Immunomodulatory Properties of Withania somnifera: Regulation of Innate Immune Cells and Clinical Implications

ABSTRACT

Withania somnifera (L.) Dunal (WS), commonly known as Ashwagandha, is a prominent medicinal herb in Ayurveda, with extensive therapeutic potential attributed to its diverse phytochemical profile. This review critically examines the immunomodulatory properties of WS, focusing on innate immune cell populations, including neutrophils, eosinophils, macrophages, dendritic cells, and natural killer (NK) cells. Bioactive constituents, particularly withanolides and withaferin A derived mainly from roots and leaves, orchestrate immune responses through modulation of cytokine networks, enhancement of phagocytic activity, and attenuation of oxidative stress. WS constituents regulate key signaling pathways, including ERK, NF‐κB, and JAK/STAT, at transcriptional and translational levels. WS exhibits a dual immunoregulatory role by enhancing innate immune cell functionality while suppressing pathological inflammation in allergic and chronic inflammatory disorders. However, critical gaps persist in elucidating its molecular mechanisms, establishing phytochemical standardization, and defining context‐specific dosing regimens. Collectively, current evidence positions WS as a promising candidate for the management of autoimmune, infectious, and inflammatory diseases. However, it further highlights the need for rigorous mechanistic studies and well‐designed clinical trials to enable its integration into evidence‐based therapeutics.

Abbreviations

8‐OHdG

8‐hydroxy‐2′‐deoxyguanosine

AP‐1

activator protein 1

BAL

broncho‐alveolar lavage

BMDCs

bone marrow‐derived dendritic cells

Caspase‐3

cysteine‐aspartic acid protease‐3

CAT

catalase

CIA

collagen‐induced arthritis

COPD

chronic obstructive pulmonary disease

COX‐2

cyclooxygenase‐2

DCs

dendritic cells

ESR

erythrocyte sedimentation rate

GC–MS

gas chromatography–mass spectrometry

G‐CSF

granulocyte colony‐stimulating factor

GM‐CSF

granulocyte‐macrophage colony‐stimulating factor

GSH

glutathione

HDM

house dust mite

HLA‐DR

human leukocyte antigen–DR isotype

HPLC

high‐performance liquid chromatography

IFN‐γ

interferon gamma

IgE

immunoglobulin E

IgG

immunoglobulin G

IL‐10

interleukin‐10

IL‐12

interleukin‐12

IL‐13

interleukin‐13

IL‐1α

interleukin‐1 alpha

IL‐1β

interleukin‐1 beta

IL‐23

interleukin‐23

IL‐4

interleukin‐4

IL‐6

interleukin‐6

IL‐8

interleukin‐8

JAK–STAT

Janus kinase–signal transducer and activator of transcription

KCs

Kupffer cells

LPS

lipopolysaccharide

MAPK

mitogen‐activated protein kinase

MCAO

middle cerebral artery occlusion

MCP‐1

monocyte chemoattractant protein‐1

MHC Class I

major histocompatibility complex class I

MIP‐α

macrophage inflammatory protein alpha

MIP‐β

macrophage inflammatory protein beta

MPO

myeloperoxidase

mtROS

mitochondrial reactive oxygen species

NAFLD

non‐alcoholic fatty liver disease

NETs

neutrophil extracellular traps

NF‐κB

nuclear factor kappa‐light‐chain‐enhancer of activated B cells

NK cells

natural killer cells

NLRP3

NOD‐, LRR‐ and pyrin domain‐containing protein 3

NMR

nuclear magnetic resonance

NO

nitric oxide

NOAEL

no observed adverse effect level

NRF2

nuclear factor erythroid 2–related factor 2

OVA

ovalbumin

PARP1

poly (ADP‐ribose) polymerase 1

PMA

phorbol 12‐myristate 13‐acetate

ROS

reactive oxygen species

SOD

superoxide dismutase

SRBCs

sheep red blood cells

TGF‐β

transforming growth factor beta

TNF‐α

tumor necrosis factor alpha

UUO

unilateral urethral obstruction

WBC

white blood cells

WFA

withaferin A

WS

Withania somnifera (L.) Dunal

1. Introduction

The Ayurvedic use of medicinal plants for wound healing and the treatment of various illnesses dates back to ancient traditional medicinal texts. Acharya Charak elucidated the concept of host resistance, termed Vyadhikshmatva (immunity), as “Vyadhikshmatvam nam vyadhi bala virodhitvam vyadhyutpada prathibhandakatvam” in the Charaka Samhita, Sutrasthana 28/7. Traditional knowledge described “Bala” as a term loosely associated with strength. However, its core contextual meaning has evolved to refer specifically to individual's immunity, signifying the power to resist diseases effectively (Mukherjee et al. 2021). This concept encompasses two key aspects: vyadhi bala virodhitvam, which refers to Yogakrut Bala, the ability to combat the severity of a disease, and vyadhyutpada prathibhandakatvam, known as Sahaj Bala, which refers to the capacity to prevent the occurrence or recurrence of diseases, supporting the modern immunology concept of disease resistance and prevention. Sahaj Bala, that is, innate immunity, is influenced by the quality of the embryo and serves as the body's first line of defense from birth. In contrast, Yuktikrut Bala, or acquired immunity, relies on various factors such as proper nutrition, physical exercise, rest, and therapies like rasayana and vajikaran (Mahesh and Shreevidya 2014). Nonetheless, historical evidence from written records, ancient monuments, and traditional remedies in Ayurveda, Siddha, and Unani systems of medicine illustrates humanity's reliance on nature by using various medicinal plant parts, such as bark, seeds, roots, and fruit, for therapeutic purposes to combat diseases and injuries (Patwardhan 2013).

Ashwagandha or Withania somnifera (L.) Dunal belongs to the Solanaceae family and is well documented in Ayurvedic texts such as the Charaka Samhita and the Susruta Samhita (Dar et al. 2015; Kalra and Kaushik 2017). The traditional name Ashwagandha derives from the horse‐like smell of its root, known as “Ashwa,” and is associated with the vitality it is believed to confer. Traditionally, it has been known for Balya (strength enhancer), Brusya (sexual performance enhancer), Vajikari (spermatogenic), Kamarupini (libido‐enhancing), and Pustida (nourishing). Charaka Samhita classifies Ashwagandha as “Bajikarana” and “Rasayana,” emphasizing its roles in promoting health, longevity, and resistance to adverse conditions and infections (Mukherjee et al. 2021; Patwardhan 2013). WS is well described for adaptogenic properties, which help the body to adjust to stress and maintain physiological balance (Mikulska et al. 2023). WS also exhibits anti‐bacterial, anti‐tumor, anti‐diabetic, chemoprotective, neuroprotective, hepatoprotective, and anti‐aging effects, attributed to its immunomodulatory and antioxidant properties and associated signaling mechanisms (Alnuqaydan et al. 2022; Gaurav et al. 2023; Henley et al. 2017; Honnutagi et al. 2026; Kashyap et al. 2020; Lerose et al. 2024; Siddiqui et al. 2021; Yoshitomi et al. 2025). WS is prescribed in various dosage forms. It is often mixed with milk or ghee to support growth, muscle mass, and physical endurance in underweight individuals. The burnt ash of the ashwagandha plant, called ashwagandha kshara, is used to treat respiratory inflammation. The ashwagandha churna and rasayana are specific dosage preparations designed for optimal absorption and tissue‐building as rejuvenators (Patwardhan 2013).

Modern science has acknowledged the efficacy of many medicinal plants, incorporating them into pharmacotherapy as plant‐based medications and pharmaceuticals (Choudhary et al. 2015). However, transitioning traditional plant‐based systems into modern practice faces challenges, including limited understanding of their mechanisms, potential immunomodulatory roles, side effects, and clinical validation. WS has notable immunomodulatory effects, including the ability to modulate cytokines, enhance macrophage function, and increase T‐cell proliferation for improved specific immunity. Early studies showed that WS extracts boost hemoglobin levels, increase red blood cell (RBC) and white blood cell (WBC) counts, and reduce serum cholesterol and ESR levels (Hussain et al. 2017; Ziauddin et al. 1996). Furthermore, WS extracts reduce the immunosuppression caused by chemotherapy, a significant concern with conventional anti‐cancer therapies (Gupta et al. 2001). Recent clinical studies indicate that bioactive compounds in WS can improve immune profiles by modulating adaptive immune cells and immunoglobulin G (IgG) levels in healthy individuals (Tharakan et al. 2021). Although WS has traditional and modern applications for immune‐related disorders, the precise molecular mechanisms and targets in the immune system remain incompletely understood.

