Tapak liman (Elephantopus scaber

L) extract–induced CD4+ and CD8+

differentiation from hematopoietic stem
cells and progenitor cell proliferation in mice
(Mus musculus L)
Cite as: AIP Conference Proceedings 1908, 060003 (2017); https://doi.org/10.1063/1.5012736
Published Online: 29 November 2017

Muhammad Sasmito Djati, Hindun Habibu, Nabilah A. Jatiatmaja, and Muhaimin Rifa’i

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AIP Conference Proceedings 1908, 060003 (2017); https://doi.org/10.1063/1.5012736 1908, 060003

© 2017 Author(s).
 Tapak Liman (Elephantopus scaber L) Extract–Induced
CD4+ and CD8+ Differentiation from Hematopoietic Stem
Cells and Progenitor Cell Proliferation in Mice (Mus
musculus L)

Muhammad Sasmito Djati1, a), Hindun Habibu2), Nabilah A Jatiatmaja3), Muhaimin

Rifa’i1, b)

1
Laboratory of Animal Physiology, Department of Biology, Faculty of Mathematics and Natural Sciences, University of
Brawijaya, Malang, Indonesia
2
Master Program of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Malang, Indonesia
3
Faculty of Dentistry, Airlangga University, Surabaya, Indonesia

a)
Corresponding author: msdjati@ub.ac.id
b)
rifa123@ub.ac.id

Abstract. Tapak Liman (Elephantopus scaber L) is a traditional medicinal plant containing several active compounds that
potentially affecting hematopoietic stem cells, such as epifrieelinol, lupeol, stigmasterol, triacontane-1-ol, dotriacontane-1-ol,
lupeol acetate,deoxyelephan-topin, isodeoxyelephantopin, polyphenol luteolin-7, as well as various flavonoids and
glucosides. The aim of this study was to elucidate the effect of leaf extract of Tapak Liman on hematopoietic stem cells in
mice BALB/c, by observation of the relative number of cells expressing CD4/CD8, CD4/CD62L, and TER119/B220 in the
spleen, and TER119/B220, TER119/VLA-4 and TER119/CD34 in bone marrow, after being administered leaf extract for 2
weeks. This experiment used 12 female mice, which were divided into three treatment groups, P1= 0.5 g·g bw-1·day-1, P2=
1.0 g·g bw-1·day-1 and P3 = 2.0 g·g bw-1·day-1 Tapak Liman leaf extract as well as a control. The relative numbers of cells
expressing surface molecules were analyzed by flowcytometry and quantitative data were tested using one-way ANOVA.
The results showed that the leaf extract of Tapak Liman has no significant effect on erythrocyte proliferation; on the other
hand, it had a significant effect on both proliferation and differentiation of B lymphocytes (B220+) in bone marrow (p=0.044)
and increased the expression of CD4+, CD8+ molecule in B cells (p=0.026) and erythroid cells in spleen and bone marrow,
based on the estimation of cells that expressed TER119+VLA-4+, identified as important in the development pathway of
erythrocytes. An increased cell percentage of TER11+VLA-4+ occurred for treatment P2, 12% higher than the control. The
increased expression of TER119+VLA-4+ was assumed to be due to the iron content in Tapak Liman, which functioned to
stimulate the progenitor hematopoietic cells to proliferate and differentiate into a precursor of erythroid cells (TER119
+
VLA-4+). There was an increasing number of cells expressing the surface molecules TER119+ and VLA-4+. This indicated
that the Tapak Liman leaf extract with a dose of 1.0 g·g bw-1·day-1 could stimulate the proliferation of hematopoietic stem
cells into the lymphoid and erythroid pathway, in spleen and bone marrow.

