ORIGINAL ARTICLE

Encapsulation of apple polyphenols in b-CD nanosponges

Marıa Ramırez-Ambrosi • Fabrizio Caldera •

Francesco Trotta • Luis A. Berrueta •

Blanca Gallo

Received: 14 November 2013 / Accepted: 10 February 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Rutin, phloridzin and chlorogenic acid are

some of the most important and characteristic polyphenols

found in apples and their by-products (cider, apple juice,

apple pomace, etc.). Despite their antioxidant power, their

low stability under light or heating conditions restricts the

use of this kind of molecules as nutraceuticals. To deal

with this issue, encapsulation seems to be an alternative

solution. Based on the obtained results, it can be concluded

that b-cyclodextrin nanosponges (b-CD NS) are promising

agents for the encapsulation of polyphenols. Rutin, phlo-

ridzin and chlorogenic acid have been included for the first

time in b-CD NS in this work. In particular, the highest

encapsulation efficiency for rutin (83.7 %) was obtained

using 1,10-carbonyldiimidazole as cross-linker in a 1:3 ratio

(nanosponge/cross-linker). However, for smaller molecules

as phloridzin and chlorogenic acid, the nanosponge which

showed the best results was the one with HMDI in a 1:8

ratio (87.2 and 77.5 %, respectively). In vitro dissolution

studies of encapsulated polyphenols showed that rutin and

phloridzin are better dissolved in ethanol, while chloro-

genic acid is better dissolved in water. Besides, TGA, DSC,

FTIR and XRPD were used as characterization techniques.

Individual polyphenols and nanosponges, equimolar phys-

ical mixtures and synthesized complexes were character-

ized. Taking into account the obtained results, it can be

confirmed that the solid products were not physical

mixtures, but inclusion complexes. Thus, using these

encapsulating agents, other polyphenols from apple and its

by-products could be encapsulated in order to enhance their

bioavailability.

Keywords Apple � Polyphenols � Nanosponges �Encapsulation � Dissolution � Characterization

Introduction

Phenolic compounds are one of the most diverse and

widespread groups of natural constituents universally dis-

tributed among vascular plants.

In apple fruit, five major classes of phenolic compounds are

present: flavan-3-ols [monomeric flavan-3-ols (catechins) and

polymeric flavan-3-ols (procyanidins)], dihydrochalcones

(phloretin glycosides), flavonols (quercetin glycosides),

hydroxycinnamic acid derivatives, and, in the peel of red

varieties, anthocyanins (cyanidin glycosides) [1, 2].

In recent years, interest in phenolic compounds has been

increasing due to compelling evidence of their health bene-

fits and their impact on food quality [3]. Several epidemio-

logical studies have associated the consumption of phenolic

compounds, and more specifically flavonoids, with lower

risks of different types of cancer and cardiovascular diseases,

and have shown that they posses antioxidant, anti-inflam-

matory and anti-ageing activity [4]. In particular, rutin has

been reported to have cardio-protective, anti-inflammatory,

asthma-reducing, cholesterol- lowering, anti-cancer and

neuronal protective properties [5]. Phloridzin is a nutraceu-

tical with good prospects in food and pharmaceutical

industries, and can exhibit antioxidant, antiinflammatory,

immunosuppressive effect, antitumor, antimutagenic, anti-

diabete, antiobesity and membrane permeability properties

M. Ramırez-Ambrosi � L. A. Berrueta � B. Gallo (&)

Departamento de Quımica Analıtica, Facultad de Ciencia y

Tecnologıa, Universidad del Paıs Vasco/Euskal Herriko

Unibertsitatea (UPV/EHU), Apdo 644, Bilbao, Spain

e-mail: blanca.gallo@ehu.es

F. Caldera � F. Trotta

Dipartimento di Chimica, Universita di Torino, Via P. Giuria 7,

Torino, Italy

123

J Incl Phenom Macrocycl Chem

DOI 10.1007/s10847-014-0393-7

[6]. Chlorogenic acid presents a high antioxidant activity [7].

Furthermore, some in vitro and in vivo pharmacological

activities of chlorogenic acid, such as hypoglycemic, anti-

viral, hepatoprotective and immunoprotective activities

have been reported [8]. However, their low stability under

light or heating conditions restricts the use of this kind of

molecules as nutraceuticals. To deal with this issue encap-

sulation seems to be an alternative solution.

