Submitted : 29-03-2024 Revised : 09-07-2024 Accepted : 19-09-2024 Majalah Obat Tradisional (Trad.
Med.
January-April 2025 Vol.
, p 67-73 ISSN-p : 1410-5918 ISSN-e : 2406-9086 Immunostimulatory Effects of Zingiber ottensii Rhizome Extract on Mouse Lymphocytes and Peritoneal Macrophages Hanif Nasiatul Baroroh1*.
Warsinah2.
Masita Wulandari Suryoputri1.
Heny Ekowati1 1 Department of Pharmacology and Clinical Pharmacy.
Pharmacy.
Faculty of Health Sciences.
Jenderal Soedirman University.
Purwokerto, 53122.
Central Java.
Indonesia 2 Department of Pharmaceutical Biology.
Pharmacy.
Faculty of Health Sciences.
Jenderal Soedirman University.
Purwokerto, 53122.
Central Java.
Indonesia
ABSTRACT
Zingiber ottensii .
angle hant.
is a member of the Zingiberaceae family, known for its pharmacological effects.
Several studies have demonstrated immunomodulatory potential of Zingiberaceae rhizomes, including Zingiber officinale.
Zingiber zerumbet, and Zingiber cassumunar.
This study aimed to evaluate the immunomodulatory effects of Z.
ottensii extract on mouse peritoneal macrophages and The extract was prepared using a maceration method with 96% ethanol .
and the extract compound was observed by phytochemical screening with thin layer chromatography (TLC).
Phagocytotic macrophage activity was quantified using a phagocytotic assay, which use mouse peritoneal macrophages.
Lymphocyte proliferation was assessed with the MTT assay and absorbance measured at 595 nm.
TLC
results, showed that Z.
ottensii extract tested positive for flavonoids and terpenoids.
The Z.
ottensii extract stimulated macrophage phagocytosis activity, significantly increasing the phagocytotic index at 25 and 50 AAg/mL concentrations compared to the control.
Additionally, significant lymphocyte proliferation was observed with the treatment of the extract.
The Z.
ottensii extract may be developed for adjuvant therapy to enhance the immune responses, offering a promising natural approach to immunomodulation.
Keywords: immunomodulatory effect.
Zingiber ottensii INTRODUCTION Infectious agents are frequently introduced to the human body, leading to illness.
The immune system can prevent these infectious pathogens from getting infiltrating an causing disease.
The immune system plays a crucial role in protecting body cells from infections and chronic illnesses.
immunomodulator that can affect both the humoral and cellular immune systems is required Immunomodulators were developed in response to the prevalence of immune system disorders, which affect both humoral and cellular immunity (Abbas et al.
, 2.
Currently, research and development of immunomodulators are encouraged and can be utilized as complementary therapies (Parham.
Numerous immunomodulators derived from natural sources, including an extract from Echinacea purpurea, have demonstrated the ability to enhance the production of Th1 and Th2 cytokines (Aida Rahmadani & Lestari, 2021.
Owen et al.
, 2.
Furthermore, using Phyllanthus niruri water extract as an immunostimulant can boost *Corresponding author : Hanif Nasiatul Baroroh Email : hanif.
baroroh@unsoed.
neutrophil activation, antibody responses, cell proliferation, and macrophage phagocytosis (Putri et al.
, 2.
One traditional medicine that can be developed as an immunomodulatory agent is the rhizome of bangle hantu (Zingiber ottensi.
which has been proven to have anti-cancer activity on MCF-7 cells (Sinaga & Wiryanti, 2.
The rhizome of Z.
ottensii contains the flavonoid antiinflammatory and anti-cancer activity (Chien et al.
Han et al.
, 2018.
Imran et al.
, 2.
Given these properties.
ottensii rhizomes have the potential to be developed as phytotherapy.
Several studies have demonstrated that Zingiberaceae rhizomes possesses immunomodulatory potential.
For instance.
Zingiber zerumbet has been shown to exhibit immunomodulatory activity (Hidayah & Indradi, 2.
, and the zerumbon compound Zingiberaceae immunomodulatory properties (Keong Yeap et al.
This investigation focuses on the immunomodulatory effects of Z.
ottensii rhizome on the cellular immune response, specifically lymphocyte proliferation.
The rhizome of Z.
may have a positive impact on human health and Majalah Obat Tradisional, 30.
, 2025 | DOI: 10.
22146/mot.
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.
0 International License.
Hanif Nasiatul Baroroh the prevent illnesses related to the immune This study aims to provide a more comprehensive understanding of Z.
pharmacodynamic action, generating scientific data that could support the findings of previous
MATERIALS AND METHODS
Materials Bangle hantu (Zingiber ottensi.
rhizomes were collected from Lembang.
