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Crudeextractfromtaro(Colocasiaesculenta)asanaturalsourceofbioactiveproteinsabletostimulatehaematopoieticcellsintwomurinemodels

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Crude extract from taro (Colocasia esculenta) as anatural source of bioactive proteins able tostimulate haematopoietic cells in two murinemodels

Patrícia R. Pereira a, Joab T. Silva a, Mauricio A. Verícimo b,Vânia M.F. Paschoalin a,*, Gerlinde A.P.B. Teixeira b

a Instituto de Química, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, 21941-909 Brazilb Instituto de Biologia, Universidade Federal Fluminense, Rio de Janeiro, 4020141 Brazil

A R T I C L E I N F O

Article history:

Received 26 November 2014

Received in revised form 16 July

2015

Accepted 21 July 2015

Available online

A B S T R A C T

Besides its nutritional value, taro (Colocasia esculenta) is traditionally used as a medicinal

plant and provides bioactive compounds with important biological properties. This study

evaluated the in vitro and in vivo immunomodulatory potential of the protein extract from

taro corms on the haematopoietic cells of C57BL/6 and BALB/c mice. The crude taro extract

(CTE) stimulated the in vitro proliferation of mice splenocytes in a dose-dependent manner

in both mice strains. The intraperitoneal inoculation of CTE induced splenomegaly and the

proliferation of total spleen and bone-marrow cells. Additionally, CTE promoted in vivo B220+

splenocyte proliferation, which was accompanied by a reduction of mature (B220+ IgM+) and

immature (B220+ IgM−) B cells in the bone marrow. The production of Antibody by B220+

splenocytes indicated the activation of B1 cells by the CTE only in C57BL/6. CTE represents

a powerful source of immunostimulatory proteins, new candidates as additives for food and

pharmaceutical industries.

© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the

CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords:

Colocasia esculenta

Crude taro extract

Immunostimulatory proteins

Lectin

Spleen and bone-marrow cell

proliferation

Mature and immature B cells

1. Introduction

To address increasing demand for natural products to benefithuman health, bioactive compounds have been extracted froma variety of natural sources including animals, plants, algae,microalgae, microorganisms, food and food by-products(Herrero, Cifuentes, & Ibanez, 2006). Plants are a rich source

of bioactive phytochemicals, which can occur in different forms,such as proteins or peptides, carotenoids, polyphenols,isoflavones and sterols among others (Abuajah, Ogbonna, &Osuji, 2015).

In plants, such bioactive compounds are used for defence,exerting pharmacological or toxicological effects in animals and/or humans. Despite their anti-nutritional value, the beneficialeffects of bioactive compounds in providing protection for the

All authors contributed equally to this work.* Corresponding author. Instituto de Química, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, 21941-909 Brazil. Tel.: +55 21

3938 7362; fax: +55 21 3938 7266.E-mail address: paschv@iq.ufrj.br (V.M.F. Paschoalin).

http://dx.doi.org/10.1016/j.jff.2015.07.0141756-4646/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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health of living beings have stimulated the application of thesemolecules in the agricultural and pharmaceutical fields(Bernhoft et al., 2010; Gupta, Tuohy, O’Donovan, & Lohani, 2015).

Since it is well known that dietary habits are directly relatedto human health maintenance, the application of bioactive com-pounds as functional food ingredients has met recent consumerdemand for products of natural origin. The consumption ofthese products can promote health and well-being by modu-lating metabolic processes, protecting humans against healthdisorders (Mitsuoka, 2014; Plaza, Cifuentes, & Ibáñez, 2008). Allthese benefits are triggered by native bioactive compounds and/or by the addition of new natural compounds, which are nowsubstituting conventional synthetic ones (Lagos, Vargas, deOliveira, da Aparecida Makishi, & do Amaral Sobral, 2015). Phar-maceutical industries have also taken advantage of bioactivecompounds, which offer a source of powerful candidates tosolve the problems faced by the use of several current drugs,such as undesirable side effects and microbial resistance(Bernhoft et al., 2010; Sasidharan, Chen, Saravanan, Sundram,& Latha, 2011).

Immunostimulators, also called immunomodulators, are aninteresting class of bioactive compounds that can be thor-oughly used as food additives, at the same time reinforcinginnate immune system responses in human beings. Exten-sively found in plants, these molecules exert theirpharmacological activity by mimicking cytokine functions,which are naturally occurring stimulators produced by the cellsof the immune system (Gupta et al., 2015; Kumar, Gupta,Sharma, & Kumar, 2011). They cause biochemical and func-tional modifications in these immunocompetent cells(Shanmugham et al., 2006), playing a critical role in the in-duction of proliferation, differentiation and cell death (Tulin& Ecleo, 2007). These compounds can specifically interact withcertain sugars on the cell surface of one or several glycopro-teins, leading to cell proliferation of different lymphocyte clones(Shanmugham et al., 2006).

