ARTIKEL UTAMA

Changes in Immunological Parameters

by Standardized Phyllanthus niruri Extract
in the Pre-Clinical and Clinical Studies

Abstract. One of traditional herbs used as medicine in many countries for various
uses is the herb Phyllanthus niruri L. Many pre-clinical studies have explored
its activities on the immune system. Further pre-clinical studies were done to
determine its safety and immunomodulating characteristics on mice and rats.
The results of these studies suggested that Phyllanthus niruri L extract is able
to modulate the immune system, via proliferation and activation of T- and B-
lymphocytes, secretion of specific cytokines such Interferon-gamma, Tumor
Necrosis Factor-alpha, and several interleukins, activation of the complement
system, activation of phagocytic cells such as macrophages and monocytes, as
well as increase of cytotoxic cells such as the Natural Killer cells. Finally, clinical
studies were designed to search its immunomodulating effects in patients on
various clinical conditions. Similar results from pre-clinical and clinical studies
were found on the immunological parameters, suggesting that Phyllanthus niruri
L extract can work as an immunomodulator, providing the benefits when used as
an adjunct therapy for infectious diseases.

Raymond R Tjandrawinata*, Key words: Phyllanthus niruri L, standardized extract, immunomodulator

Suprapto Ma'at**, Dwi Nofiarny*
* Department of Medical Affairs
Dexa Medica Group, Jakarta
Introduction

T
** Lab./Installation of Clinical Pathology
Faculty of Medicine University of Airlangga/ he herb Phyllanthus niruri L, known as Meniran in some of
Dr. Soetomo District General Hospital, Surabaya Indonesian area, is recognized and used in many countries for
over 2,000 years. There are many applications of this herb in the
use of the traditional setting of many cultures. These include jaundice,
gonorrhea, urinary tract infection, stomachache, toothache, anti-pyretic,
kidney stone and disease, diuretic, frequent menstruation, diabetes, as well
1,2
as dysentery.
There are, at least, two kinds of meniran that easily found and used as
traditional medicine. One plant has a pale green stem Phyllanthus niruri,
2,3
while the other is characterized by its reddish stems Phyllanthus urinaria.
In many countries, the name Phyllanthus niruri is also used to identify
the different species of genus Phyllanthus. In Middle and South America,
plant that recognized as P. niruri is actually a P. amarus. In Africa and Asia,
P. niruri name is used interchangeably to refer to P. amarus, P. debilis, P.
fraternus, or P. rotundifolius. In the Indian traditional medicines, Phyllanthus
urinaria is often used to refer to other types otherwise known as P. niruri,