West Java.
Indonesia, and authenticated by the Laboratory of Environment.
Faculty of Biology.
Jenderal Soedirman University.
This study has received an ethical clearance from the Health Research Ethics Commission.
Faculty of Health Sciences.
Jenderal Soedirman University with number 1123/EC/KEPK/2023.
Two 8-week-old BALB/c mice were obtained from the Faculty of Pharmacy.
Universitas Muhamadiyah Purwokerto.
Reagents and materials for the in vitro experiment included Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma MCLS), fetal bovine serum (FBS) of non-USA origin sterile-filtered suitable for cell culture .
Sigma MCLS), amphotericin B solution .
AAg/mL.
Elabscienc.
, penicillinstreptomycin solution .
Elabscienc.
, latex beads (Sigm.
DulbeccoAos phosphate buffer saline (DPBS) (Elabscienc.
, phosphate buffer saline (PBS), (PHA)(Gibc.
, stopper sodium dodecyl sulfate (SDS) (Merc.
MTT .
,5-dimethylthiazol-2-y.
-2,5diphenyltetrazolium bromid.
(Biobasi.
All other reagents and chemicals used were of the purest commercial grade available.
Methods Preparation of the Z.
ottensii rhizome extract The maceration extraction method was used to obtain the extract from Z.
ottensii rhizome.
Initially, 2 kg of Z.
ottensii rhizome simplicia powder was soaked in 12 L of 96% ethanol .
for 24 hours at room temperature.
The mixture was then filtered to collect the filtrate.
The remaining precipitate was subjected to two additional rounds of maceration using an identical quantity of 96% ethanol each time.
The combined filtrates were concentrated using a vacuum rotary evaporator followed by a water bath until a thick extract was obtained.
This thick extract was then weighed and underwent further separation for subsequent analysis.
Phytochemical screening with thin layer chromatography (TLC) The Z.
ottensii extract was applied to a GF254 silica gel plate, which served as the stationary The plate was then placed in a chamber containing a mobile phase mixture of n-hexane and ethyl acetate in an 8:2 ratio.
After elution process, the plate was removed from the chamber and allowed to air dry until the eluting liquid The plate was subsequently sprayed with stain reagentsAicitroboric for flavonoids and Liebermann-Burchard for terpenoids.
The plate was heated at 110AC for 10 minutes.
The stains formed were observed under visible light.
UV 254, and UV 366 nm.
Immunomodulatory peritoneal macrophages Two 8-week-old male BALB/c mice were housed with a pelleted basal diet (BR-.
and water ad libitum in an animal room under a 12-hour light/dark cycle at a temperature of 22AC (A 3AC) and a humidity of 60%.
The mice were sacrificed by cervical dislocation.
To isolate macrophages, 10 mL RPMI 1640 medium was injected into the peritoneal cavity of each mouse.
The peritoneal fluid was then centrifuged at 2000 rpm and 4 oC for 10 minutes.
After the supernatant was removed, the precipitate containing macrophage was resuspended in a medium containing 10% FBS, 2% penicillin and 2% streptomycin, 5% fungizone, and 30% RPMI 1640.
The density of macrophages was adjusted to 2,5 x 106 cells/mL.
For the phagocytotic activity assay, macrophages were cultured in a 24-well culture plate with coverslips, with 1 mL of cell suspension per well, and incubated at 37oC for 24 hours.
The wells were then washed with 1 mL of RPMI 1640 medium and filled with varying concentration of Z.
ottensii extract.
After a one-hour incubation at 37oC, 50 AAL of latex beads were added to each well, and the plate was incubated for another hour at The wells were washed three times with 1 mL of PBS, fixed with methanol, and stained with 20% Giemsa.
The coverslips were examined under a binocular microscope to assess phagocytotic macrophage activity, calculating both the phagocytotic index and capacity.
The phagocytotic index is calculated as the ratio of latex bead particles phagocytosed by activated macrophages, while the phagocytotic capacity was calculated as the ratio of activated macrophages capable of consuming latex particles to 100 macrophages Immunomodulator effect on lymphocyte Mice were sacrificed by cervical dislocation, and their spleens were removed.
The lymphocytes were suspended by injecting 10 mL of RPMI 1640 Majalah Obat Tradisional, 30.
, 2025 Immunostimulatory Effects of Zingiber ottensii Rhizome Extract Figure 1.
The TLC profiles of Z.
ottensii extract.
(A) Flavonids observed under .
visible light, .
UV 254 nm, .
