studies.

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2.3. Plant sample preparation
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The air-dried, powdered plant material (100 g) was subjected to soxhlet extraction

using petroleum ether to remove the lipophilic substances. The residues were dried in a hot
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air oven (40 .C) and 98.25 g of this dried matter was further extracted using 300 mL of each

of the solvents such as chloroform, acetone and methanol The different solvent extracts were
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evaporated using a rotary vacuum-evaporator (Yamato RE300, Japan) at 50 ℃ and the

remaining water was removed by lyophilization (VirTis Benchtop K, USA). Further, the
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fresh fruit pulp was directly collect from the fresh fruits and the collected fruit juice were
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freeze dried by lyophilization (VirTis Benchtop K, USA) for further analysis. Also the

extracts thus obtained were used directly to assess the antioxidant potential through various in
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vitro assays.

2.4. Chemical profiling by UHPLC-QqQ- MS/MS analysis

2.4.1. Preparation of the extracts

P. subpeltata fruit pulp (2 g) was extracted with 15 mL of ethanol solution (70 %).

The solution was heated at 35°C on the ultrasonic bath for 30 minutes and centrifuged at 5000
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g at room temperature. Further, supernatant was evaporated and methanol was used to re-

suspend the extracts the in a concentration of 50 mg/mL and 2 µL were injected in the HPLC

analysis which was filtered through 0.22 µM cellulose membrane (Merck).

2.4.2. LC-MS conditions

The phenolic profile analysis was performed using a UHPLC system model 1290

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Infinity coupled to a 6490 Triple Quadrupole mass spectrometer equipped with an

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electrospray ionization (Agilent Technologies, Palo Alto, USA), according to methodology

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described by Andrade et al., (2017). The chromatographic separation was performed on

Ascentis Express F5, 2.7 µm particle size and 150 x 2.1 mm i.d. column (Sigma Aldrich,
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Saint Louis, MO, USA). Aqueous formic acid (0.1% v/v) solution (A) and acetonitrile (B)

consisted of mobile phase, with a flow rate of 0.2 mL/min, under gradient elution at 40°C. The
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gradient profile was as follows: 0-1 min, 15% B; 1-7 min, 25% B; 7-9 min, 25% B; 9-13 min,

30% B; 13-16, 30% B; 16-21 min, 40% B; 21-23, 40% B; 23-25, 45% B; 25-28, 50% B; 28-33,
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60% B; 33-37, 75% B; 37-38, 15% B. The injection volume was 2 µL. The LC-MS data were

acquired using Mass Hunter software (Version B 7.0, Agilent Technologies).

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Mass spectrometry conditions were as follows: source temperature: 200ºC; gas flow: 12
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L/h; nebulizer: 20 psi; sheath gas temperature: 400°C; sheath gas flow: 11 L/min; capillary:

3500 V; nozzle: 500 V; acceleration cell voltage: 5 V; dwell time: 9.8 ms. Two Selected
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Reaction Monitoring (SRM) transitions were optimized for each compound identified; one

being the most intense transition for quantification, and the second for confirmatory purposes.

Electrospray ionization source was operated in positive mode, except for CAPE, which was

monitored in negative mode.

2.4.3. Identification and determination of polyphenolics by UHPLC-MS/MS method

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The linearity of LC-MS method was determined with calibration curves prepared at 8

different levels over a concentration range of 20-1000 ng mL-1 of the standards, as reported in

our earlier work (Andrade et al., 2017). The lower limit of quantitation (LOQ) was

considered the concentration at the first level of calibration curve (20 ng mL-1 ) with the

accepted criteria that the precision and the accuracy for three samples had variability of less

than 20 %. For the analysis of target phenolic compounds in the Passiflora fruit extracts,

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external calibration curve samples were run between the fruit samples.

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2.5. Nutritional analysis

2.5.1. Proximate composition

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The contents of moisture, ash and crude fiber were determined according to the

methods defined in Association of Official Analytical Chemists (AOAC, 1995). The protein,
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carbohydrate and starch contents were estimated by the method of Sadasivam and Manickam,

(2008). The results were expressed on dry weight basis

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2.5.2. Estimation of amino acids

Amino acids contents of fruit pulp were determined following the method of Ishida et
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al., (1981). Extracted samples were filtered through a 0.45 µm membrane filter and 20 µL of
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the filtrate was injected in a HPLC (model LC 10 AS, Shimadzu, Mount holly, New Jersey)

equipped with a cation exchange column packed with a strongly acidic cation exchange resin,
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viz., styrene divinylbenzene co-polymer with sulphonic group. The instrument was equipped

with a Shimadzu fluorescence detector (FL 6A; Shimadzu Scientific Instruments, Japan) and

a Shimadzu Chrompac recorder (CR 6A; Shimadzu Scientific Instruments, Japan). The

mobile phase of the system consisted of two buffers (phosphate buffer and borate buffer) and

a gradient system was used for effective separation of amino acids. The oven temperature

was maintained at 60o C with a total run time of 60 min. The amino acid analysis was
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according to the non-switching flow method using fluorescence detection after post-column

derivatization with O-phthaldehyde. Amino acid standards were used to calculate amino acid

concentrations in samples.

2.5.3. Mineral quantification

Amount of total nitrogen (N) content was estimated through micro Kjeldahl method;

phosphorus (P) by treating the samples with ammonium molybdate and freshly prepared

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ascorbic acid and analyzed by spectrophotometer read at 420 nm (Hitachi U-2001 Japan);

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Potassium (K), Sodium (Na), and Calcium (Ca) were determined by Flame Photometer by the

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method of Allen, (1989). The microelements (Fe, CO, Cu, Mg, Mn and Zn) were determined

through Atomic Absorption Spectrophotometer Tee et al., (1996).

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2.6. Quantification of bioactive secondary metabolites
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The quantification of bioactive secondary metabolites in the plant extracts was done

by the standard UV spectrophotometric methods. The total phenolic and tannin contents of
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different solvent extracts and fresh pulp of P. subpeltata fruits were determined according to

the method described by Siddhuraju and Becker, (2003) and the results were calculated as
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gallic acid equivalent (GAE). The method of Zhishen et al., (1999) was followed to quantify
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the total flavonoid content and the results are expressed in terms of rutin equivalent (RE).
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2.7. Antioxidant Activities

2.7.1. DPPH radical scavenging activity

Radical scavenging activity of different solvent extracts and fresh pulp of P.

subpeltata fruit was measured by DPPH radical scavenging method, described by Blois,

(1958). IC50 values of the extract, i.e., the concentration of extract necessary to decrease the
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initial concentration of DPPH by 50%, was calculated. A lower IC 50 value indicates higher

activity.

2.7.2. ABTS cation radical scavenging activity

ABTS cation radical decolorization assay was measured according to the method

described by Re et al., (1999). The total antioxidant activity (TAA) was expressed as the

concentration of trolox having equivalent antioxidant activity in terms of µmol/L/g sample

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extract.