Cyclodextrins (CDs) are crystalline, water soluble,

cyclic, non-reducing, oligosaccharides built up from six,

seven, or eight glucopyranose units. The most interesting

feature of these oligosaccharides is the ability to include

guest molecules inside their internal cavity. This fact

enables the modification of the physicochemical properties

of the included molecule (i.e., physical state, stability,

solubility and bioavailability) [9]. Therefore, CDs are used

in many industries, such as agro-food, cosmetology, phar-

macy and chemistry [10].

Cyclodextrins [11] are capable of including compounds

whose geometry and polarity are compatible with those of

the cavity. Nevertheless, native CDs are incapable of

forming inclusion compounds with certain molecules, such

as hydrophilic or high-molecular-weight molecules. As a

consequence, many chemical modifications of CDs have

been studied to overcome their drawbacks and to improve

their technological characteristics. At present, CD-based

nanosponges (NS) can easily be obtained through the

reaction of selected CD with a suitable cross-linking agent.

This agent may be a diisocyanate [12], diarylcarbonate or

carbonyldiimidazole [13], carboxylic acid dianhydride

[14], and 2,2-bis(acrylamido)acetic acid [15].

So far, NS have been used for the encapsulation of

resveratrol, a stilbene found in wines [16, 17]. However,

encapsulation of other polyphenols in NS has not been

studied yet. Other encapsulation agents and/or techniques

have been applied. For example, quercetin has been

encapsulated in liposomes [18], quercitrin has been loaded

in polylactide by emulsification-solvent removal method

[19] and molecular inclusion method has been applied to

myricetin using 2-(hydroxy-propyl)-b-cyclodextrin [20],

among others.

The objective of the study was the evaluation of NS as

encapsulating agent for major polyphenols found in apples.

In this preliminary investigation three representative

compounds from apple main phenolic families were cho-

sen: rutin (a flavonol), phloridzin (a dihydrochalcone) and

chlorogenic acid (a hydroxycinnamic acid) (Fig. 1).

Materials and methods

Materials

b-Cyclodextrin (b-CD) was provided by Roquette (Les-

trem, France). Rutin, phloridzin, chlorogenic acid, 1,10-carbonyldiimidazole (CDI), hexamethylene diisocyanate

(HMDI), 1,4-diazabicyclo[2,2,2]octane (DABCO), dime-

thyl formamide (DMF), dimethyl sulfoxide (DMSO) and

ethanol were purchased from Sigma Aldrich Chemie

(Steinheim, Germany). Milli Q water (Millipore) was used

throughout the studies.

Methods

Synthesis of CDI–NS

Briefly, 11.45 mmol of anhydrous b-CD (dehydrated in

oven at 120 �C for 12 h) was added to 78 mL anhydrous

Fig. 1 Chemical structures of a rutin, b phloridzin and c chlorogenic acid

J Incl Phenom Macrocycl Chem

123

DMF in round bottom flask to achieve complete dissolution

by observing transparent solution. Then a suitable amount

of CDI was added as a cross-linker. This reaction mixture

was heated up to 90 �C in an oil bath. After approximately

20 min the gelation process occurred. The temperature was

then maintained for further 3 h, in order to increase the

cross-linking reaction yield. The reaction was carried out in

three different molar ratios of b-CD and cross-linker (CDI)

1:3, 1:4 and 1:8. Once the condensation reaction was

completed, the transparent block of cross-linked CD was

ground and filtered with excess of deionized water to

remove excess of DMF. Lastly, residual by-products or

unreacted reagents were completely removed by Soxhlet

extraction with ethanol. After purification, CDI–NS were

dried by exposure to air and then stored at ambient tem-

perature until further use [12].

Synthesis of HMDI–NS

Concisely, 3.44 mmol of anhydrous b-CD (dehydrated in

oven at 120 �C for 12 h) was added to 16 mL DMSO in

round bottom flask to achieve complete dissolution by

observing transparent solution. Then 7.13 mmol of DAB-

CO was added as catalyzer. Once the catalyst was com-

pletely solubilized, a suitable amount of HMDI was

incorporated as a cross-linker. The mixture was allowed to

react for 1 h at room temperature. The reaction was carried

out in two different molar ratios of b-CD and cross-linker

(HMDI) 1:4 and 1:8. Once the condensation reaction was

completed, the transparent block of cross-linked CD was

ground and filtered with excess of deionized water to

remove excess of DMSO. Lastly, residual by-products or

unreacted reagents were completely removed by Soxhlet

extraction with acetone. After purification, HMDI–NS

were dried by exposure to air and then stored at ambient

temperature until further use.