DEXA MEDIA No. 3, Vol. 18, Juli - September 2005 89

 ARTIKEL UTAMA
P. amarus, P. debilis and P. fraternus. In Indonesia, P. niruri and
P. urinaria have been used interchangeably to indicate the
same herb, having the same local name, meniran and used
for the same purpose to treat diseases.4 Thyagarajan has since
successfully isolated three active substances from P. amarus,
which have activities against replication of hepatitis B virus,
improve immune system, and protect the liver.5
We found previously that a specific Phyllanthus niruri
extract (subsequently abbreviated as PNE) could enhance
activities and function of immune system component, both
humoral and cellular immunity.6 This paper explains the
evidence that PNE can increase immunological parameters,
and that there is a tight correlation between the results
seen in the animal studies and the various clinical studies
performed in patients having different diseases.
Materials and Methods
Specific PNE used in our studies have been subjected to
various standardized herbal processes from growing, harvesting,
post-harvest process, and manufacturing process as have
been laid out by various Indonesian government regulations.
Based on Rules of Ministry of Health of Republic of Indonesia
no. 760/Menkes/PER/IX/1992 regarding Phytopharmaca
and Guidelines of Good Traditional Medicines Clinical
Practices, there are 4 steps to prove the efficacy, safety and
quality standard of traditional medicines to be used in formal
health services. Extracts that have followed these steps can
be then classified as phytopharmaca. Briefly, the four phases
involve: Phase-1, pre-clinical study for safety and efficacy
determination. Safety evidence determines by toxicities
tests and efficacy evidence determines by pharmacodynamic
studies; Phase-2, simple standardization of those traditional
medicines, Phase-3, pharmaceutical standardization to ensure
identities and produce standardize medicines, and Phase-4,
clinical studies on healthy volunteers or patients.7,8 PNE used
in our studies have been carefully formulated and produced
under a current GMP-certified plant in the form of Stimuno®
capsule and syrup.
Standardization of the Specific PNE: Production Process,
Raw Material and Finish Goods
Phyllanthus niruri were planted in an area in Banyuwangi,
East Java, Indonesia, adhering to the principles of Good
Agricultural Practice (GAP), were subsequently processed
and cultivated in a dedicated area in the Tradimun plant in
Gresik, East Java, Indonesia. PNE was obtained by maceration
with alcohol. It is a dark brown powder having a bitter taste
and a specific aroma. Considering that there are various
types of Phyllanthus and preparation process to produce the
extract, standardization plays important roles toward the
quality of herbal pharmaceutical products. Standardization
is a pivotal step to ensure reproducible result starting from
herbs determination, cultivation, extraction process, major
substances determination, and also dosage forms preparation.
90
In this regards, the efficacy and safety of extract are directly
influenced by the standardization of extract, followed by
further processing.
Standardization was done for some physical and chemical
characteristics according to the internationally-accepted
Swiss Pharmacopoeia. For example water contents should
be very minimal, while the total flavonoid contents should
be maintained to certain amounts compared to the total
flavonoids. The processing also makes sure that general
characteristics for herbal extracts such as loss on ignition,
residual pesticide, microbe and heavy metal contamination,
and other harmful materials have been singled out during
the  subsequent  processes.  Biological/Immunological
Standardization were measured by increasing of T-lymphocyte
proliferation index and increase of IgM level, while various
cytokine measurements have been described previously.6
Because of these standardizations, the results of the studies
performed on this PNE is specific only for this extract, and
therefore the results can not be extrapolated to other PNE
that have been processed differently than that described in
this publication.
Active biological secondary metabolites contained in the
specific PNE have been characterized as flavonoids, lignan,
isolignan, and alkaloids.1,6 In this regards, the PNE used in
these studies was made from standardized herbs extract to
ensure each pharmaceutical dosage contains only a certain
amounts of a specific flavonoid.
Studies Performed on the specific PNE
To ensure its safety, efficacy and quality standard, this
product has gone through serial tests and studies:
Pre-clinical study for safety and efficacy determination
Acute, sub-acute/sub-chronic toxicity tests have been
done on mice and rats and proved that this PNE was practically
non toxic6,9,10 The PNE also showed no harm effect, as proven
on liver biopsy and level of creatinin and BUN serum of rats.
Clinical Studies
Immunomodulator especially needed for condition where
immune system status will influence patient’s condition and
disease progressiveness, such as for adjuvant therapy in cases
involving bacterial, viral or fungal infections. In this regards,
we have previously performed various studies in patients with
pulmonary TB, Acute Respiratory Tract Infections (ARTI),
Hepatitis, Chronic Obstructive Pulmonary Diseases (COPD),
Varicella Zoster, as well as Candidiasis vulvovaginitis patients.
Results of each of these studies will be published separately
under different publications. On-going trials for Hepatitis
and HIV are still being perfomed as per the date of printing of
this publication. All of our clinical studies have been carried
in different teaching hospitals, collaborating with different
investigators strictly adhering to the Good Clinical Practice.
All protocols have examined and approved by The Ethics
Committee in respective hospitals.
DEXA MEDIA No. 3, Vol. 18, Juli - September 2005
 ARTIKEL UTAMA

Immunomodulator testing on patients Pre-clinical Clinical Study

Test Parameter Research by: Case
Immunomodulator tests highly depend on the type of the study (Mice) (Human)

human disease infections. We performed tests for diseases Univ.