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2.7.3. Ferric reducing antioxidant power (FRAP) assay

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The ferric reducing antioxidant capacity of different solvent extracts and fresh pulp of

P. subpeltata fruit pulp was estimated by the method described by Pulido et al., (2000). The
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FRAP values were expressed as mmol/L Fe (II) equivalent/mg extract.
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2.7.4. Metal chelating activity

The chelation of metal ions by various extracts of fruit pulp and fresh pulp was done
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by the method of Dinis et al., (1994). The chelating activity of the extracts was evaluated

using EDTA as standard. The results were expressed as mg EDTA equivalent/g extract.
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2.7.5. Phosphomolybdenum Assay

The antioxidant activity of samples was evaluated by the phosphomolybdenum

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method (Prieto, Pineda, & Aguilar, 1999). The results were expressed as grams of ascorbic

acid equivalents per gram extract (AAE).

2.7.6. Superoxide radical scavenging activity

The assay was based on the capacity of the plant sample to inhibit formazan

formation by scavenging superoxide radicals generated in riboflavin-light-NBT system

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(Beauchamp & Fridovich, 1971). The inhibition percentage of superoxide radical generation

was calculated from the following formula:

% inhibition = [(Control OD - Sample OD)/Control OD]*100

2.8. In vitro anti-diabetic activities

2.8.1. α-Amylase inhibition assay

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Different concentrations (50–250 µL) of the fruit extracts and fresh fruit pulp (1

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mg/mL DMSO) and 500 µL of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl)

having porcine pancreatic α-amylase enzyme (0.5 mg/mL) were incubated at 25 °C for 10

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min. After incubation, 500 µL of 1 %, starch solution in 0.02 M sodium phosphate buffer (pH
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6.9 with 0.006 M NaCl) was added to the reaction mixture. Subsequently, the reaction

mixture was incubated at 25°C for 10 min and later 1.0 mL of dinitrosalicylic acid (DNS)
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was added. The reaction mixture was diluted with the addition of 10 mL distilled water, and

absorbance was measured at 540 nm (Worthington, 1993). The results of α-amylase

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inhibition activity were expressed in terms of IC 50 .

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2.8.2. α-Glucosidase inhibition assay

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Various concentrations of fruit extracts and fresh fruit pulp (50–250 µL) (1 mg/mL

DMSO) and 100 µL of rat intestinal α-glucosidase (0.5 mg/mL) in 0.1 M phosphate buffer
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(pH 6.9) solution were incubated at 25 °C for 10 min. Later, 50 µL of 5 mM p-nitrophenyl-α-

D-glucopyranoside in 0.1 M phosphate buffer (pH 6.9) solution was added. Reaction

mixtures were incubated at 25°C for 5 min and absorbance was read at 405 nm in

spectrophotometer (Apostolidis, Kwon, & Shetty, 2007). The results of α- glucosidase

inhibition activity were expressed as in terms of IC 50 .

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2.9. Data Analysis

All in vitro results were expressed as mean ± standard deviation values with triplicate

determinations (n = 3). Analysis of variance (ANOVA) and significant (p < 0.05) difference

between mean were determined by Duncan’s multiple range test.

3. Results and discussion

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3.1. UHPLC-QqQ-LC-MS/MS analysis

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The data on calibration equations, correlation coefficients (r2 ), detection limits, and

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the intra-day precisions for the analysis of phenolic compounds were established earlier in

our work (Andrade et al., 2017). The precursor and product ions with associated collision
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energies and the retention time of the phenolic compounds are presented in Table - 1 while

the chemical structure of the compounds are shown in Figure 1. A total of 15 polyphenolic
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compounds were identified in P. subpeltata fruit pulp by UHPLC-QqQ-MS/MS. The SRM

chromatogram of the two selected transitions, and their corresponding retention times were
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used for identifying the target compounds. All the results were expressed in the terms of ng/g

dry weight basis. Protocatechuic acid is the major compound found in fruit pulp of P.
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subpeltata which was quantified as 115.43 ng/g, followed by ferulic acid (55.76 ng/g),
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vanillic acid (46.01 ng/g), epicatechin (22.45 ng/g) and p-coumaric acid (20.54 ng/g).

Moreover three compounds viz. cinnamic acid (9.08 ng/g), eriodictyol (8.02 ng/g) and
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quercetin-3-glucoside (3.04 ng/g) were found in lower concentrations (Table 1). Other

compounds are reported in traces as their amounts were lower than the limit of quantification

of the method (20 ng/g).

Heterogeneous groups of phenolic compounds were produced by the plant as

secondary metabolites for their protection. Phenolic acid and flavonoid compounds are of

utmost importance as more than 10,000 compounds belonging to the group of phenolic
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compounds were already reported (Missio et al., 2016) along with their functional properties.

Polyphenols are the major antioxidants in human diets. These natural bioactive

phytochemicals specifically present in fruits and vegetables have been discovered recently

for their potential health benefit effects on the prevention of many oxidative stress related

human ailments such as bacterial infections, cancer, ulcer, diabetes, ageing, viral infections,

inflammatory disorders, atherosclerotic, cardiac, hepatoprotective activity and neurological

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disorders (Bhardwaj, Nandal, Pal, & Jain, 2014). The compounds such as protocatechuic

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acid, ferulic acid, vanillic acid, epicatechin and p-coumaric acid are the major compounds

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which were identified and quantified in the pulp of P. subpeltata fruit. These compounds

have many potential health benefits including antioxidant and antidiabetic properties which
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have been reported earlier by various workers (Li, Wang, Chen, & Chen, 2011; J. Liu et al.,

2016). Moreover, the ferulic acid was also reported for their antioxidant and antidepressant
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like activities (Lúcia, Zeni, Camargo, & Dalmagro, 2017). Other compounds such as vanillic

acid, epicatechin and p-coumaric acid were also evaluated for their biological activities
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including antioxidant, antidiabetic and anti-inflammatory properties (Hoon Kim et al., 2017;

Vinothiya & Ashokkumar, 2017). Moreover, the other Passiflora species such as P. edulis, P.
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alata, P. incarnata, P leschenaultii, P. mollissima, P. tripartita, P. ligularis and P.

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quadrangularis possess some of these compounds viz. ellagic acid, gallic acid, isoorientin,

orientin, vitexin, isovitexin and rutin which have been reported earlier with related biological
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functions (Costa et al., 2015; Saravanan et al., 2014; Saravanan & Parimelazhagan, 2014;

Viganó et al., 2016). In our earlier study on this plant leaves, we reported that this plant

possesses the compounds such as gallic acid, apigenin, catechin, cumarin derivatives and

cyanogenic glycosides which contribute for antioxidant, anti-inflammatory, analgesic and

antipyretic properties (Olafsdottir et al., 1997; Saravanan et al., 2014). Moreover, these

compounds also revealed the hepatoprotective activity (Shanmugam, Thangaraj, et al., 2016).
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3.2. Proximate composition

The data on proximate composition analysis of P. subpeltata fruit pulp are presented

in Table 2a. From these results, total carbohydrates (2.62 mg glucose equivalent/g sample),

crude protein (8.80 mg BSA equivalent/g sample), total amino acids (1.44 mg leucine

equivalent/g sample), crude fiber (5.89 g/100 g sample) and ash (1.45 g/100 g sample)

contents were found while the moisture content of the fruit pulp was 95.02%. Septembre-

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Malaterre et al., (2016) reported similar results on P. edulis fruit pulp.

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P. subpeltata fruit pulp is an excellent source of minerals including calcium (50.67

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mg/g), ferrous (1.73 mg/g), potassium (1.81mg/g) and magnesium (0.02 mg/g) (Table 2b).