8th International Conference on Global Resource Conservation (ICGRC 2017)

AIP Conf. Proc. 1908, 060003-1–060003-9; https://doi.org/10.1063/1.5012736
Published by AIP Publishing. 978-0-7354-1600-0/$30.00

060003-1
 INTRODUCTION

Traditional medicines with an herbal component have many potential indications as antioxidants, immuno-
modulators, hormonal therapies, etc.1-5 Tapak Liman (Elephantopus scaber L) is a traditional medicinal plant that
has been known for centuries, especially among pharmacologists in China. Tapak Liman contains epifrieelinol,
lupeol, stigmasterol, triacontane-1-ol, dotriacontane-1-ol, lupeol acetate, deoxyelephan-topin, isodeoxyelephantopin,
polyphenol luteolin-7, as well as various flavonoids and glucosides. Tapak Liman is used fresh, dried, or even
extracted and put into capsules. Types of diseases that can be treated with Tapak Liman include various
inflammatory diseases such as inflammation of the tonsils, eyes, and kidneys, acute and chronic inflammation of the
uterus or vaginal discharge, as well as influenza and sore throat. Additionally, Tapak Liman also serves as a
laxative of urine, an antioxidant, blood booster, and decreases fever and clears phlegm. It is also useful to overcome
chicken pox and anemia.6
Anemia is a disease caused by a deficiency of red cell volume and hemoglobin levels (Hb), thus the body is
hypoxic as a result of the reduced oxygen-carrying capacity of the blood.7 Anemia can be caused by autoimmune
diseases such as hemolytic and aplastic anemia, due to delays in the process of erythropoiesis.8 Until now, the
treatment of anemia that is caused by erythropoiesis barriers solely depends on the provision of artificial
hematopoietin. This is commonly called ESA (erythropoiesis-stimulating agent), a synthetic compound that can
stimulate the production of blood cells. However, ESA has harmful side effects such as cardiovascular
complications9 and retinopathy.10 Thu, this provides an opportunity to investigate and search for safe drugs that
could stimulate and affect erythropoiesis with no side effects, and/or boost the immune system by inducing the
erythropoiesis and lymphopoiesis proliferation and differentiation processes.
Thus, our research focused on the effects of Tapak Liman leaf extract on hematopoiesis in female mice (Mus
musculus) BALB/c. It was expected that chemical substances contained in extracts of Tapak Liman leaves could
potentially induce and influence the process of hematopoiesis differentiation and proliferation, especially
lymphopoiesis and erythropoiesis.

EXPERIMENTAL DETAILS

Preparation of Leaf Extract and Treatment

Extraction of Tapak Liman leaves was conducted in a sterilized distilled water solvent.11-12 A total of 13.8 g of
dried leaves of Tapak Liman were ground by a mortar into a fine powder. Then, 100 mL sterilized dH2O was added
to the powder and stirred at 50 °C overnight and then filtered through filter paper. The final concentration was
calculated, and the extract solution was ready for administration.
The experimental animals used in this study were mice (Mus musculus) which were randomly sampled from a
population of female mice strain BALB/c, six weeks old and healthy (actively moving and with intact fur). The
experiment used 12 female mice, which were divided into three treatment groups, P1 = 0.5 g·g bw-1·day-1, P2 = 1.0
g·g bw-1·day-1 and P3 = 2.0 g·g bw-1·day-1 Tapak Liman leaf extract, as well as a control. The administration of
Tapak Liman leaf extract was given orally. the body weight of the mice was monitored for two weeks before
administration. Each mice group was given the same standard feed and drink. The main dependent variable
measured was the relative number of cells in hematopoiesis phase with the following parameters:

1. The number of cells that expressed CD4/CD8, CD4/CD62L, and TER119/B220 in the spleen
2. The relative number of cells that expressed TER119/B220, TER119/VLA-4, and TER119/CD34 in bone marrow

Spleen isolation was done by cutting part of the spleen with surgical scissors. The spleen sample was then
washed with PBS and cleaned of attached fat. Homogenates were filtered and collected in a sterile microtube and
stored at a temperature of 4°C.
The bone marrow was obtained from the femur bone of the left and right legs of mice. Muscles were separated
from the femur, and both ends of the joints were cut with surgical scissors.13

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 Bone marrow was isolated using a flushing method, injecting PBS with a 1 ml syringe (26½ size needle) at one
end of the bone. The results of the injection were collected in a sterile petri dish, filtered and stored in a sterile
microtube at a temperature of 4°C.