UV 366 nm.
(B) Terpenoids observed under .
visible light, .
UV 254 nm, .
UV 366 nm solution into the spleen.
The suspension was centrifuged at 2000 rpm and 4oC for 10 minutes.
The resulting pellet was resuspended in RPMI 1640 and subjected to hemolysis twice using a hemolysis buffer .
mM NH4Cl, 15 mM NaHCO3, 1 mM EDTA, pH 7.
, followed by centrifugation at 2000 rpm at 4oC for 10 minutes.
After removing the lymphocyte was collected and inoculated into a FBS penicillin/streptomycin amphotericin B 0.
5 %, and RPMI 1640.
The density of lymphocytes was adjusted to 1,5 x106 cells/mL.
The cells were cultured in 96-well culture plates with 100 AAL of culture medium per well and incubated at 37oC for 24 hours.
Wells containing different concentrations of Z.
ottensii extract were then added, and the plate was incubated for 48 hours at 37oC.
Cell viability was measured using the MTT assay and read by an ELISA Reader at 595 Statistical analysis Each experiment was conducted in Data were recorded as mean A SEM.
Statistical significance across different groups was essessed using one-way analysis of variance (ANOVA) and Tukey's multiple comparison tests.
p-value of less than 0.
05 was considered statistically significant.
Statistical analysis was performed using SPSS software.
Majalah Obat Tradisional, 30.
, 2025
RESULTS
Extract and characterization The rhizome sample used was identified as Zingiber ottensii Val.
from the Zingiberaceae family.
The maceration extraction method, which is simple and preserves volatile compounds (Asworo & Widwiastuti, 2.
, was employed.
The resulting extract was a thick, brown paste weighing 90 grams with a 9% yield.
Qualitative analysis using TLC was performed to identify the compounds present in the Z.
ottensii extract.
TLC results, observed under visible light.
UV 254, and UV 366 nm, showed that the Z.
ottensii extract tested positive for flavonoids and terpenoids.
This was indicated by a yellow stain after spraying with citroboric for flavonoids and a purple-red stain for terpenoids after spraying with LiebermannBurchard (Figure .
Immunomodulatory activity of Z.
The immunomodulatory influence of Z.
ottensii extract was examined on both the innate and adaptive immune response.
The phagocytotic activity of macrophages was evaluated using a phagocytotic assay.
The terms "phagocytotic capacity" and "phagocytotic index" refer to the macrophagesAo ability to ingest and eliminate foreign objects.
Macrophages, isolated from miceAos peritonial cavities, undergo phagocytosis when latex beads adhere to them.
Latex beads, which have a diameter of about 0.
8 AAm resembling Hanif Nasiatul Baroroh Figure 2.
The phagocytotic activity of macrophages in the treatment group.
Macrophages were stained with 20% Giemsa, observing at 400x magnification.
Active macrophages are visible as purple cells .
ndicated by black arro.
Latex beads are indicated by red arrow.
the size of bacteria (Ueno et al.
, 2.
, were used as the antigen.
Macrophages recognize these beads through surface receptors, such as macrophage receptor with collagenous structure (MARCO) (Cervantes et al.
, 2.
Staining with Giemsa made the phagocytosed latex beads appear purplish, distinguishing them from non-phagocytosed ones (Figure .
The Z.
ottensii extract at concentration 25, 50, and 100 AAg/mL enhanced the phagocytotic capacity of mouse peritoneal macrophages compared to control cells, but the increase was not significantly different (Figure .
Regarding the phagocytotic index, the Z.
ottensii extract at 25 and 50 AAg/mL significantly increased the index, whereas a higher concentration .
AAg/mL) did not (Figure .
The 50 AAg/mL concentration of Z.
ottensii extract strongly enhanced the phagocytotic The immunomodulatory effect on the adaptive immune response was assessed by the proliferation lymphocytes in the presence of ottensii extract.
The ability of Z.
ottensii extract to stimulate lymphocyte proliferation was measured using the MTT assay.
Higher absorbance values indicated higher lymphocyte stimulation index The 250 AAg/mL of Z.
ottensii extract did not affect lymphocyte proliferation.
However, the extract at concentrations of 15.
625, 31.
25, 62.
AAg/mL The 125 AAg/mL concentration demonstrated the most significant enhancement in lymphocyte proliferation (Figure .
DISCUSSION
Reducing the risk of chronic illnesses involves immune system activation.
The immune response can be divided into the innate immune response and the adaptive immune response.
Although the adaptive immune response takes time to manifest after infection, it offers a highly targeted defense against pathogens.