Calcium is the foremost component for bone building and tooth development. Similarly, for
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controlling human blood pressure potassium plays a vital role (Sadia et al., 2014). Thus the

mineral contents of P. subpeltata fruits could play a protective role in human health besides
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providing strength to bone and tooth.

Owing to their biological activities, amino acids are very important since these are
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the building blocks of the human body. FAO/WHO (2007) established the pattern and

specific concentrations of essential amino acids that human life needs for daily routine
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activities. The data obtained from the results of P. subpeltata fruit pulp showed that it has
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high concentrations of glutamic acid (243 mg/g protein), arginine (115 mg/g protein),

aspartic acid (105 mg/g protein) and proline (5.02 mg/g protein) (Table 2c). In protein
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synthesis, leucine plays a vital role in growth and maintenance of the body. The P.

subpeltata fruit contained 66 mg leucine/g protein compared to FAO/WHO reference pattern

(59 mg/g protein), followed by phenylalanine (75 mg/g protein), threonine (33 mg/g protein)

and valine (55 mg/g protein). Thus the regular consumption of this fruit juice supplies the

dietary nutrient level of minerals and amino acids to the human body and also helps for the

regular metabolic reactions.

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3.3. Secondary metabolites and antioxidant properties

As shown in Table 3, the total phenolic contents of different extracts of fruit pulp

were gradually increased by polar variation of the solvents (Petroleum ether 197.14,

chloroform 175.71, acetone 553.81 and methanol 395.95 GAE mg/ g extract) while fresh

fruit pulp (724.76 GAE mg/ g extract) contained the maximum level of phenolic content

when compared to the fruit pulp extracted with solvents. The same behavior was observed in

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the tannin content of the fruit extract where the fresh fruit pulp had the maximum tannin

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content (358.57 GAE mg/ g extract) (Table 3). The maximum amount of flavonoid content

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was also found in fresh fruit pulp (4763.33 RE mg/g extract) followed by the fruit extracted

with solvent acetone (1340.00 RE mg/g extract) (Table 3). Similar results were found also in
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our earlier study on P. ligularis fruit (Saravanan & Parimelazhagan, 2014). Additionally, the

same plant leaves and seed also showed antimicrobial, and antioxidant, anti-inflammatory,
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antipyretic properties (Saravanan et al., 2014; Saravanan & Parimelazhagan, 2013). It is well

known that the consumption of phenolic and flavonoid-rich foods could protect against human
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diseases related to oxidative stress and aging problems. Many reports proved earlier that there

has been close relationship between antioxidant property and phenolic and flavonoid contents
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(Meng, Fang, Qin, Zhuang, & Zhang, 2012). Thus, the phenolic, tannin and flavonoid contents
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might be responsible for the antioxidant activity of this P. subpeltata fruit pulp.
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3.3.1. DPPH radical scavenging activity

DPPH is the stable radical pale purple in color and when reduced it turns yellow. This

reagent has been widely used to determine the free radical-scavenging activity of various

pure compounds or plant extracts. Ability to reduce DPPH radical by P. subpeltata fruit

extracts and standards were evaluated and the results are shown in Figure 2. As presented in

Figure 2, the IC50 value (5.667 µg/mL) of the fresh pulp was very close to the standard
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values of rutin (5.670 µg/mL) and BHT (6.380 µg/mL). The maximum value found in fresh

pulp is responsible for the antioxidant capacity of the pulp that scavenges the free radicals by

hydrogen donation as defined by the hypothesis reported previously (Saito & Kawabata,

2005). The major compounds protocatechuic acid and ferulic acid identified in this fruit also

are responsible for the DPPH radical scavenging activity by donating hydrogen molecule to

the radical for its stabilization in the radical form. However, there may be compounds which

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may combine with their hydrogen atom to form stable quinone. The possible mechanism

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involved in this process of compounds protocatechuic and ferulic acids on DPPH radical is

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cited below. This hypothesis was proved by Graf, (1992); Li et al., (2011).
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Reaction between DPPH• and protocatechuic acid could be explained by the following

mechanism:
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Radical can further withdraw

hydrogen atom (H•) to form stable
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Protocatechuic acid quinone

DPPH• DPPH-H
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Possible mechanism involved between the ferulic acid and DPPH•:

Ferulic acid Resonance stabilization of

DPPH• ferulic acid radical.
DPPH-H
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DPPH assay values for the P. leschenaultii leaves acetone extract were reported

(Shanmugam, Murugaiyan, et al., 2016). We also reported data on antioxidant activity of

other parts of Passiflora species viz. P. ligularis, P. foetida and P. subpeltata leaves and seed

(Saravanan et al., 2014; Saravanan & Parimelazhagan, 2014; Sasikala, Saravana, &

Parimelazhagan, 2011). Thus it is quite evident that the major polyphenolic acid compounds

such as protocatechuic, ferulic and p-coumaric acid are mainly involved in scavenging DPPH

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radical by donating their hydrogen molecule and thus attain stable condition.

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3.3.2. ABTS cation radical scavenging activity

The ABTS+• scavenging assay also is a powerful tool that has been characteristically
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used to estimate the free radical scavenging activities of pure compounds and plant extracts.

Table 3 shows the ABTS+• antioxidant activity of the different solvents and fresh pulp of P.
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subpeltata fruit samples. The ABTS+• radical scavenging capacity of fresh pulp was

considerably higher (6794.96 μM trolox equivalent/g extract) than that of the other solvents
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extracts. ABTS+• is produced by reacting ABTS with potassium persulphate and the activity

is measured spectrophotometrically at 734 nm. The addition of fruit samples and trolox react
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with ABTS+• and convert it into ABTS in a concentration-dependent manner. Further, total
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antioxidant activities of the analyzed samples were associated with the combined effect of

phytochemicals (protocatechuic acid, ferulic acid, vanillic acid and epicatechin) acting by
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additive or synergistic effects. Many phytochemical compounds, such as phenolics and

flavonoids, show a higher contribution and their content correlate positively with the

antioxidant activity (Lazarova et al., 2014). According to Re et al., (1999), this reaction

requires an electron atom to ABTS+• for stabilization. In this present study, the possible

mechanism of the protocatechuic acid and ABTS•+ are discussed. The electron atom from the

compound directly donate to the ABTS +• and this electron donation change the nature of
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ABTS+• green to ABTS yellow color which was observed in the spectrophotometer

measurement.

Possible mechanism of ABTS•+ and protocatechuic acid:

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Protocatechuic acid ABTS
ABTS+•

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Radical can further withdraw
hydrogen atom (H•) to form stable
quinone
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The same reaction of electron donation process might be possible for other
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compounds such as ferulic acid, vanillic acid and epicatechin. Thus, the synergistic effect of

the identified compounds significantly (p<0.05) increases the ABTS+• scavenging activity of
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the fresh fruit pulp and the extracts of P. subpeltata fruit.