Taro [Colocasia esculenta (L.) Schott; Araceae], an annual her-baceous plant from tropical and sub-tropical regions (Taraket al., 2011) and a member of the Araceae superfamily, is a low-cost and widely consumed staple food in the human diet (VanDamme et al., 1995). Its corms provide important nutritionalcomponents such as carbohydrates, protein, thiamine, ribo-flavin, niacin, oxalic acid, calcium oxalate, minerals, lipids,unsaturated fatty acids and anthocyanins (Ramos Filho, Ramos,& Hiane, 1997; Subhash, Sarla, & Jaybardhan, 2012). Taro hassuperior nutritional value compared with potato, sweet potato,cassava and rice. Additionally, what makes taro a valuable edibleplant is the easy digestion of its starch granules and its non-allergenic properties (Lim, 2015; Prajapati, Kalariya, Umbarkar,Parmar, & Sheth, 2011).

Additionally, taro has traditionally been used as a medici-nal plant for curative purposes. A wide variety of bioactivecompounds can be extracted from all plant parts of this species.These molecules were shown to have important pharmaco-logical activities including anticancer (Brown, Reitzenstein, Liu,& Jadus, 2005), antihyperlipidaemic/antihypercholesterolaemic(Sakano et al., 2005), anxiolytic (Kalariya, Parmar, & Sheth, 2010),wound healing (Gonçalves et al., 2013), antimelanogenic (Kim,Moon, Kim, & Lee, 2010), anti-inflammatory (Biren, Nayak, Bhatt,Jalalpure, & Seth, 2007), probiotic (Brown & Valiere, 2004),

antihypertensive (Vasant et al., 2012), antidiabetic (Eleazu,Iroaganachi, & Eleazu, 2013; Kumawat, Chaudhari, Wani,Deshmukh, & Patil, 2010; Li, Hwang, Kang, Hong, & Lim, 2014),antioxidant (Lee, Wee, Yong, & Syamsumir, 2011),hepatoprotective (Patil & Ageely, 2011), anti-inflammatory, an-timicrobial (Dhanraj, Kadam, Patil, & Mane, 2013), anti-helminthic (Kubde, Khadabadi, Farooqui, & Deore, 2010),proliferative (Pereira et al., 2014) and hypolipidaemic (Boban,Nambisan, & Sudhakaran, 2006) properties.

The purpose of this study was to determine the in vitro andin vivo immunomodulatory potential of the crude protein extractof taro corms (C. esculenta) on haematopoietic cells from thespleen and bone marrow, using two distinct murine models,the C57BL/6 and BALB/c strains. The total number of cells fromspleen and bone marrow were evaluated following crude taroextract inoculation. Additionally, spleen weight × body weightwas analysed and confronted with spleen cellularity.The in vivoeffects of the crude taro extract on mice B220+ lymphocytesfrom both spleen and bone marrow were also evaluated, as wellas the triggering of antibody production by spleen cells.

2. Materials and methods

2.1. Plant material and animals

C. esculenta (L.) Schott corms were purchased from a localgrocery store in the municipality of Niterói, Brazil. A voucherspecimen was deposited at the RFA Herbarium of theUniversidade Federal do Rio de Janeiro (UFRJ), Centro de Ciênciasda Saúde, Departamento de Botânica (Rio de Janeiro, Brazil)under the designation RFA-39.962 (Fig. S1).

Female inbred C57BL/6 and BALB/c mice (8 weeks old) wereobtained from the Animal Facility at the Universidade FederalFluminense (UFF), and were maintained under conventionalenvironmental conditions with air exhaustion, room tempera-ture at 23–25 °C, fed Nuvilab CR-1 chow (Nuvital Nutrientes S/A,Colombo, PR, Brazil) and acidified water ad libitum. The studyprotocol was approved by the Institutional Ethics Committeefor Animal Research at the Universidade Federal Fluminenseunder number 0019-08.

2.2. Crude taro extract (CTE) preparation

Sliced taro corms were dehydrated overnight at 37 °C in a ven-tilated incubator. The dried sliced corms were homogenized(1 g/5 mL) in 0.05 M sodium phosphate buffer pH 7.2 with theaid of a Waring® blender. The resultant homogenate was in-cubated overnight at 4 °C under constant shaking. Solidparticulates were removed by filtration in cheesecloth and cen-trifugation at 28,000 × g for 20 min (IEC – InternationalCentrifuge, model PR-2, Needham, MA, USA). The superna-tant, or crude taro extract, was stored at −20 °C until use(Carneiro, Rodrigues, De Castro, Da Silva, & Coutinho, 1990).

2.3. Determination of protein concentration

Protein concentration in the CTE was estimated by the Lowrymethod (Lowry, Rosebrough, Farr, & Randall, 1951), using bovineserum albumin (BSA) as a standard.

