ORIGINAL PAPER

Effects of Phyllanthus sellowianus Mull Arg. Extractson the Rheological Properties of Human Erythrocytes

Patricia Buszniez • Osvaldo Di Sapio •

Bibiana Riquelme

� Springer Science+Business Media New York 2014

Abstract Phyllanthus sellowianus extracts have been used

in Argentina since colonial times in the treatment of diabe-

tes. The in vitro biorheological and hemoagglutinant action

of different extracts of P. sellowianus bark on human

erythrocytes (RBC) were studied. RBCs were incubated

in vitro with four aqueous extracts: Maceration; Controlled

Digestion (PD); Decoction; and Infusion. Biorheological

parameters (deformability, membrane surface viscosity,

elastic modulus, and dynamic viscolelasticity) were deter-

mined with an Erythrodeformeter, and erythrocyte adhesion

was characterized by image digital analysis. Immunohe-

matological assays in RBC incubated with all the extracts

showed large globular aggregates and agglutination in

human ABO blood group system. Isolated cell coefficient

showed the increase of cell adhesion. Aggregated shape

parameters were significantly higher than normal and they

changed with the concentration, particularly of PD extracts.

Rheological results showed that the extract biorheological

action varies with the temperature used in the extract prep-

arations. The results obtained are useful to study the action

mechanism of extracts from P. sellowianus bark in order to

evaluate its use as therapeutic agent in diabetes. Immuno-

hematological Tests using ABO system showed its

agglutinant power, which is of special interest in Immuno-

hematology to be used as hemoclassifier.

Keywords Diabetes � Phyllanthus sellowianus � Red

blood cells � Hemocompatibility � Erythrocyte aggregation

Introduction

Innumerable drugs from plants found all over the world

have been documented in the literature because of their

curative potential. Sakthivel and Guruvayoorappan [1]

have indicated that medicinal plants still remain as thriving

source of life-saving drugs for the large majority of people

treating health problems. However, although remarkable

progress in medicinal plant research such as chemical

characterization, biological, pharmacological, and toxico-

logical activity of the plants has been witnessed, further

exploration for the development of new drug molecules to

elucidate the responsible mechanism for its therapeutic

action is of great importance.

Diabetes (DB) mellitus has been recognized since

antiquity, and Chang et al. [2] have recently pointed that

this pathology currently affects as many as 285 million

worldwide people and results in heavy personal and

national economic burdens. Considerable progress has

been made in conventional antidiabetic drugs. However,

new remedies are still in great demand because of the

limited efficacy and undesirable side effects of current

orthodox drugs. Moreover, Chang et al. have said that

nature is an extraordinary source of antidiabetic medicines

and up to date, more than 1,200 flowering plants have been

claimed to have antidiabetic properties. They have studied

twenty-three herbs and 5 herbal formulas and concluded

that the use of Chinese herbal medicines in DB is

P. Buszniez � O. Di Sapio � B. Riquelme (&)

Facultad de Ciencias Bioquımicas y Farmaceuticas, Universidad

Nacional de Rosario, Suipacha 535, Rosario 2000, Santa Fe,

Argentina

e-mail: bibiana_riquelme@yahoo.com.ar

P. Buszniez

e-mail: patricia.bus@hotmail.com

B. Riquelme

BioOptics Group, Rosario Institute of Physics (CONICET-UNR),

Bv. 27 de febrero 210 bis, Rosario 2000, Argentina

123

Cell Biochem Biophys

DOI 10.1007/s12013-014-0072-8

promising but still far to be probed. Also, systematic

information about the structure, activity, and modes of

action of these plants and compounds will pave the way for

the research and development of new antidiabetic drugs.

Phyllanthus sellowianus Mull Arg. (Euphorbiaceae) has

been used as herbal medicine in Argentina since colonial

times and nowadays. In the last few years, the use of

infusions of leaves, wood, and bark has notably increased

for its antidiabetic properties. Consequently, this species

has been included in the Argentinean Pharmacopeia.

Therefore, now it is important to evaluate its action

mechanism for therapeutic uses [3].

Phyllanthus sellowianus Mull Arg. belongs to the Eu-

phorbiaceae family and grows, alongside rivers and brooks,

in humid and warm regions in Argentina, and 6 varieties

have been identified [4]. This bush can reach 4 m high,

flowers appear in springtime, and fruits in summer (Fig. 1)

[5]. The name Phyllanthus comes from Greek: phyl-

lon = ‘‘leaf’’ and anthos = ‘‘flower’’, as its leaves and

flowers grow together. The name sellowianus derives from

the German naturalist Friedich Sellow, who carried out the

first description of this species.