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3.3.3. FRAP, metalchelating and phosphomolybdenum activities

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Table 3 also presents the data on ferric reducing, metalchelating and

phosphomolybdenum activities of fresh fruit pulp and solvent extracts of the P. subpeltata
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fruit. The fresh pulp had significantly stronger (p<0.05) ferric reducing (1117.15 mM Fe (II)/

mg extract), metalchelating (685.72 mg EDTA/g extract) and phosphomolybdenum (516.80

mg AAE/g extract) activities than the other solvent extracts of fruit pulp. In human body

transition metals play a vital role for the activity of many enzymes. Their unpaired electron

reacts very quickly with peroxides, resulting in the formation of alkoxyl radicals. The

chelation of transition metals by antioxidants can be considered an important mechanism in

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oxidation processes (Lazarova et al., 2014). It is well known that the flavonoids and phenolic

compounds such as protocatechuic acid, ferulic acid, vanillic acid and epicatechin efficiently

chelate metal ions and these also possess the ferric reducing antioxidant power and

phosphomolybdenum reduction properties Li et al., (2011). These authors also reported the

mechanism involved between protocatechuic acid and metalchelating activity. According to

them the chelation of metal ions may arise from the ortho-dihydroxyl group in

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protocatechuic acid. The binding process of protocatechuic acid and Fe2+ is explained as

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follows:

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Our earlier report from P. subpeltata plant leaves revealed that the acetone extract
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showed higher metal chelating activity (236.72 and 202.66 mg EDTA/g extracts) and ferric

reducing activity (3863.33 mM Fe (II)/ mg extract) (Saravanan et al., 2014). When compared
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to these results the present study revealed that the fresh pulp of P. subpeltata fruit has
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maximum ion chelation (685.72 mg EDTA/g extract) activity but not in ferric reducing

power activity (1117.15 mM Fe (II)/ mg extract).

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3.3.4. Superoxide radical scavenging activity

Superoxide anion scavenging is extremely important to antioxidant activity, known to

initiate indirectly by the lipid peroxidation as a result of the formation of H2 O2 , creating

precursors of hydroxyl radical. The data on scavenging activities of P. subpeltata fruit pulp

against superoxide anion are presented in Figure 2. Notably, fresh pulp showed significantly

(p< 0.05) stronger scavenging activity than the solvent extracts. The IC50 value (13.83µg/mL)
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of the fresh pulp was significantly close to the standards including BHT (9.51µg/ml) and

rutin (11.67µg/ml). Superoxide anion (O 2 •-) is one of the important free radicals in living

cells and it is also the precursor of all reactive oxygen species, which can be converted to

other active radicals, such as H2 O2 , ONOO -. O2 •- is closely related to the occurrence of

inflammation, aging and cancer, and hence it´s detection is important (Han et al., 2017). As

per the result of this present study, a mixture of phenolic and flavonoid compounds reacts

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with superoxide anion radical and scavenge it. Protocatechuic acid has already been reported

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earlier that this compound has superoxide anion radical scavenging activity as a dose-

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dependent manner and it also exhibits higher inhibition level than Trolox standard (Li et al.,

2011). In our recent study we reported that the species P. ligularis fruit pulp´s acetone
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extract shows the maximum superoxide anion radical scavenging activity. This study also

proved that the synergistic effects of the polyphenols could be responsible for this radical
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scavenging activity (Saravanan & Parimelazhagan, 2014). Hence, the functional compounds

and its synergistic effects might be the main reason for the fresh fruit pulp has the higher
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activities of all the tested antioxidant assays when compared to the other solvent extracts

(petroleum ether, chloroform, methanol and acetone) which have different polarity.
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3.4. Inhibition of α-amylase and α-glucosidase

α-amylase and α-glucosidase inhibitory activities of the fresh pulp and pulp extracts
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are shown in Figure 3. The IC 50 and inhibition percentage values of fresh pulp were

maximum in both α-amylase (84.66%, IC50 = 18.69 µg/mL) and α-glucosidase (86.89%, IC50

= 32.63 µg/mL) enzymes in a concentration dependent manner. These results were

significantly (p<0.05) comparable with the standard drug, acarbose for α-amylase and α -

glucosidase inhibition (89.92% and 91.39%, IC50 = 17.91 and 26.18 µg/mL, respectively).

Moreover these results were similar to the changing trend of DPPH radical scavenging
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activity. All other solvent extracts showed moderate enzyme inhibition activity. The number

of the hydroxyl groups on the phenolic and flavonoid compounds especially, on the B ring

was closely associated with the inhibitory activity of the α-amylase and α-glucosidase

enzymes (Liu, Ai, Qu, Chen, & Ni, 2017). According to this hypothesis, the fresh pulp of the

P. subpeltata fruit has 15 phenolic and flavonoid compounds which were identified in this

plant species and these may assist in inhibiting the diabetic key enzymes. Specifically the

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compounds viz. protocatechuic acid, ferulic acid, vanillic acid and epicatechin could

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efficiently involve in the process of key enzyme inhibition. The main reason behind this

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process might be the many hydroxyl groups present in those compounds. Many recent

studies prove that the polyphenols rich plants have the ability to chelate the enzymes of α-
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amylase and α-glucosidase by their alteration in structure coupled with losing of biological

functions (Nagappan et al., 2017; Saravanan & Parimelazhagan, 2014). Furthermore, the
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major compound identified in this plant, protocatechuic acid is already reported that if this

compound is orally administered, it reduces the hyperglycemia-induced advanced glycation

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end products (AGEs) both in plasma and in organs such as kidney (Lin, Tsai, Huang, & Yin,

2011). These authors also suggested possible molecular mechanism of the protocatechuic
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acid on diabetic rats by regulating mRNA expression, peroxisome proliferator-activated

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receptor α (PPARα) and PPARγ the key metabolic regulators of glucose, lipid metabolism

and clinically validated gene for therapeutic utility in T2DM. Moreover administration of
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protocatechuic acid to diabetic rats decreased the levels of cholesterol and maintains the Low

density lipid cholesterol and High density lipid cholesterol levels. Moreover, ferulic acid

treatment could improve the diabetes-induced cognition impairment. Prolonged exposure of

chronic hyperglycemia promoted inflammation release, AD-like neurodegenerative

alterations and damaged insulin signaling, which led to cognitive deficits in diabetic rats.

(Wang et al., 2017). Thus, it could be concluded that the presence of ferulic acid in P.
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subpeltata fruit pulp might be the other reason for regulating the α-amylase and α-

glucosidase enzyme processes. From these possible mechanisms and synergistic effects of

functional compounds of P. subpeltata fruit pulp and its extracts, the glucose levels in blood

could decrease which plays an important role in the inhibition of α-amylase and α-

glucosidase enzymes and thus helps to control the T2DM condition.

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Conclusion

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Potential anti-diabetic effect, antioxidant, nutritional and functional compounds

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identification of P. subpeltata in fruit pulp and its extracts were explored in this plant species.

The fresh fruit pulp revealed α-amylase and α-glucosidase activity with maximum inhibition
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percentage and minimum IC50 value. Moreover, the antioxidant properties of the fresh fruit

pulp showed maximum activity analyzed in vitro assays such as DPPH, ABTS, FRAP,
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superoxide, metal chelating and Phosphomolybdenum activities. The nutritional value

revealed that the fruit is a rich source of amino acids, minerals, carbohydrates and protein
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contents. The direct intake of this fruit pulp can supply the natural secondary metabolites

including protocatechuic acid, ferulic acid, vanillic acid, epicatechin and p-coumaric acid,
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cinnamic acid, eriodictyol and quercetin-3-glucoside. Thus, this work shows the large
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potential of P. subpeltata species for the processing of this fruit for nutraceutical applications

and in the glucose control management for Type 2 diabetic suffering humans.
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Acknowledgment

The authors acknowledge the support of Programa Nacional de Pós-

doutorado/CAPES (PNPD/CAPES - 2013) and FAPITEC –SE, Brazil for Post-Doctoral

fellowship to author, Saravanan Shanmugam and for providing financial assistance,

respectively.
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Conflict of Interest

All authors declare that there is no conflict of interest.