Preparation of polyphenol-loaded NS

Rutin, phloridzin and chlorogenic acid NS complexes were

prepared at weight ratio of 1:25 (polyphenol, b-NS w/w).

Accurately weighed quantities of each polyphenol were

suspended in 30 mL of Milli Q water using a magnetic

stirrer, then the calculated amount of the NS was added, the

mixture was shaken in a vortex and kept for 48 h under

stirring in the dark at controlled temperature (30 �C). The

suspensions were centrifuged at 2,000 rpm for 10 min.

Then, solutions were filtered under vacuum and solid

complex was cleaned with water to eliminate the free

polyphenol which was not included. Finally, it was dried

and kept into a desiccator. The batch size was kept at 0.5 g

for all batches.

Characterization of polyphenol-loaded NS

Encapsulation efficiency

An aliquot of the supernatant solution from the preparation

of polyphenol-loaded NS was analyzed by UV–Vis spec-

trophotometer (Perkin Elmer Lambda 15) at 353, 285 and

326 nm for rutin, phloridzin and chlorogenic acid,

respectively. Encapsulation efficiency was calculated by

substracting the amount of polyphenol in the supernatant

solution from the initial amount of compound. A calibra-

tion curve following the external standard method was built

for each polyphenol in water (concentrations ranging from

10-5 to 10-4 M). If necessary, dilutions with water were

done.

In vitro dissolution study

For this purpose, 50 mg of polyphenol–NS complex were

stirred in 30 mL of water or ethanol for 24 h at room

temperature. After 0.5, 1, 2, 4, 6, 8 and 24 h had passed, the

supernatant solution absorbance was measured by UV–Vis

spectrophotometry at optimal wavelengths. If necessary,

dilutions with water or ethanol were done.

Fourier transform infrared spectroscopy (FTIR)

Individual polyphenols and NS as well as polyphenol–NS

complexes were subjected to FTIR spectroscopic studies by

using a Spectrum 100 spectrophotometer (Perkin-Elmer,

USA). Spectra were recorded in the region of 4,000 to

650 cm-1 with 8 scans and a resolution of 4.00 cm-1.

attenuated transmitted reflectance (ATR) mode was

employed.

Thermogravimetric analysis (TGA)

Thermal analyses were carried out using a TGA (2050

TGA, Perkin-Elmer, USA). Samples of individual poly-

phenols and NS, and polyphenol–NS complexes of 10 mg

were weighed in aluminum sample pans and then heated at

a rate of 10 �C/min in the 40–700 �C range under a

nitrogen purge.

Differential scanning calorimetry (DSC)

Individual polyphenols and NS, and polyphenol–NS com-

plexes were subjected to DSC studies using a DSC822e

(Mettler Toledo, USA). Empty pan was used as a reference

material and samples (3–7 mg) were scanned at the rate of

10 �C/min in the range of 50–240 �C. Physical mixtures

were prepared by mixing equimolar amounts of NS and

polyphenols in a mortar.

J Incl Phenom Macrocycl Chem

123

X-ray powder diffraction (XRPD)

CDI–NS and HMDI–NS were subjected to XRPD studies

using an X’Pert Pro X-ray diffractometer (PANalytical,

Almelo, NL) with a Bragg–Brentano geometry and

sequential collection between 5� and 70� at 2h.