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2.4. Protein profile analysis by SDS-PAGE

Homogeneous 12.5% polyacrylamide gels under denaturing andreducing conditions were set up according to Laemmli (1970).Aliquots containing 10 and 15 µg of protein were loaded ontothe SDS-gels and the resolved polypeptide bands were ob-served by staining the gels with Coomassie® Brilliant Blue G-250Ultra Pure (USB Corporation, Cleveland, OH, USA) according tothe Kurien and Scofield protocol (Kurien & Scofield, 2012). Mo-lecular masses of the polypeptide bands were estimated bycomparison to the standard protein markers (PageRuler™ Un-stained Protein Ladder, Thermo Scientific, Pittsburgh, PA, USA).The gel figure displayed is representative of multiple experi-ments. Fractionation on SDS-PAGE gel was a routine stepfollowing extract preparation.

2.5. Mice treatment by inoculation of crude taro extract

One milligram of CTE protein, diluted in 0.5 mL of physiologi-cal saline solution (0.85% NaCl), was administeredintraperitoneally to C57BL/6 and BALB/c mice (n = 10 per strain).The control group (n = 5 per strain) received 0.5 mL of physi-ological saline also by intraperitoneal route (Fig. 1).

Five mice of each strain were sacrificed on the 5th day afterCTE inoculation and the 5 remaining animals from each strainwere sacrificed on the 10th day.

2.5.1. In vivo effect of CTE inoculation on the total numberof bone marrow and spleen cells from treated miceOn the 5th and 10th days after CTE inoculation, mice spleenswere removed and immediately weighed to determine the rela-tive spleen weight versus body weight. The spleens werecarefully dissociated in Hank’s balanced salt solution (HBSS)(Sigma-Aldrich, St. Louis, MO, USA) with the aid of a bolter andsyringe plunger to produce a spleen cell suspension. The bonemarrows were also removed and a cell suspension was ob-tained by percolating the femur several times with HBSS. Bothcell suspensions were washed twice in cold HBSS, centri-fuged at 258 × g at 4 °C for 7 min and the supernatants werediscarded at the end of the process. Pelleted cells were

submitted to osmotic shock with a hypotonic solution (HBSSdiluted 5× in distilled water) for 10–20 s under manual agita-tion to eliminate erythrocytes. The erythrocyte-free cellsuspensions were again washed by adding HBSS and centri-fuging at 258 × g at 4 °C for 7 min. The resulting pellet wassuspended in cold HBSS and maintained on ice.

Suspended splenocytes and bone marrow cells, in cold HBSS,were mixed 1:1 (v/v) with 0.4% trypan blue. The mixture wasincubated at room temperature for 3 min after which, viable(unstained) cells were ready for visualization and counting ona Neubauer chamber (Labor Optik, Lancing, UK) using opticalmicroscopy (Olympus BX41 – Olympus America INC, Melville,NY, USA), as described previously (Strober, 2001). The totalnumber of viable cells per millilitre was determined by mul-tiplying the number of counted viable cells by the dilution factorfor trypan blue.

2.5.2. In vivo effect of CTE on B lymphocytes proliferationTo determine the number of B lymphocytes in the spleen andbone marrow after CTE inoculation, both cell suspensions(1 × 106 cells) were, separately, incubated in phosphate-buffered saline (PBS) supplemented with 3% normal mouseserum for 20 min at 4 °C to block future unspecific antibodybinding. The mixture was then centrifuged at 560 × g for 7 minat 4 °C and the pellet was suspended in HBSS.

Anti-B220 PE (BD Pharmingen™, San Jose, CA, USA) wasadded to the splenocytes to determine the number of prolif-erating B cells. To analyse mature (B220+IgM+) and immature(B220+IgM−) B cells, anti-B220 PE and anti-IgM FITC (SouthernBiotechnology Associates Inc., AL, USA) were added to bone-marrow cells according to the manufacturer’s instructions. PBScontaining 0.001% sodium azide and 3% foetal calf serum (FCS)was added to the mixture and the cells were incubated in thedark at 4 °C for 40 min. Excess reagent was eliminated bywashing the cells with cold PBS and the cells were fixed with1% formalin. Fluorescence intensity was detected in a Flow Cy-tometer (FACSCalibur, Becton Dickinson, San Jose, CA, USA),where a gate containing only lymphocytes was selected basedon their size and granularity patterns; the results were analysed

Fig. 1 – Experimental design. Evaluation of CTE effects on haematopoietic cells in two murine models.

335J o u rna l o f Func t i ona l F ood s 1 8 ( 2 0 1 5 ) 3 3 3 – 3 4 3

using WinMDI 2.8 software (http://facs.scripps.edu/software.html).

2.5.3. Plaque-forming cell assay (PFC)The method already described by Cunningham and Szenberg(1968) with minor modifications was performed to evaluatethe effect of CTE on the activation of B lymphocytes by theproduction of antibodies against bromelain-treated erythro-cytes (anti-BrMRBC). Spleen cells from CTE-treated mice wereobtained as described previously (section 2.5.1). Briefly, mousered blood cells (MRBC) were collected from the caudal vein,conserved into an anticoagulant solution (3.8% sodium citratein physiological saline) and washed in PBS. The MRBC pelletwas mixed with an equal volume of a 10 mg mL−1 bromelainsolution (Sigma-Aldrich Chemicals), incubated for 30 min at37 °C, washed in PBS and suspended in HBSS, resultingin a 20% (v/v) bromelain-treated MRBC suspension (BrMRBC).Fresh rabbit serum, previously absorbed with BrMRBCs at4 °C for 30 min, was used as a source of complement systemfactors.