This review aims to provide a comprehensive overview of the immunomodulatory potential of WS, highlighting its effects on the functions of innate immune cells, molecular targets, and implications for disease management. Scientific studies were searched for WS and its constituents, including withaferin, with immunomodulation, immunomodulatory properties, with specific immune cell types in traditional Ayurvedic books like Charaka Samhita, Susruta Samhita, reports, research articles, and databases like PubMed, Science‐Direct, Web of Science, Wiley Online Library, and Google Scholar describing both pre‐clinical and clinical data. The key studies focusing on immunomodulatory properties of WS were selected and discussed in this review. WS and its constituents frequently influence the activity of innate immune cells, highlighting their vital role in immune functions. These include processes such as phagocytosis, the generation of reactive oxygen species, and the modulation of pro‐inflammatory and anti‐inflammatory cytokines, which also serve as biomarkers for inflammatory conditions (Morzunov et al. 2017). Additionally, these effects are linked to critical signaling pathways such as NF‐κB and MAPK. Emerging research is uncovering the pathways and molecular targets through which WS affects immune responses. However, information on its impact on innate immune cells and the specific mechanisms underlying these effects is still limited. We have focused on the effects of WS extracts and their active phytochemicals on key immune cells (Figure 1), including neutrophils, macrophages, dendritic cells, natural killer (NK) cells, and their diverse functions, survival, and immunomodulatory responses. Both immunostimulatory and suppressive characteristics have been reported for WS extracts, highlighting the need to identify potent constituents with specific effects and distinct mechanisms (Alanazi and Elfaki 2023; Gaurav et al. 2023). By examining both preclinical and clinical studies, this review highlights WS as a natural immunomodulatory agent and its potential as a modern therapeutic intervention.

FIGURE 1.

Key phyto‐constituents present in Withania somnifera . These diverse chemical constituents can present different immunomodulatory potentials. These constituents vary between roots and other aerial parts of WS. Moreover, extraction methods, including aqueous, hydro‐alcoholic, and alcoholic extracts, provide enrichment of polar and non‐polar chemical constituents.

2. Phytochemistry and Phytoconstituents of Withania somnifera

WS stands out among the 23 species of the genus Withania for its remarkable medicinal value. Phytochemistry has identified a rich diversity of bioactive compounds that contribute significantly to its therapeutic potential, including anti‐cancer, anti‐inflammatory, antimicrobial, antidiabetic, and hepatoprotective activities (Singirala et al. 2025). Both the roots and leaves of WS are widely used in traditional medicine, owing to their diverse and potent phytochemical constituents, including flavonoids, phenolic acids, steroidal lactones, alkaloids, steroids, saponins, tannins, withanolides, and mineral salts (Dar et al. 2015; Sahoo et al. 2024). Advanced techniques such as HPLC, GC–MS, and NMR have identified numerous active phytoconstituents in the roots (Sandipan et al. 2010; Singirala et al. 2025).

Among the withanolides, Withaferin‐A and Withanolide‐A have garnered the most attention due to their pronounced pharmacological activities. Other notable compounds, such as Withanone, Withanoside‐IV, Withanoside‐VI, sitoindosides IX–X, 27‐Hydroxy Withanone, 27‐Hydroxy Withanolide B, 12‐Deoxy Withastramonolide, cuscohygrine, anahygrine, and tropine, exhibit complementary effects in promoting health and combating diseases (Sandipan et al. 2010; Singirala et al. 2025). This review discusses the specific immunomodulatory actions of WS and these bioactive constituents on the innate immune system.

3. Immunomodulatory Effects of Withania somnifera

WS exhibits a biphasic immunomodulatory profile, with both immunostimulatory and immunosuppressive effects depending on the infection and inflammation context (Figure 2). This dual functionality highlights its therapeutic relevance, enabling enhancement of host defense mechanisms while limiting excessive or pathological immune responses. WS promotes leukocyte proliferation and enhances the functional activity of macrophages and natural killer (NK) cells. Concurrently, it attenuates pro‐inflammatory cytokines, including interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), and interleukin‐6 (IL‐6), while upregulating anti‐inflammatory mediators such as interleukin‐10 (IL‐10) and associated signaling pathways. These context‐dependent responses are likely driven by variations in the microenvironment, experimental conditions, and the plant's diverse chemical constituents (Figure 3). Additionally, the use of different doses, extract type, and extraction methods, such as aqueous, hydroalcoholic, and alcoholic approaches, yields distinct phytochemical profiles by selectively extracting polar and nonpolar compounds, thereby contributing to variability in immunological outcomes (Gaurav et al. 2023; Paul et al. 2021). Furthermore, WS exhibits distinct immunostimulatory responses based on disease conditions, including infection and inflammation. Among the key constituents, structurally diverse withanolides, such as Withanolide A, Withaferin A, Withanolide D, and Withanone, have demonstrated various biological activities in both in vitro and in vivo studies (Nagarajan et al. 2026; Yoshitomi et al. 2025).

FIGURE 2.

Overview of Withania somnifera ‐mediated effects on different innate immune cells. Immunomodulatory effects of WS on innate immune cells. WS reduces neutrophil infiltration, suppresses macrophage‐derived inflammatory cytokines, enhances NK cell activity, stabilizes mast cells, limits dendritic cell maturation and cytokine production, and inhibits eosinophilia.

FIGURE 3.

The immunomodulatory effects of Withania somnifera (WS) are context dependent. WS extracts and their bioactive constituents suppress excessive inflammatory responses in autoimmune and inflammatory conditions, while under immunosuppressive states, such as bone marrow stress or tumor‐associated immune suppression, they promote immune activation by increasing immune cell numbers and enhancing their functional activity.

The dosage of WS root extract in humans typically ranges from 120 to 1250 mg per day, with 600 mg daily (administered in two divided doses) the most commonly used regimen (Vaidya et al. 2024). No observed adverse effect level (NOAEL) was observed with 2000 mg/kg body weight/day in rats after repeated oral administration for 90 days (Kalaivani et al. 2023). Ayurveda recommended only the roots of WS for therapeutic usage, as safety data for leaf extracts is limited. Though several considerations are required with variation in withanolide contents (1%–35% total withanolides) in different standardized root extract preparations with diverse analytical methods. According to the W. somnifera Safety Dossier from the Indian Ministry of Ayush, the safe no observed adverse effect dose for WS root is recorded at up to 2000 mg/kg body weight for human consumption (Ashwagandha Safety Dosier 2.0, Ministry of Ayush, India 2024).

3.1. Immunostimulatory Effects of Withania somnifera

WS has demonstrated immunostimulatory effects across diverse immune model systems. This section focuses on preclinical studies, while clinical evidence is discussed in the subsequent section. Under immunosuppressive conditions, such as infections, chemotherapy, and related disorders, WS enhances immune responses. An early study reported that a methanolic extract of WS significantly increased WBC counts in BALB/c mice and protected against sublethal doses of gamma radiation; however, the specific plant part used for extract preparation was not specified (Kuttan 1996). WS has also demonstrated immunostimulatory effects in drug‐induced immunosuppression models. In a paclitaxel‐induced immunosuppression model, an aqueous extract of WS (200 mg/kg) administered to male Swiss albino mice exhibited protective effects, highlighting its potential as an adjuvant (Gupta et al. 2001). In another study, an ethanolic whole‐plant extract of WS (100 mg/kg; human equivalent dose≈500 mg/day) enhanced both humoral and cell‐mediated immune responses in Swiss albino mice with cyclophosphamide‐ and cyclosporine‐induced immunosuppression. The humoral response increased by 12% relative to the cyclophosphamide control (54%), while the cell‐mediated response improved by 19% compared to the cyclosporine control (38%). These findings underscore the potential of WS as a potent immunostimulatory agent (Verma et al. 2012). Additionally, ethanolic root extract of WS (400 mg/kg), administered orally once a week for 4 weeks, significantly increased the numbers of leukocytes, lymphocytes, neutrophils, immune complexes, and immunoglobulins in Swiss albino mice subjected to azoxymethane‐induced experimental colon carcinoma (Muralikrishnan et al. 2010).

