A Review on Eulophia nuda Immunomodulatory activity in Peritoneal macrophages and an assessment of its in-vivo activity in mice


Komal R. Thakre, Dr. V. V.  Paithnakar, Mr. J. V. Vyas, Dr. A. M. Wankhade,

Department of Pharmacology, Vidyabharti Collage of Pharmacy Amravati, Maharastra, 444602, India.

*Corresponding Author E-mail: komalthakre4441@gmail.com



The purpose of this review is to highlight the findings of research on immunomodulatory activity of eulophia nuda tubers. Taxonomically, the genus Eulophia and the genus Dioscorea are represented in Amarkand. Pharmacologically, this plant has cytotoxic, antioxidant, antiglycation, and DNA protective activities. The selection of papers was made using the most relevant databases for the pharmacological activity on the basis of their phytochemical constituents. The review also goes over different in-vitro assay techniques and animal study conformation. The effect of Eulophia nuda extracts on the release of mediators such as nitric oxide (NO), superoxide, lysosomal enzyme, and myeloperoxidase activity of isolated murine peritoneal macrophages was studied at different doses. Eulophia nuda extracts were tested biochemically, hematologically, serologically, and histopathologically. The effect of eulophia nuda extracts on delayed-type hypersensitivity (DTH), serum antibody response, and cyclophosphamide-induced myelosuppression in Swiss albino mice was studied. The current work reviews the histopathological, biological, and statistical analysis of eulophia nuda extract on animal models.


KEYWORDS: Eulophia nuda, Nitrite assay, Cellular lysosomal activity assay, DTH, Cyclophosphamide.




Eulophia nuda L., Orchidaceae family, is a perennial, terrestrial herb with underground tubers found in central and Southeast Asian regions.1 The phytochemical makeup of this plant has not been thoroughly studied. From this plant, only a small number of substances have been identified, including phenanthrene derivatives and benzylated phenanthrene derivatives. Pharmacologically, this plant has cytotoxic, antioxidant, antiglycation, and DNA-protective characteristics that are all active. The focus of research has been on the traditional knowledge as well as the diverse phytochemical and pharmacological actions of this plant.2


Eulophia nuda's qualitative phytochemical examination found that it contains a variety of phytochemically active substances such as alkaloids, flavonoids, saponins, cardiac glycosides, tepenoids, and steroids. Tubers used against tumors, scrofulous glands of the neck, bronchitis, blood diseases, rheumatoid arthritis besides as blood purifier, appetizer, vermifuge by folklores.3


The immune system (IS) is an intricately interconnected network of cells, tissues, organs, and soluble mediators that has developed to protect the organism from any external insult that jeopardises the integrity of the organism. Immunomodulation is also known as immune system modulation that is preceding changes the immune response result helps the tissue damage and immoderate response that is obtained by natural and unnatural (human-made) from.4


Immunomodulatory medications alter the immune system's reaction by raising (immunostimulators) or lowering (immunosuppressives) serum antibody production. Immunostimulants are used to boost the immune system's response to infectious infections, tumours, primary or secondary immunodeficiency, and changes in antibody transfer, among other things. Immunosuppressive medications are used to suppress the immune response to donated organs as well as to treat autoimmune illnesses such as pemphigus, lupus, and allergies.5



Figure. 1: Flower of Eulophia nuda 6    



Figure.2: Tuber of Eulophia nuda 6


Classification of Eulophia nuda:

Family: Orchidaceae

Species: Eulophia nuda

Habit: Herb

Kingdom: Plantae

Phylum: Magnoliphyta

Class: Liliopsida

Order: Asparagales

Genus: Eulophia

Flower, Fruit: May-June

Common name: Amarkand. 6


The methanolic extract of E. nuda contained five antibiotics: HygromycinA, Penicillin V, Penicillin K, Rifamycin Z, and Spiramycin 3, indicating that some bacteria may be present symbiotically in E. nuda rhizomes.6


Table.1: Phytochemical description of Eulophia nuda 6

Sr. No.

Name of Compound






5,3’dihydroxy 4,5-dimethoxy-6,3,7methylene dioxyisoflavone


5,7,2’trihydroxy,3,6,4’5’ tetramethoxy flavones


Demethyltorosoflavone D












Fulvinervin B


Epithienamycin E


Phylloquinone (vitamine K), Picrasin G (triterpenoid antifeedant)



Fresh tubers of E. nuda L. were collected. The tubers were properly cleaned and rinsed. They are then chopped into little pieces and dried at a regulated temperature of 45°C. The powder was then extracted with boiling ethanol under soxhlation to yield E. nuda ethanolic extract (ENE), and with boiling water under soxhlation to yield E. nuda aqueous extract (ENA).7