A mixture containing 20 µL HBSS, 20 µL rabbit comple-ment, 10 µL BrMRBC and 50 µL of splenocyte suspension (stocksuspension of 2 × 107 cells/mL) was loaded on a Cunningham–Szenberg chamber, sealed with paraffin and incubated at 37 °Cfor 1 h.The number of plaque-forming cells was counted underan optical microscope.

2.6. In vitro proliferation assay of mice splenocytes in thepresence of CTE

Splenocytes (4 × 105 per well) from non-inoculated mice wereobtained as described previously (section 2.5.1) and culturedin 200 µL RPMI-1640 (Sigma-Aldrich Chemicals) completemedium, supplemented with 10% foetal bovine serum, 2 mML-glutamine, 5 × 10−5 M β-mercaptoethanol, 20 µg/mL gentami-cin and increasing amounts of CTE ranging from 100 µg to0.09 µg. Cells were incubated in 96-well flat-bottom plates (TPPTechno Plastic Products AG, Trasadingen, Switzerland) for 72 hat 37 °C in a 5% CO2 atmosphere. To assess proliferation, thecells were pulsed with 0.5 mCi/well [3H]-thymidine (AmershamCo., Amersham, UK) 16 h before the end of the culture. At theend of 72 h, cell suspensions were harvested onto a glass fibrefilter (Titertek, FlowLab, Covina, CA, USA), which was dried andsubmerged in liquid scintillation mixture (Sigma-Aldrich Chemi-cals). Radioactive incorporation was measured on a beta-irradiation counter (Packard, model 1600C, Fallbrook, CA, USA).Assays were performed in triplicate and the results were ex-pressed as scintillations per minute (CPM).

2.7. Statistical analyses

A paired Student’s t test was used to compare the data fromthe cell proliferation assay and an ANOVA with Tukey post-test (Zar, 1984) was used to perform all multiple comparisonsanalyses. Significance was considered when p < 0.05, as deter-mined by the Instat GraphPad Software (GraphPad Software,Inc., San Diego, CA, USA).

3. Results

3.1. Protein profile of the crude taro extract (CTE)

Each CTE obtained from taro corms was routinely analysed by12.5% SDS-PAGE to determine its protein composition. Threemajor groups of polypeptides, with apparent molecular massesof approximately 55, 22 and 12 kDa, were visualized (Fig. 2). Eachgroup was comprised of two polypeptide bands, in agree-ment with previous results (Hirai, Nakamura, Imai, & Sato, 1993;Pereira et al., 2014).

Taro corms contain four major protein families, which arepresent exclusively in the tubers of C. esculenta, not having beenfound in other plant organs (Hirai et al., 1993). Two albumins,A1 (molecular mass 12 to 14 kDa) and A2 (molecular mass 55to 66 kDa); and two globulins, G1 (molecular mass about 14 kDa)and G2 (molecular mass about 22 kDa) are present. The G2globulin family is composed of two protein bands, one of 24 kDa(G2a) and one 22 kDa (G2b) in size, whereas G1 is composedof a large number of 12 kDa isoforms (Pereira et al., 2014;Shewry, 2003).

The semi-quantitative evaluation of the polypeptide bandintensities on the SDS-PAGE performed by densitometric analy-sis, using the GelAnalyzer 2010 software (www.gelanalyzer.com),indicated that the A2 albumin group accounts for about 13%,

Crude taro extractPP

85 kDa120 kDa190 kDa

50 kDa

60 kDa

25 kDa

40 kDa

20 kDa

25 kDa

10 kDa

15 kDa

10 kDa

1 2

Fig. 2 – A representative SDS-PAGE (12.5%) analysis of CTE.The protein profile of each crude extract obtained from tarocorms (Colocasia esculenta) was routinely analysed on a12.5% SDS-PAGE system. Lane P – molecular mass markers(PageRuler™ Unstained Protein Ladder, Thermo Scientific);lanes 1 and 2 – 10 and 15 µg of CTE protein, respectively.

336 J o u rna l o f Func t i ona l F ood s 1 8 ( 2 0 1 5 ) 3 3 3 – 3 4 3

the G2 globulin group for about 28% while the G1 globulin groupaccounts for about 59% of the total CTE protein content.

3.2. In vitro splenocyte proliferation in the presence ofCTE

CTE significantly increased the proliferation of C57BL/6 andBALB/c spleen cells (p < 0.01). For C57BL/6 cells, the optimumamount ranged from 25 µg to 0.39 µg, while in higher (100 and50 µg) or lower amounts (0.19 and 0.09 µg), no stimulatory effectwas detected. BALB/c spleen cells were shown to be more sen-sitive to CTE, since a wider range of CTE amounts, from 100to 0.19 µg, stimulated significant cell proliferation (p < 0.01)(Fig. 3).