Within the innate immune system, macrophages perform several vital functions, whereas lymphocytes play an important role in adaptive immune response.
The immunomodulatory effects of Z.
rhizome on the immune response were examined in this study.
Our findings demonstrated that Z.
ottensii extract enhanced the phagocytotic activity of mouse peritoneal macrophages compared to control cells.
While the extract did not significantly affect phagocytotic capacity, there was a noticeable Specifically, a 50 AAg/mL concentration of ottensii extract strongly increased the phagocytotic index.
Additionally, a 125 AAg/mL concentration of the extract markedly stimulated lymphocyte proliferation.
Several rhizomes in the Zingiberaceae family exhibit immunomodulatory potential.
For instance.
Zingiber officinale can stimulate Zingiber cassumunar Roxb can suppress macrophage phagocytosis through immunosuppressive activity (Mahfudh et al.
, 2.
The ethyl acetate fraction of cassumunar Roxb rhizomes can boost the expression of IL-10 and IL-14, whereas the Majalah Obat Tradisional, 30.
, 2025 Immunostimulatory Effects of Zingiber ottensii Rhizome Extract Figure 3.
Effect of Zingiber ottensii extract (ZOE) on the phagocytotic capacity of mouse peritoneal Cells were treated with ZOE at concentrations of 25, 50, and 100 AAg/mL and cultured for 1 h.
After cultivation, cultures were stained with 20% Giemsa and macrophages were Each result is represented as the mean A SEM of three independent measurements.
Significant differences compared with the control are represented as *p < 0.
Figure 4.
Effect of Zingiber ottensii extract (ZOE) on the phagocytotic index of mouse peritoneal Cells were treated with the ZOE at concentrations of 25, 50, and 100 AAg/mL cultured for 1 h.
After cultivation, cultures were stained with 20% Giemsa and macrophages were Each result is represented as the mean A SEM of three independent measurements.
Significant differences compared with the control are represented as *p < 0.
Figure 5.
Effect of Zingiber ottensii extract (ZOE) on the stimulation index of lymphocyte Cells were treated with the ZOE at concentrations ranging from 15.
625 - 250 AAg/mL and cultured for 48 hours.
After cultivation, cell viability was measured using the MTT assay and read by ELISA Reader at 595 nm.
Each result is represented as the mean A SEM of three independent measurements.
Significant differences compared with the control are represented as *p < 0.
Majalah Obat Tradisional, 30.
, 2025 Hanif Nasiatul Baroroh n-hexane proliferation and macrophage phagocytic activity (Nurkhasanah, et al.
, 2019a.
Nurkhasanah et al.
Similarly, the ethanol extract of Z.
enhance macrophage and lymphocyte activity (Masniah et al.
The essential oils of Z.
ottensii have demonstrated anti-inflammatory and anti-cancer properties (Panyajai et al.
, 2022.
Thitinarongwate et al.
, 2.
ottensii has also shown activity in increasing the proliferation and differentiation of CD8 T lymphocytes .
ytotoxic T cells/CTL) (Panyajai et al.
, 2022.
Ruttanapattanakul et al.
Increased lymphocyte proliferation activity leads to an increase in both CTL and CD4 T lymphocytes .
elper T cell.
(Abbas et al.
, 2.
These cells aid the B lymphocyte response in producing antibody that can bind to specific Another study indicated that Z.
extract at 50, 100, and 200 AAg/mL could stimulate T and B cell proliferation (Yycel et al.
, 2.
Additionally.
ottensii extract has been shown to inhibit COX-2 production (Koontongkaew et al.
Thitinarongwate et al.
, 2.
The extracts has a strong cytotoxic effect with an IC50 of 60 AAg/mL compared to other plants (Suprihatin et al.
Therefore.
ottensii extract could enhance the immune system by increasing phagocytotic index and lymphocyte proliferation, suggesting its potential as an adjuvant for immunopreventive drugs, warranting further in vivo studies.
CONCLUSION
Zingiber ottensii extract was confirmed to contain flavonoids and terpenoids.
Zingiber ottensii extract demonstrated the ability to modify lymphocyte proliferation and phagocytotic By increasing the phagocytosis index and lymphocyte proliferation stimulation index, the Z.
ottensii extract can enhance macrophage phagocytic activity.
Thus, the rhizome of Z.
can improve human health by preventing disorders linked to the immune system.
CONFLICT OF INTEREST
The authors declared no conflict of interest.
ACKNOWLEDGEMENT
The authors thank to Basic Research Grant from Jenderal Soedirman University (Grant number: 27.
97/UN23.
37/PT.
03/II/2.
for funding this research.
REFERENCES