Freund’s complete adjuvant, ovalbumin, nitroblue tetrazolium (NBT), tetramethyl benzedrine/hydrogen peroxide (TMB/H2O2), and bovine serum albumin, - penicillin, streptomycin, HEPES buffer, and Roswell Park Memorial Institute (RPMI) 1640 medium, - phytohemagglutinin-M (PHA) and fetal bovine serum (FBS). All other chemicals used were of analytical grade.8


1. Evaluation of in vitro immunomodulatory activity:

Isolation of peritoneal macrophage and culture conditions:

The mice received an intraperitoneal injection of 2 cc of 4% (w/v) fluid thioglycollate medium. Mice are subjected to peritoneal lavage with 10 ml of RPMI 1640 medium after 3 days. As a result Peritoneal macrophages were identified and removed throughout the process. With RPMI 1640, these isolated cells were cleaned. Additionally, they are given culture media made of RPMI 1640 with supplements of 100g/ml streptomycin (complete RPMI), 100u/ml penicillin, 2mM L-glutamine, and 10% FBS. The macrophage count was evaluated using a hemocytometer. The cell viability was assessed using a technique called trypan blue dye exclusion. The cells were changed to match the required cell count per millilitre. These macrophage cells were plated into a 96-well flat-bottom culture plate (Tarsons Products Pvt., Ltd., India). The plate was incubated for two hours at 37°C in an incubator with humidified 5% CO2. 9,10


Nitrite assay:

The increased accumulation of nitrite concentration in the media was a sign that nitric oxide (NO) was being produced [18]. The cell-free supernatant (50 l) was collected and combined with 50 l of Griess reagent (0.1% naphthyl ethylenediamine dihydrochloride, 2% phosphoric acid, and 1% sulfanilamide) after the macrophages (5105 cells/ml) had been incubated for 24 hours. The finished mixture was left to sit at room temperature for 10 minutes. The optical density at 540 nm was determined using a microplate reader (ELX800MS, BioTek Instruments Inc., USA). Nitrite concentrations were calculated using a standard curve of sodium nitrite in culture conditions. The stimulation index (SI) for nitrite release was calculated using the ratio of nitrite concentrations in treated and untreated macrophages.11,12


NBT dye reduction assay:

The cells (1x106 cells/ml, treated for 24 hours with MCM extract) were combined with 50 l of a 0.3% NBT solution in PBS (phosphate-buffered saline, pH 7.4), and the mixture was then incubated for a further 24 hours in a CO2 incubator. After incubation for 1 h, the adherent macrophages were washed and cleaned vigorously with complete RPMI medium, and again washed 4 times with 200μl methanol. Formazan deposits were solubilized in 120μl of 2 M KOH and 140 μl of DMSO, after air drying. The homogenization process was used. These homogenised well contents were tested for optical density estimation. A microplate reader reading at 630 nm was used to determine the optical density. SI was calculated using the optical density ratio of the control and treated macrophages.13,14


Cellular lysosomal enzyme activity:

To assess the cellular lysosomal enzyme activity of macrophages, acid phosphatase activity was measured. Macrophages were treated with MCM for 24 hours at 37°C and 5% CO2. After that, the supernatant was removed by aspiration and 20μl of 0.1% Triton X-100 (Hi-Media, India) were added to each well. After 15 min’ incubation, 100μl of 10mM p-nitrophenyl phosphate and 50μl of 0.1 M citrate buffer (pH 5.0) were added. The plates were then incubated for one hour. 0.2 M borate buffer (150μl, pH 9.8) was added to these incubated plates. The optical density at 405nm was measured with a microplate reader.15


Myeloperoxidase activity assay:

The activity of myeloperoxidase was also measured in isolated macrophages. After 3 hours of incubation, macrophages (5x105 cells/ml) were washed three times with new full RPMI media. The wells were then filled with a 100μl combination of o-phenylenediamine (0.4 g/ml) and 0.002% H2 O2 in phosphatecitrate buffer (pH 5.0). After 10 minutes, the reaction was halted with 0.1 N H2 SO4 and the optical density was measured at 490 nm. The optical density ratio of the control and treated cells was used to compute the myeloperoxidase SI.16,17


Evaluation of in vivo immunomodulatory:

Experimental animals:

Swiss albino mice use for this study. Before being employed in the tests, the animals were acclimated for a period of 10 days. They were kept in a space with a set temperature. (23–20°C) with a 12-hour cycle of light and dark. The animals were given a conventional pellet diet and unlimited access to water.18


Acute oral toxicity studies:

According to Organization for Economic Cooperation and Development Guideline Number 423, research on acute oral toxicity for ENA and ENE were conducted. 19


Detection of serum antibody response:

After 7 days of inoculation, blood was drawn from mice through the retro-orbital plexus, and serum was extracted using centrifugation. The serum antibody titers used to measure the serum IgG levels of ovalbumin were calculated by ELISA as previously mentioned. 12.5g of ovalbumin dissolved in 100μl of sodium carbonate buffer (pH 9.6) was applied to flat bottom polystyrene plates for 12 hours at 4°C. Three times with phosphate buffered saline (0.15 M, pH 7.2) containing 0.05% TWEEN-20 washes were performed on the coated plates (PBS-Tw). The wells were incubated for one hour at 37 degrees Celsius with 100μl of 1% BSA in sodium carbonate buffer. Mouse serum samples were serially diluted in PBS-Tw, and 100 μl was incubated with coated wells for 1 h at 37°C. Anti-mouse IgG conjugated with peroxidase, diluted 1:2000, was added to the plates after washing, and they were then incubated at 37°C for 1 hour. TMB/H2 O2 addition was used to determine the enzyme's activity.


The addition of 50μl of 8 N sulfuric acid stopped the enzyme process, and the absorbance was measured at 450 nm. Endpoint antibody titers were expressed as the reciprocal of the test serum samples' greatest dilution demonstrating a 3-fold increase in OD in comparison to control samples.20,21


Cyclophosphamide-induced myelosuppression:

Nine groups of six mice each were divided. The vehicle control group was given 0.5% NaCMC, while the positive control group received 50mg/kg immunosin. Negative control group got 25mg/kg cyclophosphamide, whereas the treatment groups received ENA and ENE extracts (50, 100, and 200mg/kg, p.o.) in vehicle daily for 19 days. On the 17th, 18th, and 19th days of the trial, all animals except those in the vehicle control group received cyclophosphamide (25mg/kg, i.p.) injections.22, 23


After 1 hour the extract or vehicle was administered. On day 20, blood samples were collected, and the total blood cell count was analysed using a haematology analyzer.23



Haematological parameters:

To estimate haematological parameters using an auto analyser, blood samples were obtained by heart puncture and placed in EDTA-coated heparinized vials. Hemoglobin content was one of the haematological parameters measured, along with total RBC count, total white blood cell (WBC) count, differential WBC count, and lymphocyte count.24


Histopathology analysis:

At the conclusion of the experiment, the liver, thymus, spleen, and kidneys were removed and placed in 10% phosphate buffered formalin for histological analysis. Haematoxylin-stained slides were examined at different magnifications to detect changes in the cytoarchitecture of the tissue under study.24


Delayed type hypersensitivity (DTH) response:

Mice were subcutaneously challenged 7 days after the vaccination with 25g of ovalbumin in 25 l of normal saline in the left hind footpad to measure the DTH response. A 25 l vehicle injection was administered to the right hind footpad as a control. Using a digital calliper, the increase in foot pad thickness was assessed 24 hours after the challenge.25,26


In vivo phagocytic activity by carbon clearance assay:

Each mouse received a tail vein injection of carbon ink solution 48 hours following the 20-day treatment. At 0 and 15 minutes, blood samples were taken from the orbital vein. Blood (25mL) was combined with 2 mL of 0.1% sodium carbonate before being used to measure optical densities at 660 nm.


The phagocytic index “K” was calculated by using following equation:


K = OD (In OD1- In OD2)/( t2- t1)


OD1- Optical density at t1 means blood collected at 0 min.

OD2- Optical density at t2 means blood collected at 15 min.27,28


Serum biochemical parameters:

By centrifuging the serum for 10 minutes at 3000 rpm and 4°, the serum was separated. By using the references provided in the kit literature that discuss the foundation of the techniques on which test procedures were carried out, aminotransferase enzyme levels were assessed. According to the Biuret technique, total protein and albumin to globulin (A/G) ratios were analysed. 29


Statistical analysis:

Dunnett's multiple comparisons test was used to assess the data after one-way ANOVA, with p 0.05 serving as the significant threshold. One-way ANOVA is the simplest instance. When groups of data are created based on just one component, a one-way analysis of variance is utilised. ANOVA procedure the following assumptions are required:

1.     The findings are distinct from one another.

2.     Each group's observations are drawn from a normal distribution.

3.     Each group's population variances are the same (homoscedasticity)30,31,32



The present investigation suggests that ethanol extract of the eulophia nuda tubers exhibited immunomodulating capabilities based on the following in-vitro and in-vivo experimental procedures. Additionally, a quick assessment of current understanding concerning the phytochemical found in eulophia nuda tuber. Also reviewed detailed analysis of the methods for isolating peritoneal macrophages, as well as in-vivo activity on mice and various assays for the immunomodulating action.



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Received on 06.02.2023         Modified on 01.03.2023

Accepted on 19.03.2023   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2023; 13(2):114-118.

DOI: 10.52711/2231-5691.2023.00023