Results and discussion

Encapsulation efficiency

The encapsulation efficiencies of NS formulations were

found to be 50–83 % for CDI–NS and 10–87 % for

HMDI–NS (see Table 1). In particular, CDI (1:3) seemed

to be the best choice for the encapsulation of rutin, whereas

HMDI (1:8) reached the highest encapsulation efficiencies

for chlorogenic acid and phloridzin. CDI–NS present a

very short bridge among CD units. This fact probably leads

to the formation of a network with narrow links. Conse-

quently, it can inhibit the inclusion of the guest molecule to

a certain extent, especially when high cross-linking degree

is achieved. On the contrary, HMDI–NS have a longer

chain with a significant flexibility and this behavior can

have a positive effect on the inclusion of the guest mole-

cule at higher cross-linking degree. Rutin, chlorogenic acid

and, to a less extent, phloridzin appear not to fit well the

nanocavities of HMDI–NS (1:4) because of the cited

flexibility of the HMDI chain. Thus, enhancing the cross

linking ratio (1:8) loaded molecules are better retained in a

more thick links. Nevertheless, CDI–NS have been repor-

ted to be less sensitive to the cross-linking ratio [21], being

very similar for 1:2, 1:4 and 1:8 ratios (Table 1). Taking

into account these results, HMDI–NS with a higher cross-

linker ratio (above 1:8) will be synthesized for further

studies.

In vitro dissolution study

The dissolution of the 3 polyphenols encapsulated in CDI

and HMDI–NS was tested in both aqueous and organic

media (see Fig. 2). In general, dissolution in ethanol (d–f)

happens faster and achieves a higher percentage of phenol

dissolved than in aqueous media (a–c). Obtained results

suggest that rutin and phloridzin are better dissolved in

ethanol than in water, while chlorogenic acid seems to have

better results in water, especially for CDI–NS. These dif-

ferences can be explained due to the difference in polarity

of the 3 polyphenols: chlorogenic acid has a higher polarity

than rutin and phloridzin; therefore, it is better dissolved in

water.

Fourier transform infrared spectroscopy (FTIR)

FTIR analyses of powders were performed in ATR mode

since it requires minimal or no sample preparation and

gives comparable results to transmission spectra, although

it must be taken into account that a decrease in sensitivity

may occur.

The spectrum of rutin showed two absorption bands at

1,653 and 1,596 cm-1 due to the ketone group of the ring C

(C=O stretching) and the aromatic ring (C=C stretching),

whereas CDI–NS (1:4) presents a band at 1,743 cm-1,

related to the carbonyl group of the carbonate bond

between the cross-linker and the b-CD, and a less intense

band at 1,632 cm-1 due to the deformation of O–H moi-

eties (see Fig. 3). The inclusion complex showed a similar

band as the one from rutin but at a lower frequency,

1,639 cm-1 and no change was observed in the

1,743 cm-1 signal from the NS. In the presence of rutin,

the 1,630–1,640 cm-1 band of the NS increases its inten-

sity thanks to the contribution of the two absorption peaks

originated by the polyphenol. Thus, it could be concluded

that FTIR confirms the presence of rutin in the polymer

structure. Analogous results were obtained for chlorogenic

acid and phloridzin. In the first case, 3 bands were observed

for the pure acid at 1685, 1638 and 1600 cm-1, due to the

presence of a carboxylic group and a ketone group (C=O

stretching) and an aromatic ring (C=C stretching), respec-

tively. However, the chlorogenic acid–NS complex only

showed a broad band at 1,634 cm-1, as well as a

1,742 cm-1 signal from the NS. Phloridzin spectrum pre-

sented 2 bands at 1,623 and 1,604 cm-1, related to the

presence of a ketone group (C=O stretching) and an aro-

matic ring (C=C stretching), while in the complex with

CDI–NS (1:4) appeared a signal at 1,630 cm-1 and the

same as the above mentioned was observed for the CD part

of the complex.

Thermogravimetric analysis (TGA)