Flowcytometry
Flowcytometry was performed on homogenate from spleen and bone marrow that was centrifuged at 3200 rpm,
4 °C for 2 minutes.14 The supernatant was removed and the pellet resuspended in 1 mL of sterilized PBS and then
homogenized. Homogenates were taken in aliquots of 100-200 μL by using a micropipette and inserted into new
sterile microtubes that were covered with aluminum foil. Homogenates were recentrifuged at 3200 rpm, 4 °C for 2
minutes, then the supernatant was removed and the microtube put into the icebox.
Antibody BD BioscienceTM DC4 FITC anti-mouse conjugated and PE-CD8, DC4 FITC anti-mouse conjugated
and PE-CD62L was added to all pellets in the microtubes. BD BioscienceTM antimouse TER-199/Erythrocyte Cell
FITC conjugated and PE- B220 was added to pellets isolated from spleen. To pellets from bone marrow were added
antibody BD Bioscience antimouse TER-199/Erythrocyte Cell FITC conjugated PE- B220, TER-199/Erytrocyte
Cell FITC conjugated CD49d/(VLA-4), and antimouse TER-199/ Erythrocyte Cell FITC conjugated PE-CD34 and
then incubated for 15 minutes.
Next, a flow cytometer (BD Bioscience FACS CaliburTM) was connected to a computer in the condition
Acquiring, and a program run on the computer according to expected parameters, including setting instrument
(Detector, Threshold, and Compensation) on the number of cells to be analyzed (Acquisition and storage), label of
antibody and laser excitation power, simple name and determining the grated area (R1) on the plot of histogram. The
setting plot on Acquiring mode, according to the label of antibody on the axis of Y and X (FITC or PE) and grating
area (G1=R1). Flowcytometry was ensured in the set of Low-Run. After the instrument was ready, pellets to which
had been added antibody were put into the cuvette of the flow cytometer by micropipette, to which was added 1000
μL of sterile PBS and homogenized by pipetting. The cuvette was attached to the nozzle of the flow cytometer and
data measurements acquired using the acquisition control module. Data from flow cytometry were processed with
the software BD CellQuest ProTM and displayed in the form of a histogram.

Data Analysis
Quantitative data included the relative number of progenitor and precursor cells in the development of lymphoid
and erythroid cells in the bone marrow and spleen, which was obtained from the flow cytometer. Data were
statistically analyzed by the normality test and homogeneity of variance test. Data that had a homogeneous variance
were tested by one-way ANOVA with p= 0.05. If p>0.05 then there was no significant treatment effect, whereas if
the p<0.05 then there was a significant difference between treatments. Last, treatments were compared post-doc with
the Tukey HSD (Highly Significant Difference) test. Data were analyzed with SPSS 13 for Windows.

RESULTS AND DISCUSSION

Profile of Cell Express CD4 and CD8 Molecules in Spleen
CD4 is an antigen expressed in inflammation by T cells, monocyte cells, and macrophage cells and function as
co-receptor molecules of MHC Class II, and a receptor for HIV. Otherwise, CD8 is an expressed antigen by a subset
of thymocytes, cytotoxic T cells, and has a function as a co-receptor of MHC Class I.15
The flow cytometer analysis found that in the spleen (Fig. 1), there were no significant differences between cells
that expressed CD8+CD4, CD4+CD8-, and CD4+CD8+ in each treatment group compared to the control.
Hematopoietic stem cells (HSC) were not stimulated to differentiate specifically into cells of CD4 +CD8-, CD4-CD8+,
and CD4+CD8+ (p>0.05) upon treatment with Tapak Liman (groups P1-P3).
The cell population of CD4+CD8- and cells of CD4-CD8+ in treatment P2 (1.0 g·g bw-1·day-1 Tapak Liman
extract) increased, compared to control (Figure 1). P2 appeared to increase the total number of cells isolated from
spleen. We assume that it happened due to several factors. First, the concentration of leaf extract of Tapak Liman at

060003-3
dose P2 increased lymphocytes proliferation; second, the population of P2 exposure to the disease; last, the
population of P2 had a bigger spleen (splenomegaly), i.e. the spleen ability to get and keep the erythrocytes will be
increased. Splenomegaly causes a decrease of erythrocytes, leukocytes, and thrombocytes in the blood circulation.
Figure 1 shows that the populations of T cells CD4+ and CD8+ increased in the P2 group, 7.92 x 106 cells and
8.712 x 106 cells, respectively. Otherwise, the number of T cell CD4+ and CD8+ tended to decrease with treatment
P3. It is presumed that this was negative feedback from a high dose Tapak Liman leaves extract. It may be caused
by an abundance of lupeol and flavonoid compounds in the extract, which is known as anti-inflammatories. This
dose-dependent phenomenon is in accordance with a previous study, which mentioned that herbal medicines will
stimulate or conversely suppress the degree of immunity.16