Reference.

Allen, S. E. (1989). Chemical analysis of ecological materials. (S. E. Allen, Ed.) (2nd ed.).

T
London: Blackwell Science Ltd.

P
RI
Andrade, J. K. S., Denadai, M., de Oliveira, C. S., Nunes, M. L., & Narain, N. (2017).

SC
Evaluation of bioactive compounds potential and antioxidant activity of brown, green

and red propolis from Brazilian northeast region. Food Research International, 101,
NU
129–138. https://doi.org/10.1016/j.foodres.2017.08.066

AOAC. (1995). Official Methods of Analysis of AOAC International. Gaithersburg, MD,

MA

Arlington.

Apostolidis, E., Kwon, Y.-I., & Shetty, K. (2007). Inhibitory potential of herb, fruit, and
ED

fungal-enriched cheese against key enzymes linked to type 2 diabetes and hypertension.

Innovative Food Science & Emerging Technologies, 8(1), 46–54.

PT

https://doi.org/10.1016/j.ifset.2006.06.001
CE

Balan, K., Ratha, P., Prakash, G., Viswanathamurthi, P., Adisakwattana, S., & Palvannan, T.

(2017). Evaluation of invitro α-amylase and α-glucosidase inhibitory potential of N 2 O2

AC

schiff base Zn complex. Arabian Journal of Chemistry, 10, 732–738.

https://doi.org/10.1016/j.arabjc.2014.07.002

Beauchamp, C., & Fridovich, I. (1971). Superoxide dismutase: improved assays and an assay

applicable to acrylamide gels. Analytical Biochemistry, 44(1), 276–287. Retrieved from

http://www.ncbi.nlm.nih.gov/pubmed/4943714

Bhardwaj, R. L., Nandal, U., Pal, A., & Jain, S. (2014). Bioactive compounds and medicinal
 ACCEPTED MANUSCRIPT

properties of fruit juices. Fruits, 69(5), 391–412. https://doi.org/10.1051/fruits/2014027

Blois, M. S. (1958). Antioxidant determinations by the use of a stable free radical. Nature,

181(4617), 1199–1200. https://doi.org/10.1038/1811199a0

Costa, G. M., Cárdenas, P. A., Gazola, A. C., Aragón, D. M., Castellanos, L., Reginatto, F.

H., Schenkel, E. P. (2015). Isolation of C-glycosylflavonoids with α-glucosidase

inhibitory activity from Passiflora bogotensis Benth by gradient high-speed counter-

T
current chromatography. Journal of Chromatography B, 990, 104–110.

P
RI
https://doi.org/10.1016/j.jchromb.2015.03.015

SC
Dembitsky, V. M., Poovarodom, S., Leontowicz, H., Leontowicz, M., Vearasilp, S.,

Trakhtenberg, S., & Gorinstein, S. (2011). The multiple nutrition properties of some
NU
exotic fruits: Biological activity and active metabolites. Food Research International,

44(7), 1671–1701. https://doi.org/10.1016/j.foodres.2011.03.003

MA

Dinis, T. C. P., Madeira, V. M. C., & Almeida, L. M. (1994). Action of phenolic derivatives

(acetaminophen, salicylate, and 5-aminosalicylate) as inhibitors of membrane lipid

ED

peroxidation and as peroxyl radical scavengers. Archives of Biochemistry and

Biophysics, 315(1), 161–169. https://doi.org/10.1006/abbi.1994.1485

PT

FAO, WHO, & ONU. (2007). Energy and protein requirements. Technical Report Series No.
CE

724. Geneva.

Gazola, A. C., Costa, G. M., Castellanos, L., Ramos, F. A., Reginatto, F. H., de Lima, T. C.
AC

M., Schenkel, E. P. (2015). Involvement of GABAergic pathway in the sedative activity

of apigenin, the main flavonoid from Passiflora quadrangularis pericarp. Revista

Brasileira de Farmacognosia, 25(2), 158–163. https://doi.org/10.1016/j.bjp.2015.03.009

Graf, E. (1992). Antioxidant potential of ferulic acid. Free Radical Biology & Medicine,

13(4), 435–48. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1398220

Han, J., Liu, Z., Guo, Y., Han, G.-C., Li, W., Chen, S., & Zhang, S. (2017). Determination of
 ACCEPTED MANUSCRIPT

superoxide anion radical by modified CdTe quantum dots.

https://doi.org/10.1016/j.jphotochem.2017.08.049

Hanhineva, K., Törrönen, R., Bondia-Pons, I., Pekkinen, J., Kolehmainen, M., Mykkänen, H.,

& Poutanen, K. (2010). Impact of dietary polyphenols on carbohydrate metabolism.

International Journal of Molecular Sciences, 11(4), 1365–1402.

https://doi.org/10.3390/ijms11041365

T
Hoon Kim, J., Young Kim, H., Young Yang, S., Kim, J.-B., Hyun Jin, C., & Ho Kim, Y.

P
RI
(2017). Article in press G Model Inhibitory activity of (−)-epicatechin-3,5-O-digallate on

α-glucosidase

SC
and in silico analysis. International Journal of Biological

Macromolecules. https://doi.org/10.1016/j.ijbiomac.2017.09.091
NU
Ishida, Y., Fujita, T., & Asai, K. (1981). New detection and separation method for amino

acids by high-performance liquid chromatography. Journal of Chromatography, 204,

MA

143–148. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/7217249

Jiménez, A. M., Sierra, C. A., Rodríguez-Pulido, F. J., González-Miret, M. L., Heredia, F. J.,
ED

& Osorio, C. (2011). Physicochemical characterisation of gulupa (Passiflora edulis

Sims. fo edulis) fruit from Colombia during the ripening. Food Research International,
PT

44(7), 1912–1918. https://doi.org/10.1016/j.foodres.2010.11.007

CE

Lazarova, I., Zengin, G., Aktumsek, A., Gevrenova, R., Ceylan, R., & Uysal, S. (2014).

HPLC–DAD analysis of phenolic compounds and antioxidant properties of Asphodeline

AC

lutea roots from Bulgaria and Turkey. Industrial Crops and Products, 61, 438–441.

https://doi.org/10.1016/j.indcrop.2014.07.044

Li, X., Wang, X., Chen, D., & Chen, S. (2011). Antioxidant activity and mechanism of

protocatechuic acid in vitro. Functional Foods in Health and Disease, 7, 232–244.