WS has been shown to modulate cytokines relevant to inflammatory, autoimmune, and infectious disorders. Cytokine modulation was also demonstrated by aqueous root extract of WS in immunized BALB/c mice with OVA in Freund's adjuvant (OVA‐FCA). Treatment at 50, 100, and 200 mg/kg doses promoted T‐cell proliferation, increased secretion of Th1 cytokines IL‐2 and IFN‐γ, and moderately downregulated Th2 cytokine IL‐4 (Malik et al. 2007). STAT4 activation by WS regulated Th1 differentiation and IFN‐γ secretion (Kumari et al. 2023). The aqueous‐alcoholic leaf extract of WS (1 μg/mL) enhanced IFN‐γ expression on Con A‐primed splenocytes in vitro (Bani et al. 2006; Khan et al. 2009). Beyond direct immunomodulation, glycol‐withanolides, sitoindosides IX and X, and withaferin A exhibited adaptogenic potential by increasing lysosomal enzyme activity, enhancing phagocytosis, and recruiting and activating peritoneal macrophages in Swiss mice and Wistar albino rats (Nascimento et al. 2016). Furthermore, WS methanolic root extracts (1–256 μg/mL) have been shown to enhance innate immune responses, as evidenced by a concentration‐dependent increase in nitric oxide (NO) production and nitrite levels (1 nmol/10⁶ cells) in J774 macrophages, underscoring the immuno‐boosting capabilities of WS (Iuvone et al. 2003). Nitric oxide plays a critical role in signaling, pathogen clearance, and tumor suppression, further emphasizing the therapeutic potential of WS. The studies collectively demonstrate the robust immunomodulatory potential of WS, showcasing its ability to enhance both innate and adaptive immune responses in preclinical models of drug‐induced immunosuppression, cancer, and infections. These preclinical findings underscore WS as a potent immunostimulant with broad therapeutic applications, although it may have certain limitations in clinical settings. Across various disease models, WS has demonstrated promising therapeutic effects, particularly in restoring balance in cytokine regulation, which are key regulators of immune responses. Pre‐clinical and clinical studies have not only confirmed the safety but also validated the immune efficacy of WS (Kalaivani et al. 2023; Narsing et al. 2021; Singh et al. 2022). To fully harness its potential, it is crucial to conduct more systematic scientific clinical trials that will establish WS as a valuable component in modern medicine.

3.2. Immunosuppressive and Anti‐Inflammatory Effects

Inflammatory conditions like lung inflammation, peritonitis, asthma, allergy, vasculitis, atherosclerosis, hypersensitivity, inflammatory bowel disease, sepsis, and autoimmune disorders such as rheumatoid arthritis, systemic lupus erythematosus (SLE) often result from an overactive immune response that damages host tissues and exacerbates disease severity. The immunosuppressive activity of WS offers therapeutic potential in managing immune‐related and inflammatory disorders. For instance, aqueous extracts from WS root powder (50–100 μg/mL) have been shown to inhibit inflammatory mediators, including complement activation, T‐cell proliferation, and humoral antibody responses (Rasool and Varalakshmi 2006). Administration of 1000 mg/kg WS root powder significantly reduced complement activation (11% at 72 h post‐adjuvant induction) in arthritic rats compared to untreated counterparts (86% activation), indicating its potential as an immunosuppressive agent in autoimmune disorders (Rasool and Varalakshmi 2006).

Studies have shown the efficacy of WS extracts in inhibiting over‐activated immune responses through diverse mechanisms. In a recent study, WS root extract (525 mg/kg) demonstrated potent anti‐inflammatory effects by inhibiting neutrophil, lymphocyte, and monocyte infiltration and by reducing pro‐inflammatory cytokines, including IL‐1β, TNF‐α, and IL‐6, in an endotoxin‐induced peritonitis model (Balkrishna et al. 2022). In the prostate cancer model, alcoholic WS root extract displayed dose‐dependent downregulation of JAK–STAT signaling, reducing the expression of pro‐inflammatory cytokines such as IL‐6, IL‐1β, and IL‐8, suggesting its role in mitigating chronic inflammation and oncogenesis (Aalinkeel et al. 2010). Similarly, WS has shown dose‐dependent efficacy in treating type 2 inflammation‐associated allergic disorders and arthritis, based on its anti‐inflammatory effects on pro‐inflammatory cytokines (Gupta and Singh 2014; Ali et al. 2023). The aqueous root extract of WS (200 and 400 mg/kg) protected against OVA‐induced levels of IL‐13, 8‐OhdG, TGF‐β, hydroxyproline, and peri‐bronchial inflammatory cell infiltration in a rat model of asthma (Ali et al. 2023). In vitro studies on lung cancer cell lines showed concentration‐dependent inhibition of Th2‐dependent cytokines (IL‐6, IL‐4) and neutrophil‐related cytokines (IL‐8) by 1–10 mg/mL aqueous WS root extract. Pre‐treatment with WS aqueous extracts (10–100 mg/kg) reduced neutrophil influx in lipopolysaccharide (LPS)‐treated mouse lungs, further underscoring their anti‐inflammatory potential (Sharma et al. 2023). Moreover, compounds such as Withaferin A (WFA), Withanolide A, and Withanone have been identified as having notable immunomodulatory properties (Gao et al. 2015). More specifically, WFA (20 μM) significantly reduced the expression of various inflammatory cytokines and chemokines in LPS‐primed, ATP‐activated THP‐1 cells (Dubey et al. 2018).

WS and its constituents have been identified to regulate diverse signaling pathways, including NF‐κB and AP‐1 activity, caspase‐3, PARP1, and NLRP3 activation, leading to anti‐inflammatory effects (Figure 4) (Balkrishna et al. 2022; Dubey et al. 2018; Oh et al. 2008; Xia et al. 2021). Nuclear factor‐kappa B (NF‐κB) and activator protein 1 (AP‐1) are key transcription factors that orchestrate the immune responses with activation of pro‐inflammatory cytokines. Upon stimulus exposure, NF‐κB translocates into the nucleus from the cytoplasm to turn on genes that produce pro‐inflammatory cytokines. WS and withaferin inhibited the NF‐κB translocation to the nucleus. This mechanism contributed to a marked decrease in the production of pro‐inflammatory cytokines, including IL‐1β and IL‐18 (Dubey et al. 2018). Withaferin A inhibited phosphorylation of Akt, and extracellular signal‐regulated kinases (ERKs) and further prevented IκB phosphorylation, mitigating nuclear translocation of NF‐κB, thus blocking LPS‐induced iNOS activation in macrophages (Oh et al. 2008). Moreover, Withaferin A protected against hepatitis through anti‐apoptotic activity, reducing cleaved CASP3 and PARP1, offering therapeutic protection against chronic inflammatory conditions (Xia et al. 2021). Mechanistically, WFA alleviated LPS‐induced NLRP3 activation in primary macrophages (Xia et al. 2021). Similarly, our recently published finding in neutrophils validated the effects of WS and WFA in alleviating NLRP3 and Caspase‐1 activation to suppress inflammatory IL‐1β release (Mondal et al. 2026).

FIGURE 4.

Mechanisms underlying the immunomodulatory effects of WS on immune cells. WS extracts have been reported to exert both anti‐inflammatory and pro‐inflammatory (immunostimulatory) effects depending on the biological context. WS inhibits ERK1/2 phosphorylation, JNK signaling, NLRP3 inflammasome activation, and NF‐κB nuclear translocation, thereby reducing the production of pro‐inflammatory cytokines such as TNF‐α, IL‐6, and IL‐1β, while enhancing the levels of the anti‐inflammatory cytokine IL‐10. In the tumor microenvironment, WS induces AIM2 inflammasome activation in M2 macrophages, promoting their polarization toward the M1 phenotype and thereby enhancing anti‐tumor immunity. Additionally, WS promotes a Th1‐skewed immune response by enhancing macrophage activity and increasing the secretion of IL‐12, TNF‐α, and nitric oxide, highlighting its immunostimulatory and immune‐boosting potential.

These findings suggest that WS exerts its clinical effects through modulation of key immune signaling pathways. Collectively, the literature demonstrates both immunostimulatory and immunosuppressive properties; however, the context‐specificity and underlying mechanisms remain insufficiently defined. Further research is needed to elucidate the effects of WS on specific immune cell populations and signaling pathways, particularly within the innate immune system. The following sections provide a detailed discussion of these innate immune cells and associated immunomodulatory mechanisms. Tables 1 and 2 summarize the effects of WS on innate immune cells and in vivo models, respectively.

TABLE 1.

In vitro studies on the immunomodulatory effects of Withania somnifera on innate immune cells.