3.3. The effect of CTE inoculation on the relative ratio ofspleen weight vs body weight

The intraperitoneal administration of CTE caused an in-crease in the relative ratio (spleen weight/body weight) of bothmice strains. On the 5th day after TCE inoculation, C57BL/6 miceshowed a significant increase (p < 0.001) in spleen weight/body weight (from 3.89 × 10−3 to 6.30 × 10−3), followed by asignificant reduction (p < 0.001) to basal levels (4.60 × 10−3) bythe 10th day (Fig. 4).

For the BALB/c mice, a gradual significant (p < 0.001) incre-ment of the relative ratio was observed on the 5th day (from4.44 × 10−3 to 5.88 × 10−3) and also on the 10th day (7.33 × 10−3)after CTE inoculation (Fig. 4).

3.4. In vivo effect of CTE inoculation on mice splenocytes

Interestingly, the total number of splenocytes from controlBALB/c mice (143.7 ± 19.6 × 106 cells) was significantly (p < 0.001)

higher than those from control C57BL/6 mice (48.4 ± 7.9 × 106

cells). After CTE administration, C57BL/6 mice showed a sig-nificant (p < 0.001) increase in splenocyte numbers by the 5thday (123.3 ± 15.5 × 106 cells), followed by a significant

Fig. 3 – In vitro splenocyte proliferation assay in the presence of CTE. Splenocytes (4 × 105 cells) from non-inoculatedC57BL/6 and BALB/c mice were cultured in 200 µL of RPMI complete medium in the absence (control) or presence ofdecreasing amounts of CTE, from 100 µg to 0.09 µg. Cells were pulsed with [H3]-thymidine for 16 h before the culture wasended. Proliferative activity is represented by scintillations per minute (CPM) and the values are displayed as means ± SDfrom three independent experiments. The black bars correspond to cells derived from C57BL/6 mice and the open bars, tocells from BALB/c mice. Asterisks indicate significance level *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4 – Relative ratio of spleen weight vs body weightfollowing CTE inoculation. The in vivo effect of CTE onspleen weight/body weight was evaluated afterintraperitoneal administration of CTE (1 mg) in bothC57BL/6 and BALB/c strains (n = 10, each strain). At the 5thand 10th day, 5 animals from each strain were sacrificedand their spleens were removed and weighed to determinethe relative ratio of spleen weight/body weight. The valuesrepresent the means ± SD of 5 animals from control or CTE-inoculated group. Asterisks indicate significance levelcompared to control group: *p < 0.05, **p < 0.01, ***p < 0.001.Letters also indicate significance level, a, p < 0.001compared to the 5th day.

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reduction (p < 0.05) by the 10th day (80.8 ± 9.6 × 106 cells) but,still significantly higher (p < 0.05) than control C57BL/6 mice(48.4 ± 7.9 × 106 cells). CTE inoculation also resulted in a sig-nificant increase (p < 0.001) in the number of splenocytes bythe 5th day in BALB/c mice, from 143.7 ± 19.6 to 210 ± 42.4 × 106

cells, which continued to increase up to the 10th day(214.8 ± 20.9 × 106 cells) (Fig. 5A).

3.5. Quantification of B220+ splenocytes after TCEadministration

To determine the effect of CTE on the B cell population, spleencells were probed against anti-B220 monoclonal antibodies. Asignificant increase in B220+ cells (p < 0.001) of the C57BL/6strain was observed on the 5th day (from 26.2 ± 7.8 × 106 cellsto 75.3 ± 6.8 × 106 cells) and persisted up to the 10th day(p < 0.01), when the total number of B220+ cells (31.2 ± 7.9 × 106

cells) was still significantly higher (p < 0.05) than the controlgroup. In BALB/c mice, a significant increase (p < 0.01) in thenumber of B cells was only observed on the 10th day in com-parison to the control group (from 74.2 ± 17.9 × 106 cells to108.2 ± 20.9 cells) (Fig. 5B).

3.6. The effect of CTE inoculation on antibody production

The anti-BrMRBC plaque-forming cells (PFC) were evaluated inorder to establish the frequency of antibody-secreting B cells.

Mice inoculation with CTE resulted in a significant increase ofthe anti-autologous-erythrocyte antibody-secreting cells on the10th day compared to the control group (209 ± 43.8 × 106 cellsand 7 ± 5.6 × 106 cells, respectively) in C57Bl/6 mice. No sig-nificant variation was observed in the PFC numbers in BALB/cmice (Fig. 6).

3.7. Proliferation of total bone-marrow cells and B cellpopulation after CTE inoculation

The intraperitoneal inoculation of CTE resulted in a transientbut non-significant change in the number of bone-marrow cellsin C57BL/6 mice. In BALB/c mice, a gradual increase in thenumber of bone-marrow cells was observed, with a signifi-cant increase on the 10th day (31.3 ± 1.7 × 106 cells; p < 0.001)compared to both the control (20.2 ± 4.1 × 106 cells) and the 5thday of the TCE treatment groups (21.3 ± 1.6 × 106 cells) (Table 1).