in which either extracellular or intracellular pathogens T helper 1
­2.1 x
Airlangga
–Lympho IFN-g ↔ (p=0.08) – Dr. Soetomo TBC
as the culprits. In the case that the infections were caused cyte (Th1)
(p<0.001)
Hospital
by extracellular bacteria, we tested for the immune system Surabaya
response that played a role is humoral immune system,
Univ. Indone-
including tests for antibody synthesis for IgG and IgM, or sia – Dr. Cipto
Trend ­
IgA antibody if it is a mucosal immune response; and the (p>0.05 NS)
Mangunkusu- TBC
mo Hospital
complement systems activity test. Specifically the following - Jakarta
tests were perfomed: B-Lymphocyte proliferation test and
cytokine secretion by Th2 subset (IL-4, IL-5 and IL-10) tests. Univ.
Airlangga
Additionally, for infectious diseases caused by intracellular ­35 x
– Dr. Soetomo TBC
(p<0.001)
bacteria, such as tuberculosis, the following tests were Hospital
Surabaya
performed: proliferation test for T-Lymphocyte, followed by
cytokine secretion by Th1 subset quantification (IL-2, TNF- Univ.
α, IFN-γ and IL-12). ­1.49 x
Airlangga
– Dr. Soetomo ARTI
(p<0.001)
Hospital
Results Surabaya
Based on previously done pre-clinical studies by one of
­(p<0.05)Day- Univ.
us (Suprapto Ma’at), it was found out that the specific PNE 7: 1.37 Airlangga
Candi-
diasis
could enhance activities and function of immune system xMonth-1: – Dr. Soetomo
vagi-
1.65 xMonth- Hospital
component, both humoral and cellular immunities. A nalis
3: 1.41 x Surabaya
series of clinical studies was then performed to validate the
correlation between the changes immunologic parameters Univ.
Airlangga
found in the pre-clinical and clinical phases. Comparison ­1.72 x ­1.63 x Hepa-
TNF-a – Dr. Soetomo
(p<0.001) titis
between pre-clinical study results and clinical study results (p=0.016)
Hospital
were summarized in the table below. Surabaya

Univ. Indone-
sia – Dr. Cipto
Trend ­
Mangunkusu- TBC
(p>0.05 NS)
mo Hospital
Test Pre-clinical study Clinical Study
Research by: Case - Jakarta
Parameter (Mice) (Human)
Univ. Indone-
- ­ 1.24 x
sia – Dr. Cipto
(p<0.05) with Univ. Trend ­
­1.52 x IL-6 Mangunkusu- TBC
Proliferation con-A Mitogen Airlangga (p>0.05 NS)
(p<0.05) with mo Hospital
of T-lympho- agent- ­ 1.66 x – Dr. Soetomo Hepatitis
PHA mitogen - Jakarta
cyte (p<0.001) with Hospital
agent
PHA mitogen Surabaya
agent Univ. Sri-
widjaja – Dr.
­Univ. Anda- M. Hoesin
2.4 x las – Dr. M. TBC
CD4 - TBC Hospital -
(p<0.001) Djamil Hospi- ↔ (p=0.961) Palembang
tal - Padang Candi-
Il-12 Trend ­
diasis
(p>0.05 NS) Univ.
Ratio ­Univ. Anda- vagi-
Airlangga
of las – Dr. M. nalis
- 6.4 x (p<0.01) TBC – Dr. Soetomo
CD4/ Djamil Hospi-
Hospital
CD8 tal - Padang
Surabaya
Univ. Andalas
¯ 0.7 x
– Dr. M. IL-2 -
CD8 - ↔ (p=0.73) TBC (p<0.001)
Djamil Hospi-
tal - Padang
T-helper 2
­2.85 x
↔ (p= 0.788, Lympho- IL-4 -
(p<0.001)
target cell cyte (Th2)
TGPEC ratio E:
Cytotoxicity T= 2.5:1) Univ. Sri-
of CD8 ↔ (p= 0.786, widjaja – Dr.
¯ 0.9 x Trend ¯
target cell IL-10 M. Hoesin TBC
(p=0.033) (p=0.408 NS)
TGPEC ratio E: Hospital -
T= 5:1) Palembang