Retrieved from http://www.functionalfoodscenter.net/files/46832219.pdf

Lin, C.-Y., Tsai, S.-J., Huang, C.-S., & Yin, M.-C. (2011). Antiglycative effects of
 ACCEPTED MANUSCRIPT

protocatechuic acid in the kidneys of diabetic mice. Journal of Agricultural and Food

Chemistry, 59(9), 5117–5124. https://doi.org/10.1021/jf200103f

Liu, J., Meng, C.-G., Yan, Y.-H., Shan, Y.-N., Kan, J., & Jin, C.-H. (2016). Protocatechuic

acid grafted onto chitosan: Characterization and antioxidant activity. International

Journal of Biological Macromolecules, 89, 518–526.

https://doi.org/10.1016/j.ijbiomac.2016.04.089

T
Liu, S., Ai, Z., Qu, F., Chen, Y., & Ni, D. (2017). Effect of steeping temperature on

P
RI
antioxidant and inhibitory activities of green tea extracts against α-amylase, α-

SC
glucosidase and intestinal glucose uptake. Food Chemistry, 234, 168–173.

https://doi.org/10.1016/j.foodchem.2017.04.151
NU
Lúcia, A., Zeni, B., Camargo, A., & Dalmagro, A. P. (2017). Ferulic acid reverses depression-

like behavior and oxidative stress induced by chronic corticosterone treatment in mice.
MA

Steroids, 125, 131–136. https://doi.org/10.1016/j.steroids.2017.07.006

Mathers, C. D., & Loncar, D. (2006). Projections of global mortality and burden of disease
ED

from 2002 to 2030. PLoS Medicine, 3(11), e442.

https://doi.org/10.1371/journal.pmed.0030442
PT

Meng, J.-F., Fang, Y.-L., Qin, M.-Y., Zhuang, X.-F., & Zhang, Z.-W. (2012). Varietal
CE

differences among the phenolic profiles and antioxidant properties of four cultivars of

spine grape (Vitis davidii Foex) in Chongyi County (China). Food Chemistry, 134(4),
AC

2049–2056. https://doi.org/10.1016/j.foodchem.2012.04.005

Missio, P., Silva, D., Gauche, C., Gonzaga, L. V., Oliveira Costa, A. C., & Fett, R. (2016).

Honey: Chemical composition, stability and authenticity. Food Chemistry, 196, 309–

323. https://doi.org/10.1016/j.foodchem.2015.09.051

Nagappan, H., Pee, P. P., Hui, S., Kee, Y., Ow, J. T., Yan, S. W., Kong, K. W. (2017).

Malaysian brown seaweeds Sargassum siliquosum and Sargassum polycystum: Low

 ACCEPTED MANUSCRIPT

density lipoprotein (LDL) oxidation, angiotensin converting enzyme (ACE), α-amylase,

and α-glucosidase inhibition activities. https://doi.org/10.1016/j.foodres.2017.01.023

Ngoh, Y.-Y., Lim, T. S., & Gan, C.-Y. (2016). Screening and identification of five peptides

from pinto bean with inhibitory activities against α-amylase using phage display

technique. Enzyme and Microbial Technology, 89, 76–84.

https://doi.org/10.1016/j.enzmictec.2016.04.001

T
Olafsdottir, E. S., Thorgeirsdottir, E., & Jaroszewski, J. W. (1997). European journal of

P
RI
pharmaceutical sciences. European Journal of Pharmaceutical Sciences (Vol.

SC
Supplement 1). Elsevier B.V. Retrieved from

https://www.infona.pl/resource/bwmeta1.element.elsevier-cc8efa7b-bb40-32c4-8f9c-
NU
cac9e77d2165

Porto-Figueira, P., Freitas, A., Cruz, C. J., Figueira, J., & Câmara, J. S. (2015). Profiling of
MA

passion fruit volatiles: An effective tool to discriminate between species and varieties.

Food Research International, 77, 408–418.

ED

https://doi.org/10.1016/j.foodres.2015.09.007

Prieto, P., Pineda, M., & Aguilar, M. (1999). Spectrophotometric quantitation of antioxidant
PT

capacity through the formation of a phosphomolybdenum complex: specific application

CE

to the determination of vitamin E. Analytical Biochemistry, 269(2), 337–341.

https://doi.org/10.1006/abio.1999.4019
AC

Pulido, R., Bravo, L., & Saura-Calixto, F. (2000). Antioxidant activity of dietary polyphenols

as determined by a modified ferric reducing/antioxidant power assay. Journal of

Agricultural and Food Chemistry, 48(8), 3396–3402. Retrieved from

http://www.ncbi.nlm.nih.gov/pubmed/10956123

Ratheesh Narayanan, M. K., & Anil Kumar, N. (2007). Gendered knowledge and changing

trends in utilization of wild edible greens in Western Ghats, India. Indian Journal of
 ACCEPTED MANUSCRIPT

Traditional Knowledge, 6(1), 204–216. Retrieved from

http://nopr.niscair.res.in/bitstream/123456789/908/1/IJTK 6%281%29 %282007%29

204-216.pdf

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999).

Antioxidant activity applying an improved ABTS radical cation decolorization assay.

Free Radical Biology {&} Medicine, 26(9–10), 1231–1237. Retrieved from

T
http://www.ncbi.nlm.nih.gov/pubmed/10381194

P
RI
Sadasivam, S., & Manickam, A. (2008). Biochemical Methods (3rd ed.). New Delhi: New

SC
Age International.

Sadia, H., Ahmad, M., Sultana, S., Abdullah, A. Z., Teong, L., Zafar, M., & Bano, A. (2014).
NU
Nutrient and mineral assessment of edible wild fig and mulberry fruits. Fruits, 69(2),

159–166. https://doi.org/10.1051/fruits/2014006
MA

Saito, S., & Kawabata, J. (2005). Effects of electron-withdrawing substituents on DPPH

radical scavenging reactions of protocatechuic acid and its analogues in alcoholic

ED

solvents. https://doi.org/10.1016/j.tet.2005.06.040

Saravanan, S., Arunachalam, K., & Parimelazhagan, T. (2014). Antioxidant, analgesic, anti-
PT

inflammatory and antipyretic effects of polyphenols from Passiflora subpeltata leaves –

CE

A promising species of Passiflora. Industrial Crops and Products, 54, 272–280.

https://doi.org/10.1016/j.indcrop.2014.01.038
AC

Saravanan, S., & Parimelazhagan, T. (2013). Total phenolic content, Free radical scavenging

and Antimicrobial activities of Passiflora subpeltata seeds. Journal of Applied

Pharmaceutical Science, 3(4), 67–72. https://doi.org/10.7324/JAPS.2013.3412

Saravanan, S., & Parimelazhagan, T. (2014). In vitro antioxidant, antimicrobial and anti-

diabetic properties of polyphenols of Passiflora ligularis Juss. fruit pulp. Food Science

and Human Wellness, 3(2), 56–64. https://doi.org/10.1016/j.fshw.2014.05.001

 ACCEPTED MANUSCRIPT

Sasikala, V., Saravana, S., & Parimelazhagan, T. (2011). Evaluation of antioxidant potential

of different parts of wild edible plant Passiflora foetida L. Journal of Applied

Pharmaceutical Science, 1(4), 89–96. Retrieved from

http://pesquisa.bvsalud.org/ghl/resource/pt/oai- imsear.hellis.org-123456789-150792

Septembre-Malaterre, A., Stanislas, G., Douraguia, E., & Gonthier, M.-P. (2016). Evaluation

of nutritional and antioxidant properties of the tropical fruits banana, litchi, mango,

T
papaya, passion fruit and pineapple cultivated in Réunion French Island. Food

P
RI
Chemistry, 212, 225–233. https://doi.org/10.1016/j.foodchem.2016.05.147

SC
Shanmugam, S., Murugaiyan, I., dos Santos Lima, B., Serafini, M. R., de Souza Araújo, A.