Hnatyszyn et al. in 1999 investigated the in vivo anti-

diabetic activity of P. sellowianus extracts [6]. They

identified the following chemical components in the bark

extracts: a pentacyclic triterpene (phyllanthol), a biflavo-

noid (cupressuflavone), flavonoids, coumarins (isofraxidine

and scopoletin), chlorogenic and caffeic acids; glucides and

proteins. Other studies have detected the presence of

phyllantimide in leaves and stems; phytosterols in roots

and flavonoids such as quercetin, rutin, isoquercitrin, and

xantoxilin in very small amounts.

Furthermore, hemorheological studies are considered an

important tool to detect and quantify alterations in several

pathologies, such as DB and arterial hypertension (AH).

Consequently, they are also very useful to analyze the

hemocompatibility and functionality of different chemical

agents, particularly in herbal medicine [7, 8].

Since several plant extracts can induce some biological

activities, such as agglutination of human and animal

erythrocytes and malignant cells [9], it is important to

analyze also the possible agglutinant action of P. sellowi-

anus extracts to evaluate the feasibility of its use as an

injectable solution. Furthermore, the relationship between

chemical components, hemorheological action and agglu-

tinant power could provide accurate information to eluci-

date the anti-diabetic mechanism [10].

For these reasons, the present work analyzes the in vitro

biorheological action and immunological activity of

extractive solutions of P. sellowianus bark on erythrocyte

membrane.

Materials and Methods

Plant Material

Samples of P. sellowianus Mull. Arg. were collected from

Nogoya River, in Nogoya city, Entre Rıos, Argentina, in

May 2008. Voucher specimens are stored in the Herbarium

of Plant Biology Area at the National University of Ro-

sario, Argentina. Adult plants were selected and the stems

separated to get the bark. The plant material was air-dried

at room temperature. The bark was taken out with special

bistouries [11].

Examined Herbarium Material

Samples were inquired at the National Herbaria, which are

cited according to the abbreviations listed in the Index

Herbarium [12] as following:

ARGENTINA. Provincia de Misiones. Dto. Gral. Bel-

grano, 15-III-2002, Mulgura M.E., 3371 (SI). Dto. Cai-

nguas, 21-IX-1999, Biganzoli F., 546 (SI). Provincia de

Buenos Aires. 15-XII-2003, Hurrell J.A., 5512 (MU, SI).

Morphoanatomical Characterization

Phyllanthus sellowianus’ bark and leaves were analyzed

for morphoanatomical characterization of this species.

Anatomical features of the bark were carried out from

images recorded with PM-10ADS Olympus automatic

Photomicrographic system and an optical microscope Zeiss

Axiolab attached to a Photographic system MC 80. Dif-

ferent kinds of cells were observed, identified, and mea-

sured with an optical microscope Olympus CH30-LB with

micrometric ocular.

Fig. 1 Leaves and flowers of Phyllanthus sellowianus Mull Arg. are

observed growing together

Cell Biochem Biophys

123

Leaves were previously treated according to the protocol

described by Sorribas de Lozano [13]. The microscopic

images of the leaf surfaces were obtained by Scanning

Electron Microscopy (Electronic Microscope, Leitz mod.

AMR 100, tungsten filament). An Optical Microscope

(Leica LS2, objective 409) and a digital camera (Canon

Powershost A640) attached to the microscope with a

52-mm adapter were also used for image acquisition.

Previously, leaves and bark were prepared according to two

histological techniques: Dizeo Strittmatter’ Diafanizade for

leaves and Boodle’ Macerate for bark [9].

Preparation of Extracts

Phyllanthus sellowianus bark extracts were prepared at the

following four different temperature conditions and

methods:

PM Maceration at room temperature

PD Controlled digestion at 37 �C

PI Infusion in a solvent at 100 �C

PC Decoction the bark and solvent at 100 �C

The extracts were prepared at 5 % concentration [14]

suspending 4 g of P. sellowianus bark in 80 mL of phys-

iologic solution (Rivero Cia S.A., L 107014). Finally, the

extractive solutions were filtered using 0.2 lm filters

(Acrodisc) to eliminate big particles and sterilize the

solutions. Finally, extractive solutions were stored at 4 �C

in sterile caramel glass flasks. All these steps were carried

out in sterile standard conditions [15].