Thermogravimetric analysis of polyphenols, NS and their

complexes allowed verifying the presence of an inclusion

compound and not just a physical mixture of phenol and

NS. As an example, results of TGA for phloridzin and

Table 1 Encapsulation efficiencies (%) for rutin, phloridzin and

chlorogenic acid

Rutin Phloridzin Chlorogenic acid

CDI (1:3) 83.7 80.8 67.6

CDI (1:4) 80.5 77.4 63.9

CDI (1:8) 77.9 55.0 50.3

HMDI (1:4) 10.1 38.4 9.8

HMDI (1:8) 69.8 87.2 77.5

J Incl Phenom Macrocycl Chem

123

HMDI (1:8) are shown as a derivative curve (%/�C) in

Fig. 4. On the one hand, pure polyphenol suffers a first

weight loss at 109.5 �C, which is related to its melting

process (Tm = 113–114 �C, according to reference stan-

dard specifications) and a second one at 281.1 �C, probably

due to a degradation process. On the other, HMDI–NS

(1:8) degradates after 315 �C (a peak at 326.7 �C with a

shoulder at 315.5 �C is observed) and a secondary weight

loss appears at 459.9 �C. However, complex thermal ana-

lysis revealed no presence of free polyphenol, but a slight

shift on the peaks found in the NS (323.9, 336.2 and

461.3 �C, respectively). For the other phenols included in

the NS network, similar results were obtained. In the case

of chlorogenic acid, a weak peak at 214.1 �C followed by

an intense peak at 361.6 �C was observed. Rutin showed a

weak peak at 110.3 �C and a main peak at 273.5 �C. Again,

the first one is related to the melting procedure (Tm = 210

and 195 �C, respectively) and the second one to any deg-

radation of the molecule. The peak below 100 �C is related

to residual moisture in the sample. For all NS degradation

processes of their network happen between 280–331 �C,

indicating that they have good thermal stability.

Differential scanning calorimetry (DSC)

DSC analyses were carried out in order to determine

whether the solid products were physical mixtures or

inclusion complexes. For each case, pure polyphenol, pure

NS, polyphenol–NS (1:1) physical mixture and their

inclusion complex were compared. As an example, a

% R

utin

dis

solv

ed

0

20

40

60

80

100

0 5 10 15 20 25

Time (h)

0

20

40

60

80

100

0 5 10 15 20 25

Time (h)

% R

utin

dis

solv

ed

0

20

40

60

80

100

0 5 10 15 20 25

Time (h)

% P

hlor

idzi

n di

ssol

ved

0

20

40

60

80

100

0 5 10 15 20 25

Time (h)

% P

hlor

idzi

n di

ssol

ved

0

20

40

60

80

100

0 5 10 15 20 25

Time (h)

% C

hlor

og. a

cid

diss

olve

d

0

20

40

60

80

100

0 5 10 15 20 25

Time (h)

% C

hlor

og. a

cid

diss

olve

d

CDI (1:3) CDI (1:4) CDI (1:8) HMDI (1:4) HMDI (1:8)

(a) (b) (c)

(d) (e) (f)

Fig. 2 Evolution of % polyphenol dissolved in 24 h from polyphenol–NS complexes in water (a–c) and ethanol (d–f)

CDI(1:4) Rutin

CDI(1:4)

4000 6503500 3000 2500 2000 1500 1000

cm-1

%T

Rutin

Fig. 3 FTIR spectra of rutin,

CDI (1:4) and rutin–CDI (1:4)

complex in ATR mode

J Incl Phenom Macrocycl Chem

123

description of CDI–NS (1:8) results for each phenolic

compound is given below. Rutin–CDI (1:8) thermograms

are compiled in Fig. 5. It can be observed an endothermic

peak at 157 �C, followed by another peak at 185 �C for

rutin. This is in agreement with the melting point of rutin

(195 �C). Pure NS thermogram showed a broad peak at

93 �C, as it becomes anhydrous. As it was expected,

physical mixture thermogram is a combination of host (NS)

and guest (rutin) compounds. However, in the complex

thermogram the melting peak of the guest is absent, as a

result of the interaction between the guest and the host

cavity. For the phloridzin–CDI (1:8) system the same

analyses as the above mentioned were performed (ther-

mograms not shown). Pure polyphenol melting point was

determined to be 118 �C, since an endothermic peak was

recorded at that temperature. Physical mixture thermogram

revealed the presence of both NS (broad peak at 93 �C) and

phloridzin (peak at 117 �C). As predicted, the formation of

an inclusion complex was confirmed, since only a peak at

103 �C was observed. Finally, in the case of chlorogenic

acid, the melting point was 212 �C. Physical mixture

thermogram consisted of a broad endothermic peak at

93 �C due to the NS and a narrow peak at 211 �C. Again,

the thermogram of the complex allowed confirming the

inclusion of the chlorogenic acid into the NS cavity, since a

broad peak at 94 �C was registered. Similar results were

obtained for the rest of polyphenol–NS complexes.

X-ray powder diffraction (XRPD)

XRPD analyses were performed to determine whether

carbonate and carbamate NS crystallinity were comparable.

Both NS showed a poorly crystalline (paracrystalline)

structure (Fig. 6).