Extract/compound dose	Source, characterization	Experimental cell model	Immunomodulatory effects	Mechanism of action	References
Macrophage
Root methanolic extract (1–256 μg/mL)	Laboratory Carlo Sessa (Milan, Italy) extract showed major absorption in the 200–250 nm range.	J774 macrophages	Activation and pathogen clearance	Enhanced NO production and iNOS expression	Iuvone et al. (2003)
Root ethanolic extract (0.2, 0.5, 1 mg/mL)	HPLC characterization of the collected material from BHU, India, using withaferin A as a marker compound	PBMCs, RAW 264.7 macrophage cell line	Anti‐inflammatory activity	Inhibition of LPS‐induced NO accumulation	Singh et al. (2007)
Root methanolic extract (1, 3, 10, 30, 100 μg/mL)	Patanjali Ayurveda Limited, characterization using withanolide	Human THP‐1 monocyte‐derived macrophages	Anti‐inflammatory activity	Decreased LPS‐induced IL‐6, TNF‐α	Balkrishna et al. (2022)
Leaf water extract and active chloroform fraction (0.05%–1.5%)	Guru Nanak Dev University characterization using TLC and HPLC with withaferin A and withanone	Primary microglial cells and BV‐2 microglial cell line.	Anti‐inflammatory effects on β‐amyloid and LPS activated cells	Suppression of the proliferation, reduced NFkB and AP1, decrease in TNF‐α, IL‐1β, IL‐6, RNS, and ROS production	Gupta and Kaur (2016)
Leaf alcoholic extract (10–15 μg/mL) and withaferin A (1.5 μM)	Material collected by CIMAP and processed at University of Hyderabad HPLC and LC–MS/MS characterization using withanolides	Ex vivo leishmaniasis exposure to macrophages	Anti‐leishmanial activity	Increase parasite clearance by increasing ROS, reducing IL‐10, IL‐4, and TGF‐β	Chandrasekaran et al. (2017)
Leaf and root methanolic extract (100–1000 μg/mL) Withaferin A, Withanolide A (1–10 μg/mL)	CIMAP, India, HPLC characterization using withanolides	LPS‐activated murine bone marrow‐derived macrophages	Anti‐inflammatory activity	Suppression of LPS‐induced expression of pro‐inflammatory cytokines TNF‐α and IL‐6	Purushotham et al. (2017)
Withaferin A (0.1–1.0 μg/mL)	Fisher Scientific	Diphosphoryl lipid A (DPL) stimulated macrophages/THP‐1	Inhibition of pro‐inflammatory cytokines in vitro	Attenuation of pro‐inflammatory IL‐6 and CCL5 in macrophages	Kalpana et al. (2023)
Withaferin A (0.02–0.4 μM)	Sigma Aldrich	LPS‐induced iNOS, NO and activity of NF‐κB in RAW 264.7macrophages	Anti‐inflammatory activity	Reduced iNOS, NO levels, NF‐κB. Inhibition of Akt and ERK activation	Oh et al. (2008)
Withaferin A (0.25–1.0 μM)	Sigma Aldrich	LPS/ConA induced inflammation in BALB/c mice macrophages	Macrophage polarization from M1 phenotype to M2 phenotype	Reduced pro‐inflammatory cytokines such as TNF‐alpha, IL‐1beta, and IL‐6	Alnuqaydan et al. (2022)
Withaferin A (5–20 μM)	ChromaDex	LPS + ATP induced THP‐1 macrophages activation	Anti‐inflammatory activity	Inhibition of caspase‐1, alters NLRP3 and ASC co‐localization, reduced IL‐1β and IL‐18 secretion	Dubey et al. (2018)
Withaferin A (4–10 μM)	NP	THP‐1 macrophages	Macrophage polarization from M2 to M1 phenotype	Activation of AIM2 inflammasome in M2 macrophages, activation of caspase‐1	Ngoungoure and Owona (2019)
Withaferin A (0.1–2 μM)	Biomol (Plymouth, PA, USA)	THP‐1 cells	Protects vascular barrier integrity	Inhibition of the adherence of monocytes to HMGB1‐stimulated endothelial cells	Lee et al. (2012)
Withaferin‐A encapsulated in mannosylated liposomes (ML) (10, 25, 50 μg/mL)	Natural remedies (Bangalore, India)	In vitro study on RAW 264.7 macrophages	Macrophage polarization from M1 to M2 phenotype	Reduced IL‐1β, IL‐6, IL‐23, and TNF‐α production, enhanced M2 phenotype, ablated NO, iNOS, and ROS	Neog et al. (2018)
Neutrophils
Aqueous root extract (50–100 μg/mL)	GMP facility NMPB and characterized with known withanolides	Human blood PMNs, cellular ROS and mtROS production	Reduction of ROS and mtROS production induced with various stimuli; TRLM, PMA, A23187, and ionomycin	NOX‐2 dependent inhibition of the radicals at 50 and 100 μg/mL and reduction of mtROS production	Rizvi et al. (2023)
Withaferin A (2.5–50 μM)	Cayman Chemical	Equine blood neutrophils	Anti‐inflammatory activity	Inhibition of neutrophil adhesion, migration, and respiratory burst	Bayless et al. (2022)
Withaferin A (10 μM)	ChromeDex, Irvine	In vitro study for E. coli ‐induced bacterial meningitis surrogate model	Anti‐inflammatory response in bacterial meningitis	Inhibition of E. coli ‐induced PMN transmigration across BMEC	Che et al. (2011)
Withaferin A (1.0 μg/mL)	Enzo Life Sciences	In vitro human autologous blood islet tube model	Suppression of inflammatory response	WFA inhibited neutrophil infiltration in Islet and enhanced their survival	Kanak et al. (2014)
Dendritic cells
Withaferin A (100, 250, and 500 nM)	Sigma Aldrich	In vitro study on BMDCs induced with H. pylori infection	Targets pro‐inflammatory signaling during infection	Suppressed gene expression of IL‐1β and NLRP3, reduced cleavage of caspase‐1 and IL‐1β	Kim et al. (2015)
Withaferin A (0.15 and 0.25 μg/mL)	Enzo Life Science	BALB/c mice islet transplantation model	Immunosuppressive role during allogeneic islet graft transplantation	Prolonged survival of islet allograft via inhibition of dendritic cell maturation	Kumano et al. (2021)

TABLE 2.

In vivo studies on the efficacy of Withania somnifera through modulation of innate immune cells.

Experimental/disease model	Extract/compound dose	Source, characterization	Immunomodulatory effects	Mechanism of action	References
Macrophages
Normal Balb/c mice	Methanolic root extracts (800 mg/kg)	Amala Ayurveda Hospital and Research Centre	Potent immuno‐stimulator	Enhanced phagocytic activity of peritoneal macrophages	Davis and Kuttan (2000)
SRBC immunized BALB/c mice model	Aqueous‐alcoholic root extracts (10, 30, 100 mg/kg)	Institute's experimental field, characterized using withanolides	Enhanced macrophage functions	Improved secretion of nitrite, IL‐12, and TNF‐α	Malik et al. (2007)
Experimental aspergillosis mice model	Aqueous root extract (100 mg/kg)	Characterized extract from Green pharmacy (Pune, India)	Improved pathogen clearance	Enhanced phagocytic activity and intracellular killing efficacy	Dhuley (1998)
Collagen‐induced arthritis (CIA) rats	Aqueous root extract (300 mg/kg P.O daily)	Fresh roots extract, Delhi Market, HPLC characterized withanolides	Anti‐inflammatory in arthritis condition	Decreased serum TNF‐α, IL‐1β and IL‐6. Reduced TNF‐α, IL‐1β positive synovial cells/macrophages	Khan et al. (2019)
Adjuvant‐induced rat arthritic model	Withaferin A (30 mg/kg), mannosylated‐liposomal‐WA (10 mg/kg), liposomal‐WA (10 mg/kg)	Natural remedies (Bangalore, India)	Alleviated inflammatory response in arthritis	Reduced oxidative stress (ROS and NO), promoted M1–M2 polarization	Sultana et al. (2017)
Endotoxin, diphosphoryl lipid A (DPL) induced cytokine in mice	Withaferin A (50 μg/kg)	Fisher Scientific	Inhibition of pro‐inflammatory cytokines in vivo	Decreased IL‐6, IFNγ, TNF, and VEGF in animal model	Kalpana et al. (2023)
M. tuberculosis infection model	Withaferin A (0.1–0.5 mg/mL and 2 mg/kg)	Sigma Aldrich	Enhanced macrophage activation to mitigate intracellular M. tuberculosis	Switching to M1 phenotype, increasing ROS, enhanced IL‐6, IL‐1β, and IL‐23, increased antigen presentation to T cells	Kumari et al. (2023)
Unilateral ureteral obstruction (UUO) renal injury	Withaferin A (1–3 mg/kg)	PS3 Laboratories LLP (Hyderabad, India)	Protection from inflammation	Attenuation of TGF‐β signaling pathway, Inhibition of macrophage recruitment at injury site	Peddakkulappagari et al. (2019)
Neutrophils
Paclitaxel‐induced neutropenia in male Swiss albino mice	Aqueous extract (200 mg/kg)	Dabur Research Foundation, India	Neutropenia reversal	Reversal of neutropenia similar to the available GM‐CSF and G‐CSF growth factors in the chemotherapeutic drug induced neutropenia	Gupta et al. (2001)
LPS‐induced inflammation model	Aqueous root extract (10, 30, and 100 mg/kg)	GMP facility of NMPB, characterized with withanolides	Protection against excessive inflammation	Attenuation of LPS‐induced neutrophil counts in broncho‐alveolar lavage (BAL) fluid of BALB/c mice	Sharma et al. (2023)
Endotoxin‐induced peritonitis mice model	Methanolic root extracts (175 and 525 mg/kg)	Patanjali Ayurveda Limited, characterized using withanolides	Anti‐inflammatory activity	Attenuation of neutrophils and cytokines IL‐1β, TNF‐α, and IL‐6 in the peritoneal cavity	Balkrishna et al. (2022)
Azoxymethane‐induced colon cancer mice model	Ethanolic root extract (400 mg/kg)	Co‐operative Pharmacy, Chennai, India, characterized with withanolide	Ameliorates neutropenia, enhances immune response	Increased number of neutrophils, enhanced their killing and phagocytic activity	Muralikrishnan et al. (2010)
Sheep RBC (SRBC)‐challenged footpad swelling rat model	WS Churna (200–300 mg/kg/day)	Local market, Bangalore	Potentiation of cellular immune response	Enhanced neutrophil adhesion, increased delayed‐type hypersensitivity response	Gupta et al. (2006)
UUO induced renal tubulointerstitial fibrosis model	Withaferin A (3 mg/kg)	AdooQ BioScience	Anti‐inflammatory molecule in renal fibrosis condition	Reduction of neutrophil infiltration in the kidney, curtailed expression of NF‐κB‐p65, IL‐1β, and COX‐2	Chen et al. (2020)
LPS‐induced acute lung Injury rat model	Withaferin A (2–6 mg/kg)	Sigma	Enhanced anti‐inflammatory response	Reduced neutrophil infiltration, MPO levels, MIP‐2, TNF‐α, IL‐1β and IL‐6, Upregulate TGF‐β1, and IL‐10	Gao et al. (2015)
S. aureus‐infected zebrafish model	Withaferin A (60 μM in feeding solution)	—	Antibiofilm, immunosuppressive function during bacterial infection	Decrease in the number of localized macrophages and neutrophils with antibacterial, antibiofilm and anti‐inflammatory activity	Murugan et al. (2023)
Eosinophils
Murine asthmatic Balb/c model	Aqueous extract (750 mg/kg)	Collected and prepared at Pretoria University	Anti‐inflammatory in allergic reaction	Increase in the eosinophils number in the BALF of asthmatic Balb/c mouse	Oberholtzer et al. (2008)
Natural killer cells
Ovarian cancer (OVCA) hen model	Root powder (1–2 g/kg)	Organic India USA	Prevention of tumor progression and metastasis	Increase NK cells number in the tumor vicinity, Decrease in the expression of MICA by the tumor	Barua et al. (2013)
Mast cells
Wistar rat anaphylaxis model	Methanolic extract (250, 500, 750 mg/kg)	Local market Tirupati, prepared at Department of Botany S.V. University	Anti‐anaphylactic activity	Stabilization of mast cell and reduced mast cell degranulation upon exposure to the allergen	Girish and Reddy (2017)
OVA‐ induced allergic mouse model	Aqueous root extract (200 and 400 mg/kg)	Natural remedies (Bengaluru, India) 2% withanolide content	Anti‐anaphylactic activity	Prevention of mesenteric mast cell degranulation, reduced mortality rate	Ray (2023)

