**1. Introduction**

*Phyllanthus maderaspatensis*, belonging to the genus *Phyllanthus* (Euphorbiaceae), is widely distributed in Sri Lanka, South Africa, China, and southern India. In India, the whole plant is used against kidney and urinary tract infections, digestive disorders, hepatitis, and diabetes. In Tanzania, the whole plant is used as a topical application for scabies.

**Citation:** Ilyas, U.; Katare, D.P.; Naseef, P.P.; Kuruniyan, M.S.; Elayadeth-Meethal, M.; Aeri, V. Immunomodulatory Activity of *Phyllanthus maderaspatensis* in LPS-Stimulated Mouse Macrophage RAW 264.7 Cells. *Separations* **2021**, *8*, 129. https://doi.org/10.3390/ separations8090129

Academic Editor: Ernesto Reverchon

Received: 2 August 2021 Accepted: 17 August 2021 Published: 24 August 2021

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A root decoction has been indicated to cure constipation, gastrointestinal disorders, menstrual problems, intestinal pain and diarrhea, lack of appetite, testicular swelling, chest complaints, and snakebites. Leaves are mixed with lemon juice and applied to the skin for the treatment of rheumatics. The plant is used for a variety of ailments including smallpox, syphilis, asthma, and bleeding gums, as well as in various biological activities such as chronic hepatitis B infection [1], antihepatotoxic and choleretic activities [2], adulticidal and larvicidal efficacy [3], antidiabetic activity [4], hepatoprotective and antioxidant activity [5], anti-Epstein–Barr virus [6], antiretroviral reverse transcriptase [7], and antiherpes simplex virus type 1 and type [8]. Compounds with immunomodulatory activities like flavonoids, fatty acids, triterpenes, and polysaccharides are found in many plants [9]. *Phyllanthus* genus was found to be rich in polyphenols, lignans, flavonoids, triterpenes, hydrolysable tannins, sterol, and alkaloids [10]. Nowadays, HPTLC is used instead of HPLC owing to the easiness, specificity, speed, and low cost [11].

Chronic inflammation has been concerned with various steps involved in tumorigenesis, including metastasis, cellular transformation, promotion, survival, invasion proliferation, and angiogenesis. Only a minority of all tumors are caused by germline mutation, whereas approximately 90% are linked to environmental and somatic mutation factors. Many environmental causes of tumors and risk factors are associated with some form of chronic inflammation [12]. Throughout, chronic inflammation acts as an associate accommodative multitude resistance against illness or injury and is primarily a self-limiting methodology; inadequate resolution of inflammatory responses typically ends up in numerous chronic ailments such as AIDS and cancer [13,14]. At the time of infection, the immune system goes under the attack of a large number of viruses, bacteria, and fungi [15]. The immune system is a part of the body that detects the pathogen using a specific receptor to produce an immediate response by the activation of immune components' cells, chemokines, and cytokines, as well as release inflammatory mediators [16,17]. The immune system can be manipulated by the use of immunomodulators in disease conditions by achieving immunostimulation (as in the treatment of coronavirus, cancer, and AIDS) or immunosuppression (suppression of normal or excessive immune function (e.g., the treatment of graft rejection or autoimmune disease)) [18,19]. Nitric oxide (NO) is a highly reactive and diffusible gas molecule that exerts many biological effects, which include iron homeostasis, smooth muscle relaxation, platelet reactivity, neurotransmission, and cytotoxic defense mechanism against pathogens [20]. NO is also involved in the pathogenesis of many human pathological conditions such as inflammatory disease, cancer, and neurodegenerative disorders [21]. Nitric oxide acts through the stimulation of the soluble guanyl cyclase; is expressed in the cytoplasm of almost all mammalian cells; and mediates a wide range of important physiological functions such as immunomodulation, inhibition of platelet aggregation, vasodilation, and neuronal signal transduction. Nitric oxide is also generated by phagocytes (neutrophils, monocyte, and macrophages) as part of the human immune response [22]. Phagocytes are formed with inducible nitric oxide synthase (iNOS), which is activated by interferon-gamma (IFN-γ) as a single signal or by tumor necrosis factor (TNF) along with a second signal. In this way, the immune system may regulate the armamentarium of phagocytes that play a role in inflammation and immune responses [23]. Lipopolysaccharide (LPS) is widely reported as a major inducer for the production of inflammatory cytokines, which in turn stimulate iNOS induction during the inflammatory process in RAW 264.7 cells macrophages [24]. These cytokines can be formed from macrophages in response to bacterial LPS, inflammatory stimulation, and infection. They also play an important role in the immune system by aiding cytostatic and cytotoxic effects on malignant or infected cells [25].

#### **2. Materials and Methods**

#### *2.1. Reagents and Apparatus*

We purchased reference standards for quercetin rutin, ellagic acid, catechin, and kaempferol from Natural Remedies Pvt. Ltd. (Bangalore, India). All chemicals used were of analytical grade, including mouse macrophage RAW 264.7 cells (National Centre for Cell Science (NCCS), Pune, India, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and MTT assay kit, trypsin EDTA, penicillin and streptomycin and DMSO (Sigma-Aldrich Co. LLC, St. Louis, MO, USA), Trypan blue solution, galactosamine, and absolute ethanol (Himedia Lab Pvt. Ltd., Mumbai, India). Tissue culture flasks, 96 and 24-well micro-culture plates, Eppendorf tube, inverted microscope, serological pipette, heamocytometer (Himedia Lab Pvt. Ltd., Mumbai, India), laminar flow hoods (Khera instrument, New Delhi, India), CO2 incubator (NuAire, Plymouth, MA, USA), water bath, deep freezer (−20 ◦C) were used.