FIGURE 1. Profile of T Cell CD4 and CD8; the cell expression of CD4 +CD8-, CD4+CD8+, and CD4+CD8+ in the spleen. Control
(K), P1: 0.5 g·g bw-1·day-1, P2: 1.0 g·g bw-1·day-1, P3: 2.0 g·g bw-1·day-1 Tapak Liman extract.
The results showed that mice administered leaf extract of Tapak Liman for two weeks compared to the control
group showed no significant effects on erythropoiesis in female BALB/c mice. This was most likely caused by
several factors, e.g. the extraction method was not appropriate to obtain the active substance, or a longer extraction
time was needed. However, the study indicates a significant effect on the production of B lymphocytes in the bone
marrow and increases in the number of B cell (CD62L+) in the spleen.

Profile of Cell Express CD4 and CD62L Molecules in Spleen

CD62L is an antigen expressed by B cells, T cells, monocytes and natural killer cells (NK cells). It binds CD34
which functions as a leukocyte adhesion molecule (LAM/L-selectin), GlyCAM, and has roles in the interaction of
rolling over endothelial cells.3
Analysis of the flow cytometry results (Fig. 2) showed that the treatment groups had no significant differences in
all treatment (p>0.05) in the percentage of cells that express CD4 +CD62L and CD4+CD62L+, whereas the average
percentage of cells that express CD4CD62L+ (B cell) increased for treatment P1 and P2, 6% and 3% respectively,
over the control, and decreased in treatment P3 by 5%.
Statistical analysis showed a significant effect (p<0.05) of the increased percentage of CD62L in treatment P 2.
This indicates that flavonoid compounds present may have an increasing effect on the production of IL-2, thus
increasing the B lymphocytes.
In terms of the number of cells that expressed CD4 +CD62L, CD4CD62L+, and CD4+CD62L+ (Figure 2), the
largest number of cells expressing CD4+CD62L and CD4CD62L+ was treatment P2, with 7.128 x 106 cells, and
7.524 x 106 cells respectively. The number of cells increased with an increased dosage of Tapak Liman leaf extract
but decreased in treatment group P3. The treatment with the highest number of cells that expressed CD4+CD62L+
was P3 (2.270667 x 106 cells).

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 FIGURE 2. Profile of CD4 and CD62L cells; relative cell number of CD4 +CD62L, CD462L+, and CD4+ CD62L+ in spleen.
Control (K), P1: 0.5 g·g bw-1·day-1, P2: 1.0 g·g bw-1·day-1, P3: 2.0 g·g bw-1·day-1 Tapak Liman extract.

Profile of Cell Expression of TER119 and B220 in the Spleen

B220 or CD45R antigen is expressed by B cells, a subset of T cells (naive T cell), and monocytes. It functioned
as an isoform on An exon-contained CD45.15 Analysis of flow cytometry results (Figure 3) showed that the
treatments affected the percentage of cells that expressed TER119+ B220 and TER119+B220+. Otherwise, cells that
express TER119B220+ tend to increase in treatment P2, 9% compared to control, but decreased by 6% compared to
treatment P3.
Statistical analysis showed that the percentage of TER119+B220, TER119B220+ and TER119+B220+ were not
significantly different in each treatment (p>0.05). Whereas the number of cells of TER119+ and B220+ increased in
P2 (Fig. 3), compared to other treatments and control. This indicates that the flavonoid compound increased the
production of IL-2, thus increasing the proliferation of B lymphocytes.

FIGURE 3. Profile of TER119 and B220 cells; relative cell number of TER119 + B220, TER119B220+ and TER119+B220+ in
the spleen. Description: Control (K), P1: 0.5 g·g bw-1·day-1, P2: 1.0 g·g bw-1·day-1,
P3: 2.0 g·g bw-1·day-1 Tapak Liman extract.