C57BL/6 immature B cells (B220+IgM−) showed a dynamicbehaviour where, on the 5th day after CTE inoculation, thenumber of immature B cells decreased (from 1.6 ± 0.4 × 106 cellsto 0.5 ± 0.2 × 106 cells) (p < 0.001), followed by an increase(3.4 ± 1.3 × 106 cells) on the 10th day (p < 0.001). In BALB/c mice,no significant variation of the immature B cell (B220+IgM−) popu-lation was observed after CTE inoculation.

A similar effect was observed on mature B cells (B220+IgM+)from both strains. On the 5th day after CTE inoculation, thenumber of B220+IgM+cells (0.7 ± 0.2 × 106 cells) from C57BL/6

Fig. 5 – In vivo effect of CTE inoculation on the number of total and B220+ splenocytes. The total number of splenocytes wasdetermined on the 5th and 10th day after intraperitoneal inoculation of 1 mg CTE (A) and B cells were identified using ananti-B220 as probe (B). The values represent the means ± SD of 5 animals from the control or CTE-inoculated group.Asterisks indicate significance level compared to control group: *p < 0.05, **p < 0.01, ***p < 0.001. Letters also indicatesignificance level: a, p < 0.001 between the control groups of both strains; b, p < 0.01 compared to the 5th day; c, p < 0.05compared to the 5th day.

338 J o u rna l o f Func t i ona l F ood s 1 8 ( 2 0 1 5 ) 3 3 3 – 3 4 3

mice decreased significantly (p < 0.001) compared to the con-trols (3.3 ± 0.5 × 106 cells). Additionally, a significant (p < 0.01)increase was observed on the 10th day (2.9 ± 0.9 × 106 cells) com-pared to the 5th day, but not in the control mice. In BALB/c mice,no significant variation in the number of mature B220+IgM+ cellswas observed following CTE inoculation (Table 1).

4. Discussion

Taro functional and nutritional properties have been studiedfor almost half a century with the first report in 1967 (Lim, 2015;Rao, Shurpalekar, & Sundaravalli, 1967). Although taro pro-vides mainly carbohydrates, it is an important source ofproteins, which electrophoretic profiles can change slightlyamong different cultivars (Hirai, Sato, & Takayanagi, 1989).

Herein, water-soluble proteins were extracted from tarocorms and the electrophoretic analysis revealed the pres-ence of three major groups, in accordance with typical profilesreported previously (Hirai et al., 1993; Pereira et al., 2014), whichincludes the presence of two polypeptide bands of around12 kDa, as well as two of about 22 kDa and two others of ap-proximately 55 kDa (Fig. 2).

Taro proteins have already been studied, characterized andevaluated regarding their biological properties (Shewry, 2003).The amino acid sequence of the 22 kDa polypeptide bands ishomologous to the trypsin inhibitor family found in soy-beans, winged beans, sweet potato and barley (Hirai et al., 1993)with potent biotechnological applications in the agriculturalarea to protect plants from phytophagous pests and patho-gens (Mehrabadi, Bandani, & Franco, 2012). The 55 kDapolypeptide band was identified as an albumin, with pI rangingfrom 5.5 to 6.0, although no biological activity was associatedwith this band (de Castro, Carneiro, Neshich Dde, & de Paiva,1992).The 12 kDa polypeptide bands were identified as the sub-units of the lectin from C. esculenta corms, called tarin (Bezerraet al., 1995; Pereira et al., 2014; Van Damme et al., 1995) andwere fully characterized by our research group (Pereira et al.,2015). This protein was extensively associated with impor-tant biological activities such as anti-insect (Das, Roy, Hess, &Das, 2013; Roy, Banerjee, Majumder, & Das, 2002; Roy, Gupta,Hess, Das, & Das, 2014), immunostimulatory (Pereira et al., 2014;Tulin & Ecleo, 2007) and antimetastatic (Kundu et al., 2012)properties.

Preliminary studies, performed by this research group, dem-onstrated that crude taro extract has an in vivo stimulatory effecton peritoneal macrophages and also enhances their phago-cytic activity.Those effects were accompanied by an incrementin spleen weights, suggesting that CTE can cause modifica-tions on other haematopoietic cells (unpublished results).Additionally, a previous study showed the ability of CTE pro-teins to stimulate in vitro proliferation of mice spleen and bonemarrow cells (Tulin & Ecleo, 2007).

The present study performed a further investigation re-garding not only the in vitro but also the in vivo effect of CTEon haematopoietic cells from mice spleen and bone marrow,with special attention to B lymphocytes.