A., Narain, N., Thangaraj, P. (2016). HPLC–DAD–MS identification of polyphenols

NU
from Passiflora leschenaultii and determination of their antioxidant, analgesic, anti-

inflammatory and antipyretic properties. Arabian Journal of Chemistry.

MA

https://doi.org/10.1016/j.arabjc.2016.02.008

Shanmugam, S., Sivaraj, D., dos Santos Lima, B., dos Passos Menezes, P., de Carvalho, Y.
ED

M. B. G., de Souza Araújo, A. A., Parimelazhagan, T. (2017). Polyphenols rich

Passiflora leschenaultii leaves modulating Farnesoid X Receptor and Pregnane X

PT

Receptor against paracetamol-induced hepatotoxicity in rats. Biomedicine {&}

CE

Pharmacotherapy, 88, 1114–1121. https://doi.org/10.1016/j.biopha.2017.01.156

Shanmugam, S., Thangaraj, P., Lima, B. dos S., Chandran, R., de Souza Araújo, A. A.,
AC

Narain, N., Júnior, L. J. Q. (2016). Effects of luteolin and quercetin 3-β- d -glucoside

identified from Passiflora subpeltata leaves against acetaminophen induced

hepatotoxicity in rats. Biomedicine & Pharmacotherapy, 83, 1278–1285.

https://doi.org/10.1016/j.biopha.2016.08.044

Shuayprom, A., Sanguansermsri, D., Sanguansermsri, P., Fraser, I. H., & Wongkattiya, N.

(2016). Quantitative determination of vitexin in Passiflora foetida Linn. leaves using

 ACCEPTED MANUSCRIPT

HPTLC. Asian Pacific Journal of Tropical Biomedicine, 6(3), 216–220.

https://doi.org/10.1016/j.apjtb.2015.11.006

Siddhuraju, P., & Becker, K. (2003). Studies on antioxidant activities of mucuna seed

(Mucuna pruriens varutilis) extract and various non-protein amino/imino acids throughin

vitro models. Journal of the Science of Food and Agriculture, 83(14), 1517–1524.

https://doi.org/10.1002/jsfa.1587

T
Tee, E. S., Kuladevan, R., Young, S. I., Khor, S. C., & Zakiyah, H. O. (1996). Laboratory

P
RI
procedures in nutrient analysis of foods. Division of Human Nutrition, Institute of

SC
Medical Research, Kuala Lumpur, Malaysia.

Viganó, J., Aguiar, A. C., Moraes, D. R., Jara, J. L. P., Eberlin, M. N., Cazarin, C. B. B.,
NU
Martínez, J. (2016). Sequential high pressure extractions applied to recover piceatannol

and scirpusin B from passion fruit bagasse. Food Research International, 85, 51–58.
MA

https://doi.org/10.1016/j.foodres.2016.04.015

Vilcacundo, R., Martínez-Villaluenga, C., & Hernández-Ledesma, B. (2017). Release of

ED

dipeptidyl peptidase IV, α-amylase and α±-glucosidase inhibitory peptides from quinoa

(Chenopodium quinoa Willd.) during in vitro simulated gastrointestinal digestion.

PT

https://doi.org/10.1016/j.jff.2017.06.024
CE

Vinothiya, K., & Ashokkumar, N. (2017). Modulatory effect of vanillic acid on antioxidant

status in high fat diet-induced changes in diabetic hypertensive rats.

AC

https://doi.org/10.1016/j.biopha.2016.12.134

Wang, H., Sun, X., Zhang, N., Ji, Z., Ma, Z., Fu, Q., Ma, S. (2017). Ferulic acid attenuates

diabetes-induced cognitive impairment in rats via regulation of PTP1B and insulin

signaling pathway. Physiology & Behavior, 182, 93–100.

https://doi.org/10.1016/j.physbeh.2017.10.001

WHO. (2016). Global report on diabetes WHO Library Cataloguing-in-Publication Data.

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ISBN (Vol. 978). Retrieved from http://www.who.int/about/licensing/

Worthington, V. (1993). Alpha Amylase, in V. Worthington (Ed.), Worthington Enzyme

Manual Freehold, Worthington Biochemical Corp, NJ,. In Worthington Enzyme Manual

Freehold, Worthington Biochemical Corp, NJ, (pp. 36–41). Retrieved from

http://www.worthington-biochem.com/BA/default.html

Zhang, B., Deng, Z., Ramdath, D. D., Tang, Y., Chen, P. X., Liu, R., Tsao, R. (2015).

T
Phenolic profiles of 20 Canadian lentil cultivars and their contribution to antioxidant

P
RI
activity and inhibitory effects on α-glucosidase and pancreatic lipase. Food Chemistry,

SC
172, 862–872. https://doi.org/10.1016/j.foodchem.2014.09.144

Zhishen, J., Mengcheng, T., & Jianming, W. (1999). The determination of flavonoid contents
NU
in mulberry and their scavenging effects on superoxide radicals. Food Chemistry, 64(4),

555–559. https://doi.org/10.1016/S0308-8146(98)00102-2
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Figure Captions
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Figure. 1. Chemical Structures of principal identified compounds in P. subpeltata fruit

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pulp.
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Figure. 2. DPPH and Superoxide radical scavenging activity of P. subpeltata fruit pulp

and its solvent extracts.

Values are expressed as mean ± standard deviation of 3 determinations (n = 3);

Values are expressed as the amount of sample required to decrease the initial concentration

of DPPH by 50% (IC 50 );

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Mean values in the histograms followed by different superscript letters indicate significant

statistical difference (p<0.05)

Figure. 3. α-amylase and α-glucosidase enzymes inhibition by P. subpeltata fruit pulp

and their solvent extracts.

Values are expressed as mean ± standard deviation of different aliquot determinations (n = 3).

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Mean values followed by different superscript letters indicate significant statistical difference

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(p< 0.05).

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Table 1: MS/MS parameters, retention time for selected polyphenolic compounds and their
concentrations present in P. subpeltata fruit pulp
Products (m/z) Compou
Molec Reten
Ioniza Precu C Confirm C nd
Compou ular Quantific a a tion
tion rsor E ation E concentra
nd formul ation time
a mode ion
transition
(e transitio (e
(min) tionb
V) n V) (ng/g)
Artepellin C19 H24 15 15
ESI+ 301 245 189 29.3 < LOQ
C O3
Chrologe C16 H18 15 25
ESI+ 355 163 145 3.16 < LOQ
nic acid O9

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Cinnamic 15
C9 H8 O2 ESI+ 149 103 25 131 11.5 9.08
acid

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coumaric C9 H8 O3 ESI+ 165 147 15 91 6.51 20.54

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acid
C15 H10 25 25
Daidzein ESI+ 255 199 137 11.8 < LOQ

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O4
Epicatech C15 H14 15 15
ESI+ 291 139 123 3.62 22.45
in O6
Eriodicty C15 H12 30 25
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ESI+ 289 153 145 13.1 8.02
ol O6
Ferulic C10 H10 15 20
ESI+ 195 177 134 7.47 55.76
acid O4
Gallic 15 15
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C7 H6 O5 ESI+ 171 109 125 2.17 < LOQ

acid
C15 H10 35 35
Luteolin ESI+ 287 153 135 14.7 < LOQ
O6
C15 H12 35 35
Narigenin ESI+ 273 153 119 16.0 < LOQ
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O5
Protocate 15 35
C7 H6 O4 ESI+ 155 65 137 2.80 115.43
chuic acid
Quercetin 15 35
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C21 H20
-3- ESI+ 465 303 85 7.24 3.04
O12
glucoside
Vanilin C8 H8 O3 ESI+ 153 93 15 65 35 6.51 < LOQ
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Vanillic 15 35
C8 H8 O4 ESI+ 169 93 65 4.57 46.01
acid
a
: Collision energy
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b
: <LOQ, compound presented in sample under Limit of Quantification.
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Table 2a. Proximate Composition of P. subpeltata fruit pulp.