Physicochemical properties of extractive solutions were

evaluated: pH was adjusted to 7.4 (physiologic pH) with a

solution 0.25 M of Na(OH) and osmolarity was leveled up

to 300 mOsm. The total protein content was measured by

Pyrogallol Red method. Viscosities of solutions were also

measured at 115.2 s-1 in a cone/plate viscometer (Brook-

field DV-II ?). These results are shown in Table 1.

The extractive solutions were also diluted at 25 and

50 % in physiologic solution and stored at 4 �C in sterile

glass flasks.

Human Erythrocytes

Blood samples of ABO blood group system were obtained

from healthy donors in sterile tubes, anticoagulated with

EDTA ,and processed within 45 min after collection.

Blood samples were centrifuged at 1,000 rpm, during

5 min at 25 �C. After removing plasma and buffycoat,

RBCs were washed three times with phosphate buffered

saline (PBS) (pH = 7.4, Osm = 295 mOsmol/kg).

Treatment of Human Erythrocytes

Washed erythrocytes were incubated with the extractive

solutions (PM; PD; PC), diluted at 50 and 25 % in PBS

solution at 37 8C during 60 min [16]. After washing three

times with PBS, RBCs were suspended in autologous

plasma at 40 % to measure the hemorheological param-

eters [17, 18] and 0.25 % to analyze the size, distribu-

tion, and morphology of the aggregates by digital image

analysis [19]. During the washing process, the aspect of

the supernatant was observed to confirm the absence of

hemolysis.

Immunohematological Tests

Immunohematological activity was tested in ABO blood

group with the four different extracts. To evaluate the

agglutinant capacity of the extractive solutions, two qual-

itative tests were carried out: Tube test and Plate Test.

Plate Test: One drop of the whole blood in physiologic

solution 20 % was mixed with 1 drop of the extract solu-

tion on a plate, moving it carefully to mix them and making

possible the contact for 2 min. During this time, aggluti-

nation could occur and the positive or negative result can

be observed with a tungsten lamp. This procedure was

repeated with all the extractive solutions (Table 2).

Tube Test: This test was carried out in two different

media with the four extractive solutions. First, 2 drops of

Table 1 Physicochemical analysis of extractive solutions from

Phyllanthus sellowianus bark obtained by different methods: PM, PD,

and PC

Total

Proteins mg/dL

Osmolarity

mOsm/kg

Initial pH Viscosity

cp

PM 65 ± 5 282.5 ± 0.5 5.7 ± 0.4 1.70 ± 0.02

PD 35 ± 5 278.5 ± 0.5 5.9 ± 0.1 1.47 ± 0.02

PC 63 ± 3 230 ± 1 5.8 ± 0.9 0.90 ± 0.02

Mean values ± SD

Table 2 Immunohematological

plate test showing the possible

agglutinant power of PM, PD,

and PC extract for different

groups of ABO blood groups

Blood

samples

PM PD PC

O ? (?) (?)

A ? ? - (? -)

AB ? ? - (?)

Table 3 Immunohematological

tube test showing the possible

agglutinant power of PM, PD,

and PC extract for different

samples from ABO blood

groups in Bromeline medium

Blood

Samples

PM PD PC

O ? ? ?

A ?? ? - ??

AB ??? ?? ???

Cell Biochem Biophys

123

the whole blood (O, A, B or AB group) were mixed with

1 mL of the extractive solution and 1 drop of saline solu-

tion in a Kahn tube. Then, another tube was prepared in the

same way with the whole blood and the extractive solution,

adding a proteolytic enzyme called Bromeline, to facilitate

the attraction between RBCs. After 15 min, the tubes were

centrifuged at 1,000 rpm for 1.5 min. Finally, the positive

or negative results were observed using a tungsten

lamp (Table 3).

Hemorheological Analysis

Rheological parameters of treated RBCs were evaluated

with an Erythrodeformeter [17] [20]. The use of this

equipment is based on laser diffractometry technique and

the following viscoelastic parameters of the erythrocyte

membrane can be determined [21]:

DI Deformability Index

l Elastic modulus

gm Membrane surface viscosity

where l is related to the elasticity of the citoesqueleto and

gm is related to the fluidity of lipid belayer.

These values were analyzed in RBCs obtained from B

(?) healthy donors, treated with the different extracts

diluted at 25 % and control.