4. Effects of Withania somnifera on Innate Immune Cells

4.1. Granulocytes

4.1.1. Neutrophils

Neutrophils serve as the first line of defence against infections through phagocytosis, ROS production, and neutrophil extracellular traps (NETs). Moreover, dysregulation of neutrophil function can result in various diseases, including chronic granulomatous disease, arthritis, asthma, hypersensitivity, inflammatory bowel disease, and sepsis. WS has been studied for its immunomodulatory effects, including its influence on neutrophil development and functions. The aqueous extract of WS has demonstrated efficacy in reversing neutropenia in male Swiss albino mice challenged with paclitaxel (Gupta et al. 2001). Interestingly, WS 200 mg/kg showed efficacy comparable to that of GM‐CSF and G‐CSF (Gupta et al. 2001). Similarly, ethanolic root extract of WS at doses of 200 and 400 mg/kg demonstrated increased neutrophil numbers, phagocytic activity, and enhanced their killing capacity in an azoxymethane‐induced colon cancer mouse model (Muralikrishnan et al. 2010). Over‐the‐counter WS formulations, such as Ashwagandha churna, have also exhibited immunomodulatory activity in a sheep RBC (SRBC)‐challenged footpad swelling rat model (Gupta et al. 2006). WS powder enhanced neutrophil adhesion and delayed‐type hypersensitivity responses at doses of 200 and 300 mg/kg/day (Gupta et al. 2006). These results highlight the potential of WS extracts in boosting immune responses by enhancing neutrophil function and numbers.

Conversely, WS has also been observed to mitigate elevated neutrophil counts and function in various inflammatory conditions. For example, aqueous root extracts of WS at doses of 10, 30, and 100 mg/kg attenuated LPS‐induced increases in neutrophil counts in the broncho‐alveolar lavage (BAL) fluid of BALB/c mice (Sharma et al. 2023). A study evaluating the anti‐inflammatory properties of withanolide‐rich methanolic WS root extracts in a model of endotoxin‐induced peritonitis demonstrated their anti‐inflammatory effects (Balkrishna et al. 2022). Doses of 175 and 525 mg/kg reduced neutrophil, lymphocyte, and monocyte migration into the peritoneal cavity and decreased the release of IL‐1β, TNF‐α, and IL‐6 by targeting the NF‐κB/activator protein 1 pathway (Balkrishna et al. 2022). Together, WS displays enhanced neutrophil production and function in immunosuppressive conditions; in contrast, it mitigates neutrophil infiltration in inflammatory conditions like peritonitis and lung injury.

In our in vitro study on human neutrophils, WS aqueous root extract offered protection against phorbol 12‐myristate 13‐acetate (PMA)‐induced reactive oxygen species (ROS) and mitochondrial ROS (mtROS) production. The extract inhibited free radical production by 19% at 50 μg/mL and 30% at 100 μg/mL, with a 19% reduction in mtROS production. Notably, the antioxidant effect on PMA‐induced ROS was more pronounced than on ionophore‐stimulated cells, suggesting WS extracts as NOX‐2‐targeted effector molecules (Rizvi et al. 2023). We further validated the effects of WS and withaferin A on NOX‐2‐dependent ROS generation in our recent study (Mondal et al. 2026). Further studies on other neutrophil functions and mechanisms may provide better insights into the anti‐inflammatory effects of WS.

The bioactive component withaferin A (2.5–50 μM) has shown inhibitory effects on neutrophil adhesion, migration, and the respiratory burst. In equine neutrophils, withaferin A induced apoptosis in GM‐CSF‐primed neutrophils as well as neutrophil adhesion induced by interleukin‐8, leukotriene B4, platelet‐activating factor, and PMA, highlighting its potential clinical benefits in resolving inflammation mediated by neutrophils (Bayless et al. 2022). Withaferin A (10 μM) also inhibited E. coli ‐induced PMN transmigration across the blood–brain barrier by targeting vimentin, a protein involved in bacterial meningitis pathogenesis (Che et al. 2011). In the renal tubulointerstitial fibrosis model, withaferin A (3 mg/kg) reduced inflammation and neutrophil infiltration and suppressed NF‐κB‐p65 phosphorylation, IL‐1β, and COX‐2 expression, thereby attenuating inflammation and fibrosis (Chen et al. 2020). Additionally, withaferin A improved islet graft survival in an instant blood‐mediated inflammatory response model by inhibiting neutrophil infiltration (Kanak et al. 2014). In the neonatal lung injury model, withaferin A (2, 4, and 6 mg/kg) reduced neutrophil infiltration, myeloperoxidase (MPO) levels, and inflammatory cytokines such as TNF‐α, IL‐1β, and IL‐6, while enhancing anti‐inflammatory markers such as TGF‐β1 and IL‐10 (Gao et al. 2015). Withaferin A also protected against oxidative damage, making it a potential candidate for treating lung injuries (Gao et al. 2023). Similarly, withaferin A decreased localized macrophage and neutrophil numbers at doses of 2, 4, and 6 mg/kg in S. aureus ‐induced zebrafish infection model (Murugan et al. 2023). Together, these studies suggest that WS and its active constituent withaferin A mitigate excessive inflammation by inhibiting neutrophil chemotaxis and effector functions. Concurrently, WS appears to restore neutrophil counts in neutropenic conditions, potentially through the regulation of granulopoiesis, although this mechanism requires further investigation. The antioxidant and anti‐inflammatory properties of WS extracts highlight their therapeutic potential in conditions such as arthritis, sepsis, and other inflammatory disorders. However, further studies are needed to elucidate the precise mechanisms underlying WS‐mediated effects on neutrophils to optimize their clinical application.