The results confirmed that CTE is able to stimulatehaematopoietic cells both in vitro and in vivo. Not surpris-ingly, the in vitro proliferation assays (Fig. 3) resulted in a dose-

Fig. 6 – Number of antibody-secreting B cells caused by CTEinoculation. One milligram of CTE was administered toC57BL/6 and BALB/c strains (n = 10, each strain) byintraperitoneal route. On the 5th and 10th day afterinoculation, 5 animals from the control and CTE-inoculatedgroup were sacrificed and their spleen cells were retrievedto perform the plaque-forming cell (PFC) assay. The valuesrepresent the means ± SD of 5 animals from control or CTE-inoculated group. Asterisks indicate significance level:*p < 0.05, **p < 0.01, ***p < 0.001.

Table 1 – Phenotype analysis of bone marrow B cells from CTE-inoculated mice.

Mice strain Time point Total bone marrow (×106) cells B220+ IgM− (×106) cells B220+ IgM+ (×106) cells

C57BL/6 Day 0 24.8 ± 5.8 1.6 ± 0.4 3.3 ± 0.3Day 5 23.0 ± 6.2 0.5 ± 0.2*** 0.7 ± 0.3***Day 10 29.5 ± 6.8 3.4 ± 1.3*,a 2.9 ± 1.1b

BALB/c Day 0 20.2 ± 4.1 5.7 ± 2.2 2.7 ± 1.0Day 5 21.3 ± 1.6 4.9 ± 1.3 3.7 ± 1.4Day 10 31.3 ± 2.9***,a 4.5 ± 0.8 1.7 ± 0.5

In vivo effect of CTE on bone marrow B cells. Mice (C57BL/6 and BALB/c) were inoculated with 1 mg of CTE by intraperitoneal route. Five andten days after inoculation, bone-marrow cells were obtained from the femoral bone. The number of total cells were counted and probed againstanti-B220 and anti-IgM antibodies. The values represent the means ± SD of 5 animals from control or CTE-inoculated group. Asterisks indicatesignificance level: *p < 0.05 or ***p < 0.001. Letters indicate significance level: ap < 0.001 compared to the 5th day, bp < 0.01 compared to the 5thday.

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dependent curve and are in accordance with the literature(Tulin & Ecleo, 2007). In this study, a wider range of CTEconcentrations was used (from 100 to 0.09 µg), whereconcentrations ranging from 0.78 to 25 µg stimulatedsplenocyte proliferation, while higher concentrations, between50 and 100 µg, inhibited the proliferative activity in C57BL/6mice.

Immunostimulatory effects have been constantly associ-ated to active plant proteins classified as lectins (Ashraf & Khan,2003) for at least 5 decades (Nowell, 1960). These molecules arecharacterized by their ability to bind specifically to carbohy-drates, triggering a variety of biological properties includingthe stimulatory effect on the immune system (Hivrale & Ingale,2013). As aforementioned, a lectin, named tarin, is present inthe CTE preparation and accounts for 58% of the total visual-ized proteins. Previous studies revealed that CTE showshaemagglutinating activity (unpublished results), which typi-cally detects the presence of lectin activity, suggesting theparticipation of such protein in the biological response de-scribed herein. In a previous study, the purified form of tarolectin was also able to induce in vitro splenocyte proliferationin a dose-dependent manner (Pereira et al., 2014), as also shownin the present study. Typically, immunostimulatory lectins, alsocalled mitogenic lectins, generate dose-dependent curves(Eijsvoogel, 2012), consistent with the results obtained herein.The best-known immunostimulatory lectins are phytohae-magglutinin (PHA) from the kidney bean (Phaseolus vulgaris),concanavalin A (Con A) from the jackbean (Canavalia ensiformis)and pokeweed mitogen (PWM) from the roots of pokeweed(Phytolacca americana). While the first two lectins mainly acti-vate T lymphocytes, the latter activates both T and B cells(Shanmugham et al., 2006).

Intraperitoneal inoculation of CTE caused an increase ofthe spleen weight/body weight index (Fig. 4) in both mousestrains, reflecting the increment in total splenocytes andB220+ splenocytes (Fig. 5), but the dynamics of this CTE effectdiffered within the strains. While in C57BL/6 mice thesealterations were transient, reaching a peak on the 5th dayand declining by the 10th day, in BALB/c mice theseparameters increased gradually until the 10th day. This dis-similarity is, probably, caused by a difference in the cytokineprofile of both mice strains inherent to their distinct geneticbackground.

The proliferative potential of the purified lectin from tarohas also been previously studied, showing that this protein isalso able to increase significantly the number of totalsplenocytes and B220+ cells on the 5th day after the intraperi-toneal inoculation of C57BL/6 mice (Pereira et al., 2014). Thesefindings reinforce the participation of lectin in the immune re-sponse shown herein.

Interestingly, after CTE inoculation in C57BL/6 mice,splenocytes were stimulated to a significant production of an-tibodies on the 5th day, when the number of B220+ splenocyteswas significantly increased. B lymphocytes were not just stimu-lated to a proliferative state but were also activated by CTE,leading to antibody release.