Analysis of Erythrocyte Aggregation

Erythrocyte aggregation parameters were studied by Digital

Image Analysis. Briefly, treated RBCs and Control were

suspended in autologous plasma at 0.25 % and pushed in an

excavated slide [22] [23]. After 5 min, each sample was

observed using an Inverted Optical Microscope (Union

Optical, Japan; objective 409) with a digital camera (Canon

Powershost A 640) attached by means of a 52-mm adapter.

The images of each RBC sample were obtained in triplicate.

The erythrocytes were incubated with the extractive

solutions (PM, PD, PC) diluted at 25 and 50 % in PBS

solution to get small aggregates to obtain clear and precise

results.

The aggregates were classified according to the four

following categories to analyze the aggregate size, distri-

bution, and morphology [24]:

a- Individual cells (IC)

b- Aggregates with 2, 3 or 4 cells

c- Aggregates with 5 or more cells

d- Networks of big aggregates (Amas)

Isolated cell coefficient (CCA for their acronym in

Spanish) obtained by digital image analysis [25] is defined

as the difference between individual cell number in control

sample (CAi) and individual cell number of samples where

the erythrocytes were treated with the extractive solutions

(CAf).

CCA¼CAi � CAf

CAi

According with this equation, when the CCA is equal

or near to zero, there is no variation in the isolated cell

number after extract activity. However, if the aggrega-

tion increases after the treatment, the number of isolated

cells decreases, and as a consequence CCA will be near

to 1.

The images obtained were also analyzed to characterize

the morphology of the erythrocyte aggregates [17]. The

projected area (A) and the perimeter (P) of each erythro-

cyte aggregate were measured by means of ImageJ Soft-

ware to calculate the Aggregate Shape Parameter (ASP) by

the following equation:

ASP ¼ 4pA

P2

This parameter is a measure of circularity of erythrocyte

aggregates because ASP is near 1 for globular aggregates,

and ASP is approximately 0.5 for normal aggregates called

‘‘rouleaux’’ (similar to a rectangular area).

ASP was calculated by digital image analysis averaging

three images for each sample, using diluted extractive

solutions at 25 % (PM, PD and PC). ASP could not be

measured at higher concentrations using pure extractive

solutions and PI because of the presence of very large

network of aggregates.

Statistical Analysis

Statistical calculations were carried out with the SPSS 10.0

for Windows software package (Statistica). Results are

expressed as the Mean ± standard deviation of 5 inde-

pendent experiments. Student’s t test was used for statis-

tical analyses; P values \ 0.05 were considered to be

significant.

Results

Morphoanatomical Characterization

The images of leaves obtained by scanning electron

microscopy (SEM) showed the stomas, its distribution and

stomatic cells (Fig. 2). Parasitic stomas were clearly

observed distributed in the leaf surface with irregular ori-

entation. The values of the stoma sizes varied between 20

and 30 lm according to the microscope scale in the image.

Cell Biochem Biophys

123

The analysis of bark images (Fig. 3) provides the fol-

lowing bark anatomical features:

Outer bark anatomical features: The rhytidome was

comprised only by one periderm, exfoliating, consisting of

12–15 layers of cells with varying dimensions. Super-cells

were generally quadrangular with wavy anticlinal, slightly

thickened, and smooth walls. The large air contents were

observed. Pheloderm was multi-layered with thin cell

walls. The thickness was 300 lm.

Inner bark anatomical features: The cortical paren-

chyma was abundant and homogeneous with very few

calcium oxalate druses. Small groups of fibers were

observed at the edge of the phloem. Functional phloem was

divided into 1-2-3-seriate radio with marked widening of

the distal region. Abundant styloids in axial parenchyma

were observed. The thickness varied between 800 and

1000 lm.

Immunohematological Tests

Experiences with ABO system showed that the agglutinant

capacity of the erythrocytes could be observed when they

Fig. 2 Image of adaxial face of a leave obtained by Scanning

Electron Microscopy, where can be observe the epidermis, stomas,

and stomatic cells. Scale: 100 lm

Fig. 3 Phyllanthus sellowianus Mull. Arg. Transverse section of the

bark. cp cortical parenchyma, fi fibers, ph functional phloem, ra radio,

su super. Scale: 100 lm

Table 4 Mean values of stationary viscoelastic parameters for

human erythrocytes treated with three extractive solutions diluted at

25 %

RBCs DI l 10-3 N/m gsup 10-4 N.s/m

Control 0.58 ± 0.05 4.8 ± 0.7 1.9 ± 0.1

Treated with PM� 0.55 ± 0.05 6.1 ± 0.2 2.8 ± 0.4

Treated with PD� 0.58 ± 0.05 7.8 ± 0.7 3.6 ± 0.9

Treated with PC� 0.53 ± 0.05 6.4 ± 0.5 2.3 ± 0.1

Mean values ± SD

control PM 1/4 PD 1/4 PC 1/40,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