4.1.2. Eosinophils and Mast Cells

Eosinophils are crucial in combating parasitic infections and participating in the development of allergic diseases such as asthma and allergic rhinitis. A study examined the effect of WS whole‐plant water extract on eosinophils in an OVA‐induced asthma model (Oberholtzer et al. 2008). This study demonstrated that WS (600 mg/kg) decreases eosinophil counts in bronchial lavage fluid and blood smears compared to the untreated asthma group, suggesting the potential of WS as an anti‐inflammatory agent for allergic diseases (Oberholtzer et al. 2008). Similarly, another study investigated the anti‐allergic effects of withaferin A administration at a dose of 2 mg/kg in a pulmonary eosinophilia model (house dust mite, HDM extract‐induced eosinophilic airway inflammation). This study reported a significant reduction in airway eosinophil counts and decreased peri‐bronchial inflammation compared with the HDM control. The effect was partially attributed to lower IL‐25 levels, a cytokine involved in recruiting eosinophils to allergen‐injured sites (Agner et al. 2025). Though the direct role of IL‐25 and the signaling involved needs further investigation. In another study, WS extract (200 and 400 mg/kg) attenuated ovalbumin‐induced airway inflammation in allergic bronchial asthma in Wistar albino rats (Ali et al. 2023). This was associated with a decrease in BALF eosinophils, IgE, IL‐4, TNF‐α, and oxidative/nitrosative stress (Ali et al. 2023). Limited studies conducted so far thus suggest the potential of WS as an anti‐allergic agent, offering promising implications for managing allergic conditions such as asthma and eosinophilic airway inflammation.

Mast cells play a key role in allergic reactions and inflammatory responses by releasing mediators, including histamine, cytokines, and proteases, upon activation. WS has been shown to modulate mast cell activity, particularly reducing degranulation and stabilizing the cell membrane, thus inhibiting the release of pro‐inflammatory mediators. One study demonstrated the effects of WS aqueous root extract (200 and 400 mg/kg) pre‐treatment on allergic inflammation using an acute systemic anaphylaxis mouse model. The results revealed significant mast cell‐stabilizing and anti‐anaphylactic effects in the OVA‐induced allergic mouse model, with a reduction in mortality from 75% in the allergen (OVA) group to 37% at 400 mg/kg and 50% at 200 mg/kg, respectively (Ray 2023). Furthermore, the anti‐anaphylactic activity of methanolic and aqueous whole plant extracts of WS was demonstrated on rat peritoneal mast cells. The extracts were administered at doses of 250, 500, and 750 mg/kg, with the 500 mg/kg methanolic extract showing the greatest inhibition of antigen‐induced histamine release in mesenteric mast cells, comparable to the standard drug prednisolone (Girish and Reddy 2017). These findings collectively highlight the potential of WS as a therapeutic agent for managing allergic reactions, particularly through its mast cell‐stabilizing properties, reduced eosinophil infiltration, antigen‐induced histamine release, antibody production, and protection against anaphylaxis. However, the mechanisms and molecular pathways underlying these effects require further investigation. Addressing these gaps will be crucial to developing WS as a reliable anti‐allergic treatment, advancing from preclinical models to clinical applications.

4.2. Monocytes and Macrophages

Monocytes, a key component of the circulating innate immune system, play a central role in pathogen recognition and clearance. Upon migration into tissues and encountering pathogens, monocytes differentiate into macrophages, potent phagocytic cells essential for host defence. Numerous studies have demonstrated the capacity of WS, including its root extracts and active compounds, to modulate monocyte and macrophage functions. Whole plant alcoholic extract at 100 mg/kg mitigated chemotactic inhibition in experimental aspergillosis model in mice, while enhancing TNF‐α and IL‐1α production and phagocytic activity of peritoneal macrophages, leading to prolonged survival of infected mice (Dhuley 1997, 1998). Similarly, Davis and Kuttan (2000) reported increased macrophage phagocytic activity following administration of methanolic WS root extract (20 mg/dose/animal), further supporting its role as an effective immune stimulant. While oral administration of aqueous WS root extract (300 mg/kg) significantly reduced TNF‐α, IL‐1β, and IL‐6 levels and the number of macrophages and synovial fibroblasts expressing these cytokines in collagen‐induced arthritis (CIA) rats (Khan et al. 2019). Additionally, Withaferin A inhibited inflammatory cytokines in CIA rats and human RA‐FLS (Sheng et al. 2023). In a recent study, WS roots neutral polysaccharide, AP‐1 (100–400 μg/mL), significantly enhanced CD86 expression and M1 polarization of murine macrophage RAW264.7 cells. This was associated with NF‐κB activation and increased pro‐inflammatory mediators, including reactive oxygen and nitric species, TNF‐α, IL‐1β, and IL‐6, toward immune enhancement (Li et al. 2025).

Interestingly, macrophages exhibit dynamic reprogramming between a pro‐inflammatory killing‐M1 state and an anti‐inflammatory healing‐M2 state, regulating inflammatory vs. resolution phases. Alnuqaydan et al. (2022) showed that Withaferin A (WFA 0.25–1.0 μM) suppressed pro‐inflammatory cytokines (TNF‐α, IL‐1β, IL‐6), while enhancing IL‐10 levels in LPS/ConA‐stimulated immune cells, promoting macrophage polarization from the pro‐inflammatory M1 to the anti‐inflammatory M2 phenotype. These findings were corroborated in RAW 264.7 macrophages, where WFA at 25 μg/mL reduced pro‐inflammatory cytokine production (Neog et al. 2018). However, aqueous‐alcoholic root extracts of WS (30 mg/kg) in SRBC‐immunized BALB/c mice induced a Th1‐skewed response by enhancing macrophage activity and the secretion of IL‐12, TNF‐α, and nitrite, indicating immune stimulation under certain conditions (Malik et al. 2007).

In vitro experiments have further elucidated the dual actions of WS. Methanolic root extract (1–256 μg/mL) increased nitric oxide production and iNOS expression in J774 macrophages in a dose‐dependent manner, demonstrating transcriptional‐level immunostimulant (Iuvone et al. 2003). Conversely, ethanolic extract at 1 mg/mL inhibited LPS‐induced NO accumulation in RAW 264.7 cells, suggesting an anti‐inflammatory response (Singh et al. 2007). The same study found that WS inhibited nuclear translocation of NF‐κB and AP‐1, and phosphorylation of IκBα, in PBMCs from both healthy donors and rheumatoid arthritis patients, and suppressed TNF‐α, IL‐1β, and IL‐12p40 production (Singh et al. 2007). These findings highlight a dose‐ and context‐dependent, biphasic immunomodulatory effect of WS.

The biphasic behavior has also been observed with WFA. Oh et al. (2008) demonstrated that WFA (400 nM) downregulated iNOS and NO production in LPS‐stimulated RAW 264.7 macrophages through inhibition of Akt and ERK phosphorylation, thereby blocking NF‐κB activation. In periodontal inflammation models, WFA (50–250 nM) reduced IL‐6, TNF‐α, iNOS, and NO production in response to Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans , suggesting its potential for treating inflammatory diseases such as periodontitis (Noh et al. 2016). Likewise, WFA and withanone at sub‐micromolar concentrations suppressed LPS‐induced cytokines (TNF‐α, IL‐6) in bone marrow‐derived macrophages, confirming their efficacy even at lower doses (Purushotham et al. 2017). Kalpana et al. (2023) showed that WFA (0.1–1.0 μg/mL) activates immune signaling without biasing toward the MyD88‐dependent TLR‐4 pathway, instead engaging the TRIF pathway, thus avoiding the toxic pro‐inflammatory effects associated with LPS. Dubey et al. (2018) reported that WFA (5–20 μM) reduced IL‐1β and IL‐18 secretion in nigericin‐stimulated THP‐1 cells by disrupting NLRP3‐ASC colocalization and inhibiting caspase‐1 activation. In the tumor microenvironment, where M2 macrophages can facilitate tumor progression, WFA (4 μM) induced AIM2 inflammasome activation in M2 macrophages, resembling the M1 phenotype and promoting anti‐tumor immunity (Ngoungoure and Owona 2019). Furthermore, Neog et al. (2018) employed a mannosylated liposomal delivery system to administer WFA (25 μg/mL) to RAW 264.7 macrophages, which suppressed M1‐associated cytokines (IL‐1β, IL‐6, IL‐23, TNF‐α) and enhanced M2 polarization via inhibition of NO, iNOS, and ROS, suggesting WFA regulates macrophage repolarization. The same group demonstrated therapeutic benefits of ML‐WA in adjuvant‐induced arthritis models, where 10 mg/kg WFA reduced inflammation, oxidative stress, bone erosion, and cartilage degradation, while promoting M1 to M2 macrophage transition as indicated by increased CD162 and decreased CD86 expression (Sultana et al. 2017).