Moreover, CTE was shown to have a distinct effect on B1lymphocytes, which differ from B2 lymphocytes by theirability to produce natural antibodies including, those againstsenescent erythrocytes (anti-BrMRBC) (Hardy & Hayakawa,

2005). The results presented herein indicate that CTE wasable to induce the proliferation of B2 cells (Fig. 5) derivedfrom both C57BL/6 and BALB/c, while acting differently on B1lymphocytes (Fig. 6). The fact that CTE had a strong effect onB1 cells from C57BL/6 but not from BALB/c mice (Fig. 6)suggests that the genetic background of the mice strainsmay affect B-cell activation, and points to the importance offurther examination of B-cell behaviour in the context ofplant immunostimulators.

The effect of CTE on total bone-marrow cells occurred after10 days for both strains, although the increase was only sig-nificant for BALB/c mice. A transient reduction of B220+IgM−

(immature B cells) and B220+IgM+ (mature B cells) in the bonemarrow with a concomitant increase of B220+ lymphocytes inthe spleen of both strains was observed.These changes suggestthat CTE acts on the transient cell populations during the dif-ferentiation process, and/or on the compartmental mobilizationof B220+ cells to the spleen. However, the distinct dynamics thatoccurred within the same time frame do not allow for the con-clusion of how and where the reduced bone marrow B220+ cellswould be destined.

Another recent study by our research group showed thatthe lectin from taro binds with low or no affinity to freemannose, but is able to strongly bind to complex and high-mannose N-glycans. Among the ligands that best interact withthis lectin are the epitopes designated Lewisy/CD174 (Fucα1-2Galβ1-4[Fucα1-3]GlcNAcβ1-) and H2/CD173 (Fucα1-2Galβ1-4GlcNAcβ1-) antigens (Pereira et al., 2015). These antigens arefound in human CD34+ haematopoietic progenitor cells (Cao,Merling, Karsten, & Schwartz-Albiez, 2001) and in peripheralblood granulocytes (Dettke, Palfi, & Loibner, 2000). Glycans withfucosylated sites play an important role in cell–cell and cell–matrix interaction, differentiation, proliferation and apoptosis.Both antigens (CD174 and CD173) allow the recirculation of lym-phocytes from the bloodstream into lymphatic organs and theinteraction between haematopoietic progenitor cells and bone-marrow stromal cells (Cao et al., 2001). The expression of CD34on the surface of haematopoietic progenitor cells character-izes a heterogeneous population with multipotent capacity toreconstitute the myelo-lymphopoietic system in a supralethalirradiated host (Stella et al., 1995).

The immunostimulatory effects observed in this studyare, possibly, the result of the participation of tarin throughthe specific binding to CD34+ progenitor cells, favouring Blymphopoiesis. The participation of other factors and/orcompounds, however, cannot be discarded. However,further investigation should be conducted to determine thereal contribution of each component to the B proliferationresponse.

Taro corms are, undoubtedly, a potent natural source ofimmunostimulatory molecules.The stimulation of the immunesystem by CTE can positively contribute to health and well-being maintenance, since it can be applied to reverse animmunosuppressive status caused by infectious agents, che-motherapeutic agents or even psychological conditions.Moreover, CTE can confer protection against several diseasessince it stimulates the proliferation of total haematopoietic andB cells, contributing to the improvement in the immune re-sponse. Such molecules represent powerful candidates as newadditives for food and pharmaceutical industries.

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5. Conclusions

Taro corms are a natural source of bioactive proteins, which actas immunostimulators on total bone marrow and spleen cellsfrom distinct mice strains.The proliferation of mice B220+ lym-phocytes of both organs was also stimulated by CTE inoculation.The increment in B220+ cells from the spleens was accompa-nied by a decrease of mature (B220+IgM+) and immature (B220+IgM−)lymphocytes in the bone marrow, suggesting the early mobili-zation of these cells to the spleen. The production of primaryantibodies by B220+ splenocytes indicated the activation of B1cells by CTE. Total splenocytes were also stimulated in vitro byCTE in a dose-dependent manner. The in vivo effects of CTE inboth murine models encouraged us to test taro active proteinson the recovery of the immune status of immunosuppressedmice. Future studies need to be conducted in order to explorethe mechanisms of CTE-induced cell activation.

Author contributions

PRP – as a Graduate student, was involved in the entire studydesign, and data interpretation; MAV – participated in the studydesign and coordination and helped draft the manuscript; JTS– participated in the study design; VMFP and GAPBT – helpedin the data interpretation and in the drafting of the manu-script. All authors read and approved the final manuscript.

Conflicts of interest

The authors declare no conflict of interest and the foundingsponsors had no role in the study design, data collection, analy-sis, or interpretation, in the manuscript preparation writing orin the decision to publish the results.

Acknowledgments

We would like to acknowledge Dr. Marcus A. Nadruz Coelhoat the Botanical Garden of Rio de Janeiro, Brazil for confirm-ing the identification of the studied taro plant, and all thosewho directly or indirectly contributed to this study.

Appendix: Supplementary material

Supplementary data to this article can be found online atdoi:10.1016/j.jff.2015.07.014.

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