DEXA MEDIA No. 3, Vol. 18, Juli - September 2005 91

 ARTIKEL UTAMA

Discussion
Pre-clinical Clinical Study
Test Parameter Research by: Case
study (Mice) (Human)
Based on the table above, it can be inferred that the results
­1.5 x
1.67 x (p<
Univ. of majority of pre-clinical studies done on experimental
Proliferation of B (p<0.07) Airlangga
lymphocyte with LPS
0.001) with
– Dr. Soetomo
Hepa- animals had a high degree of correlation to those done in
LPS mitogen titis
­ mitogen
agent
Hospital clinical studies in patients. Brief interpretation of tests and
agent Surabaya
results on immunologic parameter changes of PNE studies
Univ. suggest that the specific PNE acts by activating both humoral
Airlangga
IgM
­8.6 x ­1.22 x
– Dr. Soetomo ARTI and cellular immune system.11 - 14
(p<0.001) (p<0.05)
Hospital On the the humoral immune system, PNE can be seen to
Surabaya
increase the antibody synthesis in both animal and human
Univ.
Airlangga
studies, particularly the IgG and IgM, while the IgA increase
­16,2 x ­1.24 x
IgG
(p<0.001) (p<0.001)
– Dr. Soetomo ARTI can be seen in one of the clinical studies. This suggests that
Hospital
Surabaya
PNE fights on intracellular and extracellular pathogens by (a)
neutralization of toxin; (b) increasing the phagocytosis process
Univ.
Airlangga by phagocytes through opsonization; (c) increasing the activity
­1.21 x
IgA
(p<0.05)
– Dr. Soetomo ARTI of the complement system due to antigen-antibody complex
Hospital
Surabaya (Ag-Ab complex) through the classical pathway, and finally,
Univ.
(d) inhibiting proliferation of pathogens by forming Ag-Ab
Phagocytosis
Macrophage/ ­2.96 x 2.34 x
Airlangga
Hepa-
complex. With regards to increased complement activation
– Dr. Soetomo
monocyte (p<0.01) (p<0.01)
Hospital
titis through the classical pathway brought about by the PNE, it
­
Surabaya can be inferred that PNE also help in the destruction (lysis)
TNF-a Secretion ↔ (p=0.363
of pathogens invaded by MAC (membrane attack complex)
- -
by monocyte NS) formed by the various complement fragments, particularly
Persahabatan
C5 to C9. Further, PNE also helps in the augmentation of
Complement 1.38 x Trend ­
­ (p<0.001) (p>0.05 NS)
Hospital COPD phagocytosis process by neutrophils, macrophages and other
- Jakarta
immunocompetents cells, since C3b complement fragment
Chemotaxis of
2.37 x could act as antibody through the opsonization process, while
neutrophyl - -
­
(p<0.001) the C3a and C5a fragments could act as chemoattractants.11-14
Chemotaxis of Results of the studies shown above also explain that the
1.56 x
macrophage
(p<0.006)-
- - specific PNE acts as an immunomodulator via activation
­
of the cellular immune system. Specifically, PNE acts to
­1.41 x
activate neutrophils, macrophages/monocytes, and T- and B-
(p<0.001) Univ.
Cytotoxicity of (E:T = 12.5 Airlangga lympocytes. The increase in the process of phagocytosis by
1.56 x Hepa-
NK cell : 1)­ 1.69 x
(p<0.05)
– Dr. Soetomo
titis neutrophils suggest a speed up eradication process of invading
­ (p<0.001) Hospital
(E:T = 25 Surabaya microbes, viruses or fungi, especially the extracellular foreign
: 1) pathogens and clean them from the body. On the other
Note: ↑ increase; ↓ decrease; ↔ no changes; NS= no statisti- hand, the increase of phagocytic profile of monocytes /
cally significance difference between treatment and control groups. macrophages by PNE can be interpreted as that PNE speeds
up the lysis of cells infected by the intracellular microbes such
It can be inferred from the table above that there is a strong the Mycobacterium tuberculosis, and renders the microbes
correlation between changes in immunological parameters exposed to other components of the immune systems in the
brought about by the specific PNE found in the animal studies extracellular compartments. In relation to this aspect, it is
versus those found in the clinical studies done in patients, interesting that PNE can also expedite the lyses of cancer
with regards to expression of certain cytokines, proliferations cells or other mutated cells. While, the increase cytokine
of T and B cells, production of specific antibodies, activation expression and secretion, such as IFN-g, TNF-a, IL-4, IL-6,
of macrophages and the process of phagocytosis, activation IL-12, and reduction of IL-10 can be interpreted that PNE
of the complement system, as well as activation of cytotoxic can also influence the reactions involving cellular immune
Natural Killer cells. Although the scope of this publication system.
is limited to the discussion of changes in the immunological The specific PNE can also affect proliferation of specific
parameters, however various investigators have found that T-lymphocytes. Increase in T-lymphocytes by PNE suggests
these changes are also correlated positively to the decrease of the involvement of T-helper cells or T-cytotoxic cells in the
disease progression studied in the different clinical conditions neutralization of pathogens. Previous studies suggest that
and settings as well as the different patient types. cytokine secretion of T-helper lymphocytes (CD4) can be