Parameters Value
Moisture (%) 95.02 ± 2.45
Crude Fiber (g/100g sample) 5.89 ± 0.56
Ash Content (g/100g sample) 1.45 ± 0.29
Total Protein (mg BSA equivalent/g sample) 8.80 ± 0.48
Total Carbohydrates (mg glucose equivalent/g sample) 2.62 ± 0.69
Total Amino acids (mg Leucine equivalent/g sample) 1.44 ± 0.59
Values are expressed as mean ± standard deviation of three determination (n= 3);
BSA: Bovine serum albumin.

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Table 2b: Minerals quantification of P. subpeltata fruit pulp
Minerals (mg/g dry weight basis)

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P K Na Ca Fe Cu
0.10±0.08 1.81±0.27 0.02±0.00 50.67±5.58 1.73±0.35 ND

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Mg Mn Zn Si B Mo
0.02±0.00 0.06±0.01 0.19±0.07 ND 0.54±0.04 ND
Values are expressed as mean ± standard deviation of three determination (n=3);
ND – Not Detected
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Table 2c. Amino acids profile of P. subpeltata fruit pulp.
Reference pattern
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Amino acids
Fruit pulp FAO/ WHO (2007)
(g/100g protein)
(mg/g protein)
Alanine 57.00 -
Arginine 115.00 -
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Aspartic acid 105.00 -

Cystine 5.00 6.00
Glutamic acid 243.00 -
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Glycine 54.00 -
Histidine 21.00 15.00
EP

Isolucine 33.00 30.00

Leucine 66.00 59.00
Lysine 27.00 45.00
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Methionine 3.00 16.00

Phenylalanine 75.00 38.00
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Proline 8.00 -
Serine 56.00 -
Threonine 33.00 23.00
Tryptophan 5.00 6.00
Tyrosine 15.00 38.00
Valine 55.00 39.00
Values for each amino acid of the fruit pulp is expressed relatively to the reference
pattern of FAO/ WHO (2007).
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Table 3. Total phenolics, tannins and flavonoids contents and ABTS+•, FRAP, Metal
chelating and Phosphomolybdenum assays of P. subpeltata fruit pulp and extracts.
Metal
Total Flavonoi
Tannin ABTS+• FRAP chelating Phosphomo
Solvents phenolics ds
(GAE (μM trolox (mM Fe Activity lybdenum
used for (GAE (RE
mg/ g equi/g (II)/ mg (mg (mgAAE/g
extraction mg/ g mg/g
extract) extract) extract) EDTA/g extract)
extract) extract)
extract)
Petrolem 197.14±1.8 57.62±2. 620.00±3.3 917.99±
216.37±2.95b 27.56±2.11c 58.40±1.39d
Ether* 9c 51c 3c 25.48c
Chlorofor 175.71±1.8 25.95±2. 583.33±8.8 1444.49±
210.54±2.11b 253.54±1.44b 74.00±0.69c

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m* 9c 30d 2c 5.85b

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553.81±1.0 206.19±1 1340.00±3. 4589.97±
Acetone* 9a .65a 33a 5.85a
999.72±8.10a 571.47±0.80a 306.53±1.62a

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395.95±1.6 169.05±3 1096.67±3. 3952.10±15.4
Methanol* 641.65±0.70a 527.56±1.05a 213.47±0.23ab
5b .22b 33ab 7ab
Fresh 724.76±5.7 358.57±5 4763.33±1 6794.96±20.6 1117.15±13. a a
685.72±1.38 516.80±3.49

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pulp 7a .71a 5.28a 2
a
16
a

*
indicates that fruit pulp extracted with solvent;
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Values are expressed as mean ± standard deviation of triplicate determinations (n = 3);
GAE - Gallic Acid Equivalents; RE - Rutin Equivalents; TE - Trolox Equivalents; EDTAE -
Ethylenediamine Tetra Acetic acid Equivalent; AAE - Ascorbic Acid Equivalent;
Mean values followed by different superscript letters indicate significant statistical difference
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(p< 0.05).
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Figure 1

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Epicatechin Eriodictyol

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Cinnamic acid p-coumaric acid
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Protocatechuic acid Ferulic acid

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Quercetin-3-glucoside Vanillic acid

 Pu
lp
-P
Figure. 2.
et
ro IC50 of DPPH (g/mL)
l iu
m

0
20
40
60
80
Pu
lp Et
-C he
hl r 50.76d
o ro
Pu fo
lp rm
- 64.21e
A
ce

A
Pu t on
lp e
-M 13.32b

C
et
h

C
an
DPPH

Fr ol 20.93c

E
es
h
Pu

P
lp
5.667a
T R
E ut
in 5.67a
D
B
H
T 6.38a
M A

Pu
lp
-P
N

et
ro IC50 (g/mL)
liu
U

m
0
50
100
150
200
250

Pu
lp Et
S

-C he
hl r 207.8e
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or
C

of
Pu or
lp m
R

-A 107.4d
I

ce
Pu to
P

lp ne
-M
42.18c
T

et
ha
no
Fr l 21.33b
Superoxide

es
h
Pu
lp
13.83a
R
ut
in
11.67a
B
H
T
9.51a
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Figure. 3.

100
IC50g/mL
-amylase inhibition (%)

80 Pulp-Petrolium Ether 28.93c  0.85

Pulp-Chloroform 28.15c  0.72
60 Pulp-Acetone 21.85b  0.33
Pulp-Methanol 25.15b  0.51
40 Fresh Pulp 18.69a  0.49
Acarbose 17.91a  0.28

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0 50 100 150 200 250 300
Concentration of sample (g/mL)

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IC50g/mL
-glucosidase inhibition (%)

100
Pulp-Petrolium Ether 92.34e  2.27
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80
Pulp-Chloroform 78.19d  3.55
60 Pulp-Acetone 46.35b  2.91
Pulp-Methanol 63.62c  1.62
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40 Fresh Pulp 32.63a  1.85

Acarbose 26.18a  1.58
20
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0
0 50 100 150 200 250 300
Concentration of sample (g/mL)
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Highlights
 15 phenolic compounds were identified in P. subpeltata fruit for the first time.

 Different in vitro biological activities of P. subpeltata pulp were analyzed.

 Protein and essential amino acids were detected in fruit pulp.

 Fruit pulp serves as potent inhibitors of diabetic key enzymes.

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