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 Profile of Cell Expression of TER119 and B220 Molecules in Bone Marrow
Bone marrow is the primary or central lymphoid organ where the process of hematopoiesis occurs. It includes
the lymphopoiesis for maturation, differentiation, and proliferation of T cells and B cells into lymphocytes that
recognize the antigen. Bone marrow is also where erythropoiesis takes place.
In this study, isolated cells from the bone marrow to which was added antibody antimouse TER-119/Erytrocyt
Cell conjugated to FITC and antimouse B220 conjugated to PE (BD Bioscience©). TER-119 is a marker for the
erythroid cell development in erythropoiesis from proerythroblasts to mature erythrocytes. Otherwise, B220 (CD45)
is a molecular marker for B cell and all hematopoietic cells, which function for phosphatase thyroxine, increasing
signal through the antigen receptor of B and T cells.
Flowcytometry results (Figure 4) showed a difference in the average percentage of B220 + (CD45) between
treatment groups compared to the control. The percentage of cells that expressed TER119B220+ increased 9% over
the control for P1, while for treatment P2, significantly increased 16% compared to the control. Otherwise, the
number of cells expressing TER119+B220 was not significantly different, similar to TER119+B220+. Referring to
these data, the administration of doses of Tapak Liman leaf extract increased the B cell population (B220 +/CD45+).

FIGURE 4. Profile of TER119 and B220 cells; relative cell number of TER119+B220, TER119B220+ and TER119+ B220+ in bone
marrow. Control (K), P1: 0.5 g·g bw-1·day-1, P2: 1.0 g·g bw-1·day-1, P3: 2.0 g·g bw-1·day-1 Tapak Liman extract.

The highest cell population of TER119B220+ was found in P2 (24.546666 x 106 cells). This demonstrated
proliferation and differentiation of hematopoietic stem cells had occurred, which tend to the production of B cells
(B220+/CD45+). The increase in B cells is assumed to be due to the flavonoid and lupeol compounds contained in
Tapak Liman.

Profile of Cell Expression of TER119 and VLA-4 Molecules in Bone Marrow

VLA-4 (Very Late Integrin Antigen-4) or CD49d is an antigen expressed by B cells, thymocytes, granulocytes
and dendritic cells. It functions as an integrin a4, connected to CD29 (leukocytes), and binds fibronectin, MadCAM-
1, and VCAM-1 (Vascular Adhesion Molecule-1).
The number of cells that expressed the TER119VLA-4+ and TER119+VLA-4+ are presented in Figure 5. The
percentage of cells that expressed TER119VLA-4+ decreased by 11% for P2, but increased by 12% in
TER11+VLA-4+, compared to the control.
As shown in Fig. 5, the highest cell population of TER119+VLA-4+ was in P2, i.e. 7.536 x 10 6 cells. The
increased expression of TER119+VLA-4+ in the P2 group treatment is assumed to be due to the iron content in
Tapak Liman, which functioned to stimulate the progenitor hematopoietic cells to proliferate and differentiate into
the precursors of erythroid and lymphoid cells into TER119+VLA-4+, which express nucleated erythrocyte cells, and

060003-6
other cells. Besides that, the increased expression of TER119VLA-4+ may be caused by adhesion molecules that
interact between cells with the extracellular matrix, which is needed for the activation of T cells for leukocytes
maturation. The increasing number of cells expressing surface molecules of TER119 + and VLA-4+ indicates that the
leaf extract of Tapak Liman at 1.0 g·g bw-1·day-1 was adequate to stimulate the proliferation of hematopoietic stem
cells into lymphoid and erythroid cells. The protein VLA on the surface of T cell functions to channel activation
signals through the T cell receptor. Under normal conditions, leukocyte only attaches to endothelial cells, but under
inflammation stimuli, adhesion between leukocyte and endothelial cells is enhanced.13

FIGURE 5. Profile of TER119 and LA-4 cells; relative cell number of TER119 VLA+ and ER119+VLA-4+ cells bone marrow.
Control (K), P1: 0.5 g·g bw-1·day-1, P2: 1.0 g·g bw-1·day-1, P3: 2.0 g·g bw-1·day-1 Tapak Liman extract.