4,5

5,0

[10

-4dy

ne.s

/cm

]

Samples

control PM 1/4 PD 1/4 PC 1/40

2

4

6

8

10

[10

-3dy

ne/c

m]

Samples

ημ

(a)

(b)

Fig. 4 Mean values of the elastic modulus (l) and membrane surface

viscosity (gm) obtained with the Erythrodeformeter from RBCs

treated with 3 extractive solutions

Cell Biochem Biophys

123

were incubated with P. sellowianus extractive solutions.

Different quality and quantity of agglutinates were devel-

oped depending on each solution. The highest agglutinant

activity was reached by the extract solutions prepared by

decoction procedure (PC).

Results obtained from Plate-Test with ABO system

groups are shown in Table 2.

The most significant results obtained from Tube-

Test are shown in Table 3. The Tube-Test essays

showed no significant results because all the extract

solutions had the same agglutinant power on RBCs in

saline medium. However, the agglutinates changed in

quality with the addition of Bromeline, particularly

with AB group.

Hemorheological Analysis

The average values of stationary hemorheological param-

eters: ID, l, gsup for samples treated with three diluted

extractive solutions and obtained by duplicate are shown in

Table 4 and Fig. 4. l and gsup showed significant differ-

ences (p \ 0.02) in RBCs treated with all the extractive

solutions against the control sample. The ID presented

similar values in all the essays (p [ 0.05). The most sig-

nificant alteration was observed in l and gsup with the

erythrocyte treated with PD extractive solution (p \ 0.01).

Digital Image Analysis

Several examples of the images obtained from blood

samples (group B, Rh positive) control and the RBC treated

with pure extracts PM, PD, and PC are shown in Fig. 5.

The normal erythrocyte arrangement called ‘‘rouleaux’’ can

be observed in the control sample (Fig. 5a), while different

globular aggregates are presented in treated RBC samples

(Figs. 5b–d).

Isolated cell coefficient, numbers, and percentages of

cells were calculated with 3 images for each sample and

average values were obtained. CCA was first calculated by

incubating RBCs with extractive solutions diluted at 50 %.

(a) Control (b) PM

(c) PD (d) PC

Fig. 5 Images of adhered RBCs for the control and samples after incubating with the PM, PD, and PC extractive solutions

Cell Biochem Biophys

123

In this case, there was no significant difference (p [ 0.05)

between the values in relation to the prepared extractive

solutions. Every sample showed strong aggregation and all

the values were near 1 (Table 5). On the other hand, sig-

nificant differences in CCA (p \ 0.02) were observed with

extract solutions diluted at 25 % (Table 6) and the fol-

lowing order of aggregation power could be established:

18 Infusion and Decoction (the highest erythrocyte

aggregation)

28 Maceration and Digestion (the lowest erythrocyte

aggregation)

The average values of CCA of all the extractive solutions

diluted at 50 and 25 % were also calculated and compared

(Fig. 6).

These results suggest a relationship with the working

conditions since the extractive solutions obtained by mac-

eration and digestion were prepared at room temperature

and 37 8C, respectively, while the solutions obtained by

infusion and decoction were prepared at 100 8C (solvent

boiled temperature). Therefore, work temperature can be

responsible for these alterations since the quantity and

quality of the extracted active components were affected.

Images RBCs incubated with P. sellowianus extractive

solutions (PM, PD and PC) at different concentrations

(100 % and diluted at 25 %) showed a possible agglutinant

activity. Different sizes and quantities of ‘‘rouleaux’’ could

also be observed (Fig. 7a).

Assays were carried out with ABO blood group incu-

bating the RBCs with all the extracts, which produced large

globular aggregates (Fig. 7b–g) probably induced by its

agglutinant power.

To evaluate this possible agglutinant activity, the images

were studied and ASP average values for each extractive

solution and concentration were calculated. RBCs treated

with pure solutions or dilutions lower than 25 % could not be

analyzed because of the presence of macroscopic aggluti-

nates, which represented an obstacle to measure ASP. Only

images of solutions diluted at 25 % could be analyzed

(Table 7). A similar situation could be observed when ASP

was calculated with erythrocytes incubated with extractive

solutions obtained by Controlled Digestion at 37 �C and at

different concentrations (Table 8). ASP average values of

PD extracts were measured and the results showed that the

biorheological action depends lineally on the concentration

of the extractive solutions (Fig. 8).