The immunostimulatory potential of WFA was also observed in infectious disease models. For example, WFA (0.1–0.5 mg/mL) treatment to Mycobacterium tuberculosis ‐infected macrophages promoted M1 polarization, increased pro‐inflammatory cytokines (IL‐6, IL‐1β, IL‐23), and enhanced T‐cell activation, contributing to greater host resistance (Kumari et al. 2023). In a Leishmania infection model, alcoholic WS leaf extracts and WFA significantly reduced parasite load in peritoneal macrophages, with a concurrent downregulation of IL‐10 and increased IFN‐γ, favoring parasite clearance (Chandrasekaran et al. 2017). Reno‐protective effects of WFA have also been linked to macrophage modulation. In a model of unilateral urethral obstruction (UUO), WFA (1–3 mg/kg) inhibited macrophage‐derived pro‐fibrotic and chemotactic markers (MCP‐1, MIP‐α, MIP‐β), as well as TGF‐β, thereby reducing renal fibrosis (Peddakkulappagari et al. 2019).

Together, these findings demonstrate the complex, context‐dependent immunomodulatory effects of WS and its active constituents (Figure 2). WS exhibits dual response, stimulating immune activity under certain conditions while suppressing it in diverse inflammatory settings, based on its diverse array of phytoconstituents and other undefined factors. While these preclinical findings are promising, further mechanistic studies and in vivo validations are necessary to fully elucidate the therapeutic potential of WS and facilitate its translation into clinical applications.

4.3. Microglial and Kupffer Cells

Microglia, the resident macrophages of the central nervous system (CNS), actively participate in immune surveillance, synaptic remodeling, and respond to injury or infection by releasing inflammatory mediators and phagocytosing cellular debris (Gao et al. 2023; Muzio et al. 2021). WS has demonstrated modulating microglial cell activity and protecting against neurodegenerative processes largely through its antioxidant and anti‐inflammatory properties (Gupta and Kaur 2019). The ethanolic WS root extract (EWSR) and neuroprotectants alone and in combination have successfully restored the levels of antioxidants glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) in rotenone‐induced neurodegeneration in the brain of the rat (Epuri et al. 2023). It further showed reduced microglial activation in the whole brain, indicating the efficiency of EWSR as a neuroprotectant (Lerose et al. 2024). These mechanisms directly contribute to its potential in mitigating neurodegenerative disease progression. WFA has been particularly noted for its ability to attenuate microglial activation and inhibit endothelial cell apoptosis following traumatic brain injury. Treatment with WFA leads to a significant reduction in neuroinflammatory cytokines, including IL‐1β, IL‐6, and TNF‐α, thereby dampening microglial overactivation and preserving neuronal function (Zhou et al. 2020). This highlights WFA relevance not only in trauma‐related injury but also in chronic neuroinflammatory conditions. Withaferin A (0.5 μM) was shown to inhibit LPS‐induced COX‐mediated inflammation in microglial cells by suppressing transcription and translation of pro‐inflammatory mediators and prostaglandin E2 production, extending its relevance to neuroinflammatory conditions (Min et al. 2011). These extracts and their active fractions also restore microglial‐neuronal communication, which is essential for neuronal survival and function (Gupta and Kaur 2019).

In scopolamine‐induced models of neurotoxicity, WS water extract and withanone reversed cytotoxic effects in IMR32 neuronal and C6 glioma cells by normalizing neuronal markers (PSD‐95, GAP‐43) and reducing oxidative stress and DNA damage indicators such as ROS and γH2AX (Zahiruddin et al. 2020). In a middle cerebral artery occlusion (MCAO) model of focal ischemia, prior treatment with a hydro‐alcoholic root extract of WS (300 mg/kg) significantly reduced IL‐6, TNF‐α, and MCP‐1 levels. WS pre‐supplementation was also observed to inhibit MCAO‐induced increase in COX‐2; NF‐kβ proteins and reduce the glutamate levels (Sood et al. 2024). Collectively, these findings underscore the multifaceted role of WS in regulating microglial cell function, reducing neuroinflammation, and offering neuroprotection. The neuroprotective effects of WS and its constituents in MCAO model are promising, but need more investigations with long‐term efficacy and safety to further establish its translational relevance and as a promising adjunct therapy.

Kupffer cells (KCs), the resident macrophages of the liver, play a central role in the immune surveillance of hepatic sinusoids (Zhao et al. 2025). Kupffer cells also mediate inflammatory responses during hepatic pathologies (Dixon et al. 2013; Naito et al. 2004). Non‐alcoholic fatty liver disease (NAFLD), characterized by steatosis, that is, accumulation of fat in the liver, presents increased infiltration of KC, initiating the cascade of inflammatory signaling and disease progression (Dixon et al. 2013). WS has shown considerable therapeutic potential across a range of neurodegenerative and hepatoprotective conditions. WFA (10 mg/kg) protected against GalN/LPS‐induced fulminant hepatitis by inhibiting NLRP3 inflammasome activation, but not NRF2 signaling in macrophages (Xia et al. 2021). Their dual role in both promoting liver damage and supporting tissue repair underscores their significance in hepatic homeostasis and pathology. Future research should aim to clarify the precise molecular mechanisms underlying these effects and further explore the broader clinical applications of WS as hepatoprotective.

4.4. Dendritic Cells

Dendritic cells (DCs) are specialized antigen‐presenting cells that play a pivotal role in initiating and regulating immune responses, serving as the bridge between the innate and adaptive immune systems. Dysregulation of dendritic cell function can contribute to the development and progression of various diseases, including autoimmune disorders, cancers, and chronic infections. Kim et al. (2015) studied the effects of withaferin A on H. pylori ‐induced infection and its impact on IL‐1β production in murine bone marrow‐derived dendritic cells (BMDCs). Withaferin A (100, 250, and 500 nM) decreased IL‐1β production in a dose‐dependent manner. Moreover, withaferin A suppressed H. pylori ‐induced IL‐1β and NLRP3 gene expression. This anti‐inflammatory effect was further supported by suppression of IκB‐α phosphorylation, caspase‐1 cleavage and IL‐1β in BMDCs (Kim et al. 2015). This suggests the potential of withaferin A for conditions such as peptic ulcers, chronic gastritis, and gastric cancer, all of which are associated with H. pylori infection. In another study, the immunosuppressive effects of withaferin A were investigated in the context of allogeneic islet graft transplantation to protect the transplanted grafts. Withaferin A extended islet allograft survival by 80% at concentrations of 0.25 and 0.5 μg/mL, primarily through the inhibition of lymphocyte proliferation and dendritic cell maturation, marked by reduced CD83 expression and HLA‐DR (Kumano et al. 2021). Collectively, these findings suggest that WS has the potential for improving transplantation outcomes by modulating lymphocyte proliferation and dendritic cell maturation. The precise molecular mechanisms through which withaferin A modulates dendritic cell function need to be further elucidated.

4.5. Natural Killer Cells

NK cells, while cytotoxic, are distinct from cytotoxic T cells and are part of the innate immune system. These cells play a crucial role in the immune system through their ability to recognize and eliminate virus‐infected cells and tumor cells without prior sensitization. NK cells also shape the adaptive immune response, making them vital in defending against infections, tumors, and other pathological conditions.

In one study, WS root powder, when administered as a dietary supplement, increased NK cell frequency in the tumor vicinity, reduced the incidence and progression of ovarian tumors in hens, a spontaneous pre‐clinical model of human ovarian cancer (OVCA). The increase in NK cell population was associated with a decrease in the expression of MHC class I chain‐related protein A by the tumor cells. Notably, WS (2 g/kg body weight) was effective in preventing tumor progression and metastasis to distant organs (Barua et al. 2013). This study offers promising evidence of the enhanced antitumor activity of NK cells with WS supplementation. However, the research on this subject is currently limited; the effect of WS on NK cell functions, such as activation, cytokine production, and interactions with other immune cells, remains largely unexplored. The mechanistic link between increased NK cells and reduced MHC class I expression needs a better understanding. The broader immunomodulatory effects of WS on NK cell function, especially across various disease contexts, warrant for therapeutically utilized NK cell activation by WS in preclinical and clinical studies.

5. Immunomodulatory Efficacy of Withania somnifera: Clinical Insights

WS is well known for its adaptogenic and therapeutic properties, which depend on its immunomodulatory and antioxidant properties (Dipankar et al. 2025). More specifically, WS helps the body manage stress by reducing cortisol through the hypothalamus‐pituitary–adrenal (HPA) axis and balancing the immune system (Pandit et al. 2024). Besides its therapeutic applications in Ayurveda for multiple diseases, WS has been the subject of more than 60 clinical trials, as recorded in a clinical trial registry at https://clinicaltrials.gov and Clinical Trials Registry—India (CTRI) and summarized elsewhere (Mikulska et al. 2023). These trials have focused on the effect of WS on a variety of conditions, including rheumatoid arthritis, anxiety, insomnia, hypothyroidism, chronic stress, adaptogenic effects, and more. Here, we have focused on the clinical trials of WS evaluating effects on immune cell number and functions (Table 3). These clinical studies validate the potential of WS to modulate immune responses, reduce inflammation, and boost overall immunity.