0

0.20

0.40

Der

iv. W

eigh

t(%

/°C

)

0

20

40

60

80

100

Wei

ght(

%)

0 100 200 300 400 500 600 700

Temperature (°C)

0 100 200 300 400 500 600 700

Temperature (°C)

0 100 200 300 400 500 600 700

Temperature (°C)

0

0.50

1.00

1.50

Der

iv. W

eigh

t(%

/°C

)

0

20

40

60

80

100

Wei

ght(

%)

0

0.20

0.40

0.60

0.80

1.00

1.20

1.40

Der

iv. W

eigh

t(%

/°C

)

0

20

40

60

80

100

Wei

ght(

%)

(a)

(b)

(c)

Fig. 4 TGA thermograms of a phloridzin, b HMDI (1:8) and

c phloridzin-HMDI (1:8) complex

50 100 150 200

(c)

(b)

(a)

(d)

Temperature (ºC)

Hea

tflo

w

Fig. 5 DSC thermograms of a CDI (1:8), b rutin, c physical mixture

and d inclusion complex

J Incl Phenom Macrocycl Chem

123

XRPD pattern decomposition shows some broad

reflections similar for both NS. Areas and intensity vs.

FWHM (Full Width at Half Maximum) ratio are indicated

in Table 2 for each one of the 3 peaks. It was found that

both NS exhibit a very similar pattern of diffractogram on a

whole, but they differed mainly in the areas of the peaks as

well as in the I/FWHM ratio. Taking into account the

results for XRPD analyses, it could be concluded that

HMDI–NS crystallinity is similar to that of CDI–NS.

Conclusions

It has been shown that rutin, phloridzin and chlorogenic

acid can be successfully encapsulated within b-CD NS with

a high degree of retention and protection. FTIR, TGA and

DSC analyses were performed for the characterization of

the inclusion complexes. Taking into accounts obtained

results, NS with lower (1:1 or 1:2) and higher (1:10, 1:12,

etc.) b-CD/cross-linker ratios will be synthesized, for the

encapsulation of bigger and smaller molecules, respec-

tively. Studied compounds are some of apple major poly-

phenols and representative of phenolic families found in

apple. Thus, using these encapsulating agents, other poly-

phenols from apple and its by-products could be encapsu-

lated in order to enhance their bioavailability. Given the

antioxidant potential and health benefits of flavonoids,

further studies of these solid inclusion complexes will be

developed so as to be used either for the formulation of

functional foods or as a food supplements.

Acknowledgments Authors want to express their thanks to Diparta-

mento di Chimica—University of Torino. This research was supported

by Gobierno del Paıs Vasco/Eusko Jaurlaritza (Dpto. de Educacion,

Universidades e Investigacion, ref. IT413-10), Ministerio de Ciencia y

Tecnologıa (Project number CTQ2009-08390) and Plan Nacional de

I?D?I 2008–2011 (Project number RTA2012-00118-C03-03). Marıa

Ramırez-Ambrosi thanks Gobierno Vasco/Eusko Jaurlaritza for her

Ph.D. grant. Technical and human support provided by the Molecules

and Materials unit of the General X-ray Service of SGIker (UPV/EHU,

MICINN, GV/EJ, ESF) is gratefully acknowledged. Authors also want to

thank Dr. Rodrıguez Bengoechea for the DSC analyses.

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Inte

nsit

y(a

rb. u

nits

)380

330

280

230

180

130

80

30

-20

-70

-120

Inte

nsit

y(a

rb. u

nits

)

2θθ (°)

2θ θ (°)

5 12 19 26 33 40 47 54 61 68 75

5 11 17 23 29 35 41 47 53 59

380

330

280

230

180

130

80

30

-20

-70

-120

(a)

(b)

Fig. 6 XRPD pattern decomposition for: a CDI–NS and b HMDI–NS

Table 2 Peak position recurrence between CDI and HMDI–NS

sample and intensity vs. FWHM ratios

CDI–NS HMDI–NS

Peak

position

(2h)

Area I/FWHM Peak

position (2h)

Area I/FWHM

11.74 706.78 56.76 11.32 322.65 39.70

19.27 1,354.94 42.69 19.77 1,734.27 44.50

26.88 2,044.9 4.99 25.5 762.23 4.64

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