Profile of Cell Express the TER119 and CD34 Molecules in Bone Marrow
A cluster of differentiation 34 (CD34) is an antigen expressed by hematopoietic precursor cells and in capillary
endothelial cells functions as Ligand CD62L (L-selectin). The average cell percentage of TER119CD34+,
TER119+CD34, and CD34+TER119+ in the bone marrow, as measured by flow cytometry, is presented in Figure 6.
The percentage of cells expressing TER119D34+ decreased compared to control, as well as the percentage of
TER119D34+ cells. While the percentage of cells that expressed CD34+TER119+ increased by 4% in treatment P1, it
tended to decrease in treatment P2 and P3, compared to control.

FIGURE 6. Profile of TER119 and CD34 cells; relative cell number of TER119 +CD34+, TER119CD34+ and TER119+ CD34+
in bone marrow. Control (K), P1: 0.5 g·g bw-1·day-1, P2: 1.0 g·g bw-1·day-1, P3: 2.0 g·g bw-1·day-1 Tapak Liman extract.

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 Based on the absolute number of cells, the TER119+CD34cell population did not increase after being treated
with Tapak Liman leaf extract. However, it tended to increase in P 2, compared to the other two treatments.
Otherwise, the number of TER119CD34+ and CD34+TER119+ cell population tended to increase in treatment P1
compared to control (Figure 6).
Statistical analysis showed no significant difference (p>0.05) between treatments. This is assumed to be due to
several factors: 1. The solvent for leaf extraction was not optimal to obtain sufficient active compound, and 2. the
time of samples extraction was not sufficiently long.
The treatment dose used in P2 (1.0 g·g bw-1·day-1) showed a significant effect (p=0.026) on increasing the
percentage of cell CD62L+ in the spleen and B220+ (p=0.044) in the bone marrow, compared to the 0.5 and 2 g·g
bw-1·day-1 doses as well as the control group. It indicates that there was increased proliferation and differentiation of
hematopoietic cells into lymphocytes, i.e. CD4+, CD8+ and B220+ T cells. This may have been caused by flavonoid
compounds present in the extract that potentially stimulate the activity of IL-2, thus increasing the proliferation of
lymphocyte cells.
Based on the cell numbers from all of the treatment groups, it is shown that the cell populations increased with
the addition of Tapak Liman leaf extract dose of 1.0 g·g bw-1·day-1 (P2) compared to the control group. Otherwise,
the cell population decreased with the 2.0 g.g bw.-1day-1 dose (P3). It appears the flavonoid compounds present,
besides having an effect as an immunostimulant, it also has the effect of immunosuppressant at higher doses.12
Moreover, it appears the cytotoxic effects of compounds in Tapak Liman, which serve as an immunosuppressant,
also allow the resistance progenitor cells in the lymphoid and erythroid pathway to proliferate and differentiate into
lymphocytes and erythrocytes.
The increased cell expression percentage of TER11+VLA-4+, 12%, seen in treatment P2, compared to control,
was important because it identifies the pathway for development of erythrocytes. The increased expression of
TER119−VLA-4+ is likely caused by adhesion molecules interacting with the extracellular matrix, which is needed
in the activation of T cell in the maturation process for leukocytes. Furthermore, the increased expression of
TER119+VLA-4+ was assumed to be due to the iron content in Tapak Liman, which functions to stimulate
progenitor hematopoietic cells to proliferate and differentiate into a precursor of erythroid cells (TER119+VLA-4+),
which further develop into nucleated erythrocyte cells and others. There was an increasing number of a cell
expressing the surface molecules TER119+ and VLA-4+, with doses of 1.0 g·g bw-1·day-1 Tapak Liman leaf extract,
stimulating the proliferation of hematopoietic stem cells into lymphoid and erythroid cells.

SUMMARY
The administration of 1.0 g·g bw-1·day-1 of Tapak Liman leaf extract stimulated proliferation of lymphocytes
and erythrocytes lineage (TER119+VLA-4+), in spleen and bone marrow.

ACKNOWLEDGEMENT
The author would like to thank Prof. Dr. Ir. Siti Chuzaemi, MS, the head of Institute of Research and
Community Services (LPPM), University of Brawijaya and also the Project Leader of Graduate Grant, Directorate
General of High Education DEPDIKNAS.

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