Discussion

Results show that biorheological action of extracts changed

with respect to temperature conditions used during the

extract preparation. Biorheological activities with PC and

PI extractive solutions showed higher agglutination than

PM and PD extractive solutions.

In all the extractive solutions studied in the present work,

ASP was greater than normal values, which indicates the

atypical globular shape of erythrocyte aggregates. There-

fore, these extractive solutions could not be used as inject-

able without altering the normal erythrocyte adhesion.

Table 5 Mean values of total cell number (Ct), isolated cell num-

ber (Ca), and isolated cell coefficient calculated from the images of

RBCs of control and samples treated with the extractive solutions

diluted at 50 % in PBS

RBCs Ct Ca %Ca CCA

Control 66 ± 1 37 ± 1 56 ± 2 –

PM 97 ± 1 8.0 ± 0.5 8 ± 1 0.85 ± 0.07

PD 87 ± 1 4.0 ± 0.5 5 ± 2 0.9 ± 0.1

PC 104 ± 1 12 ± 1 11 ± 1 0.8 ± 0.1

Mean values ± SD

Table 6 Mean values of total cell number (Ct), isolated cell num-

ber (Ca), and isolated cell coefficient calculated from the images of

RBCs of Control and samples treated with the extractive solutions

diluted at 25 % in PBS

RBCs Ct Ca % Ca CCA

Control 37 ± 1 24 ± 1 65 ± 4 –

PM 42 ± 1 22 ± 1 52 ± 3 0.2 ± 0.1

PD 51 ± 1 23 ± 1 45 ± 3 0.3 ± 0.1

PC 53 ± 1 12 ± 1 23 ± 2 0.7 ± 0.1

PI 44 ± 1 15 ± 1 34 ± 3 0.5 ± 0.1

Mean values ± SD

-- -- -- --0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

PIPCPDPM

Agg

rega

tion

Coe

ffici

ent

Samples

Dilution 1/4 Dilution 1/2

Fig. 6 Mean values of CCA of the extractive solutions diluted at 50

and 25 % in PBS

Cell Biochem Biophys

123

(a)

(b) (c)

(d) (e)

(f) (g)

Control

PM 100% PM 25%

PD 100% PD 25%

PC 100% PC 25%

Fig. 7 Images of RBCs treated with extractive solutions (100 and 25 % in PBS). Different morphologies of adhered red blood cells can be

observed

Cell Biochem Biophys

123

Hemorheological tests showed significant differences in

l and gsup in RBCs treated with all the P. sellowianus

extractive solutions against the control samples, and more

specifically with PD extractive solutions.

Conclusion

Results obtained in the present work are useful to study the

action mechanism of extracts from P. sellowianus bark in

order to evaluate its use as a therapeutic agent in diabetes

and give information about its hemocompatibility. This

study could contribute to analyze and optimize the proto-

cols for pharmaceutical administration and suitable con-

centrations, without promoting microcirculation alterations

commonly found in vascular pathologies. Moreover,

immunohematological tests with ABO blood group show

the possible agglutinant power of P. sellowianus extracts,

which could be useful in Immunohematology to be used as

hemoclassifier in the future.

Acknowledgments Authors thank to Pablo Dıaz from Instituto de

Fısica Rosario (CONICET-UNR) for scientific and technical sup-

porting in the acquisition of SEM images.

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Table 7 Mean values of ASP from Control and samples treated with

Phyllanthus sellowianus extractive solutions diluted at 25 % in PBS

Control PM PD PC

0.65 ± 0.03 0.76 ± 0.03 0.67 ± 0.04 0.77 ± 0.03

Mean values ± SD

Table 8 Mean values of ASP from Control and samples treated with

Phyllanthus sellowianus extractive solutions, prepared by controlled

digestion at different concentrations

Control PD 25 % PD 50 % PD 100 %

0.65 ± 0.03 0.67 ± 0.04 0.73 ± 0.02 0.80 ± 0.02

Mean values ± SD

20 30 40 50 60 70 80 90 100 1100,0

0,2

0,4

0,6

0,8

1,0

AS

P

PD concentration [%]

Fig. 8 Mean values (dots) and linear fit (line) of ASP versus the

different dilutions of PD extractive solutions. R2 = 0.95782

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