92 DEXA MEDIA No. 3, Vol. 18, Juli - September 2005


differentiated by subpopulations of T-helper 1 and T-helper
2, which is summarized in the following table:11,14
T-helper 1 (Th 1) T-helper 2 (Th 2)
IL-2 IL-4
TNF-a IL-5
IFN-g IL-10
IL-12
In this case, interpreting T cells cytokine secretion should
be done carefully. Principally, if cytokine secretion is dominated
by Th1 cells, then the immune response is geared towards the
cellular immune system, on the other hand if it is dominated
by Th2 cells then the response is geared towards the humoral
immune system.11,13,14 As can be seen above, PNE affects the
two subpopulations of T-helper cells, because it expresses
the different cytokines associated with the different T-helper
cell subpopulation (Table). In this case, PNE can increase
the secretion of IL-4 in the animal study showing its role in
the increased proliferation of B-lymphocytes, regulation of
antibody secretion by the B-lymphocytes, as well as antibody
isotype switching. It is interesting to see that PNE decreases
the secretion of IL-10 by Th2 cells in both animal and human
studies. IL-10 has been previously implicated in the reduction
of activation of monocytes/macrophages, which in turn, can
also reduce the activation of Th1 cells.11,14 Our data suggest
that PNE is able to reverse the deactivation of macrophages/
monocytes, thereby increase the secretion of IL-1, IL-12,
chemokines and TNF-a. PNE also increases the expression
of the class II MHC, and the subsequent activation of T-
lymphocytes.
Specific PNE can also activate cytotoxicities brought about
by T-cytotoxic (CD8) Lymphocytes and those brought about
by the Natural Killer cells. With regards to the former, it has
been previously shown that action of T-cytotoxic lymphocytes
is MHC-1 restricted, meaning that it will only work if antigen
expressed with class-1 molecule.12,14 Cells that are infected
by fungi, intracellular bacteria, viruses or even cancer cells,
which express the class-1 MHC, will be bound and lysed,12,14
therefore the fact that the specific PNE can activate the T-
cytotoxic lymphocytes suggest that this extract participates in
the process of pathogen killing. Action of NK cells is similar
with T-cytotoxic (CD8), however they specifically do not
require the presence of class-1 MHC molecule.11,14 Rather,
they will directly stick to target cells and lyses them.11,14
On the other hand, the results reflecting on the increase of
chemotaxis reaction mediated by administration of specific
PNE (Table) suggest that the faster the inflammatory cells
(neutrophils, monocytes and lymphocytes) reach the injury
site; the faster the rest of immunology processes will occur.
We have shown in this publication evidence suggesting
that specific PNE acts on the body as an immunomodulator,
DEXA MEDIA No. 3, Vol. 18, Juli - September 2005
ARTIKEL UTAMA
and that a positive correlation exists in the various
immunological parameters between the results of pre-clinical
and clinical studies. Because the development this specific
PNE have followed the prescribed guidance of the Indonesian
government,11,14 the Indonesian National Administration of
Drug and Food Control has granted the classification of the
specific PNE used in our studies as a phytopharmaca. By its
definition,11,14 this specific phytopharmaca extract can now
be used by physicians as an agent in the therapy or prevention
of infectious diseases. The use of this specific PNE by health
professionals in a naturalistic setting is yet another evidence
that herbal extracts which have undergone extensive scientific
studies can be complementing or substituting any chemically-
derived drug with the same indication in the formal medical
treatment.
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