TABLE 3.

Clinical trials evaluating the efficacy of Withania somnifera .

Experimental/disease model	Extract/compound dose	Major outcomes	References
Randomized, placebo‐controlled, double‐blind study as complementary medicine for COPD	WS root powder (250 mg twice per day) for 12 weeks of intervention	Significantly reduced inflammation Decreased neutrophils (70% ± 5.5% to 63% ± 6.4%) and MPO activity. Increased NO levels (67 ± 13 –88 ± 16 μM) decreased serum IL‐6 levels, demonstrating increased anti‐inflammatory activity. No effect on serum TNF‐α levels.	Singh et al. (2022)
Randomized, double‐blind, and placebo‐controlled study, an open‐label extension	WS ethanolic root extracts (60 mg) on healthy participants up to 60 days	A significant increase in the frequency of CD16+/CD56+ NK cells (19%) after 30 days of administration Longer 60 days administration led to a significant increase in CD45+, CD3+, CD4+, CD8+, CD19+, and CD16+/56+ counts	Tharakan et al. (2021)
Smaller clinical trial	WS alcoholic root extract (6 mL twice per day) over 96 h	320% increase in CD69 receptor expression, indicating an enhanced activated state of CD56+ NK cells, along with an increase in CD4+ T‐cell population	Mikolai et al. (2009)
Randomized, double‐blind, placebo‐controlled	WS root extract (300 mg twice per day) Safety study after 8 weeks of supplementation	No significant changes in hematological parameters, liver function, or thyroid function No significant changes in neutrophil and platelet counts	Narsing et al. (2021)
Placebo‐controlled, randomized double‐blind pilot clinical trial	WS tablets (500 mg twice per day) along with other herbal formulations containing Tinospora cordifolia, Swasari Ras, and Ocimum sanctum for 7 days	Reduced serum levels of IL‐6 and TNF‐α, likely mitigate the inflammatory cytokine storm	Devpura et al. (2021)
Healthy participants	WS aqueous ethanolic root extracts (500 mg per day) with T. chebula fruit for 28 days	Increased counts of T cells, CD4 cells, NK cells, and an improved CD4:CD8 ratio Enhanced serum levels of IFN‐γ and IgG	Sadhupati et al. (2024)

In a randomized, placebo‐controlled, double‐blind study, WS root powder (250 mg twice daily for 12 weeks) was tested as a complementary medicine for the treatment of chronic obstructive pulmonary disease (COPD) patients. WS add‐on therapy improved pulmonary functioning with reduced chronic inflammation and systemic oxidative stress in COPD patients. WS administration acted as a strong antioxidant, significantly reducing inflammation by lowering neutrophil counts and MPO activity. It also increased plasma NO levels and decreased serum IL‐6 levels, demonstrating increased anti‐inflammatory activity (Singh et al. 2022).

In another clinical trial, a randomized, double‐blind, and placebo‐controlled study was conducted to evaluate the immunomodulatory effects of WS ethanolic root extracts (60 mg) on healthy participants (Tharakan et al. 2021). The study found a significant increase in the frequency of T Cells (CD45+, CD3+, CD4+, CD8+), B cells (CD19+), NK cells (CD16+/CD56+) after 30 days of administration, whereas the placebo group showed a decrease. An open‐label extension up to 60 days led to a significant increase in T cells, B cells and NK cell counts, even in the placebo group (Tharakan et al. 2021). In a smaller clinical trial, the administration of WS alcoholic root extract (6 mL twice per day) over 96 h increased the CD69 receptor expression, indicating an enhanced activated state of NK cells (Mikolai et al. 2009). Consistent with this, the CD4+ T‐cell population was also increased in the participants who received the WS extract (Mikolai et al. 2009). These findings suggest that WS plays a prominent role in enhancing NK cell activity and improving the function of both innate and adaptive immune cells.

A 8‐week randomized, double‐blind, placebo‐controlled clinical study conducted in an Indian population to assess the safety of WS root extract (300 mg twice per day). The study showed no significant changes in hematological parameters, liver function, or thyroid function (Narsing et al. 2021). Additionally, there were no significant changes in neutrophil and platelet counts after 8 weeks of supplementation (Narsing et al. 2021). A placebo‐controlled, randomized double‐blind pilot clinical trial involving COVID‐19 positive patients examined the effects of WS tablets (500 mg twice daily) in conjunction with other herbal formulations (Devpura et al. 2021). The results indicated that patients in the treatment group experienced faster recovery compared to those in the placebo group at Day 7. This was associated with a significant reduction in the levels of IL‐6 and TNF‐α in serum in treatment group after 7 days (Devpura et al. 2021). This suggests that WS supplementation may help mitigate the inflammatory cytokine storm often associated with severe COVID‐19. Another clinical trial by Sadhupati et al. (2024) evaluated the effects of a mixture of T. chebula fruit and WS aqueous ethanolic root extracts (500 mg per day) on healthy participants. The results showed improved functionality of both cell‐mediated and humoral immune responses, including increased counts of T cells, CD4 cells, NK cells, and CD4:CD8 ratio. The formulation also enhanced serum levels of IFN‐γ and IgG, further supporting WS's role in modulating immune activity (Sadhupati et al. 2024). Although the effects of other herbs were not specified in the above trials. In a recent prospective, randomized, double‐blind, placebo‐controlled study, WS root extract (300 mg twice daily) improved overall health and quality of life in elderly men and women over 8 weeks, although its immunomodulatory effects were not investigated (Honnutagi et al. 2026).

These clinical evidences to date suggest that WS has the potential to modulate immune responses, reduce inflammation, and enhance immune cell function, particularly NK cells, CD4+ T‐cells, and other lymphocyte populations. While these clinical studies indicate a promising role for WS in modulating immune responses, they primarily focus on generalized markers such as immune cell counts and cytokine levels. Further research is needed to understand how WS influences immune cell activation, cytokine profiles, and signaling pathways, as well as to assess its long‐term effects on the human immune system. The immune‐boosting function of WS makes it a promising candidate for therapeutic applications in inflammatory conditions, cancer prevention, and viral infections. Detailed studies on immune cell signaling, effector molecule pathways, and the synergistic effects of WS with other therapies will be crucial to unlocking its full therapeutic potential in immune‐related diseases.

6. Current Limitations and Future Directions

WS is one of the most widely utilized herbs in Ayurveda and other traditional texts. In the post‐COVID era, there has been a marked increase in the use of adaptogens, including WS. This growing demand has expanded the commercial utilization of the whole plant and aerial parts, which may exhibit considerable phytochemical variability and raise safety concerns compared with the traditionally recommended root extracts in Ayurveda. Recent preclinical studies using whole‐plant and leaf preparations have reported toxicity, potentially attributable to differences in extraction methods and the use of non‐traditional plant parts (e.g., leaves and stems) (Brendler et al. 2025; Sher et al. 2024). In contrast, consistent with Ayurvedic recommendations, several recent clinical studies have demonstrated that WS root extracts are safe and well‐tolerated (Brendler et al. 2025; Vaidya et al. 2026). In a double‐blind, placebo‐controlled trial, WS root extract (200 mg twice daily for 6 months) was well tolerated, with no adverse effects and no clinically significant changes in hematological, hepatic, renal, or lipid parameters (Vaidya et al. 2026). Similarly, a 12‐month multicenter observational study confirmed the safety of WS root extract (300 mg twice daily), reporting improved health‐related quality of life, reduced serum cortisol levels, and no significant changes in lipid or glucose profiles (Salve et al. 2025).

A major challenge in the global standardization of WS is the variation in quality standards across pharmacopeia, including the Indian Pharmacopoeia (IP), British Pharmacopoeia (BP), and United States Pharmacopeia (USP) (Avula et al. 2025). The lack of comparative data for globally sourced raw materials further complicates quality assurance. Notably, approximately 40%–60% of commercial WS products fail to meet these standards. Additionally, withanolide content varies widely (1%–35%) among standardized extracts due to differences in plant parts and analytical methods. These include low‐concentration extracts (1%–5%), medium‐concentration extracts (e.g., KSM‐66, standardized to 5%–10%), and high‐concentration extracts (e.g., Shoden, including leaf components, standardized up to 35% withanolide glycosides). Importantly, safety data for leaf‐based extracts remain limited. Given that the biological activity of WS is largely driven by withanolides, such variability may contribute to inconsistent efficacy and potential adverse effects. In response to these concerns, the Ministry of Ayush has issued a safety dossier recommending a no‐observed‐adverse‐effect level (NOAEL) for WS root of up to 2000 mg/kg body weight for human consumption (Ashwagandha Safety Dosier 2.0, Ministry of Ayush, India 2024). These findings underscore the urgent need for harmonized global standards addressing plant parts, extraction methods, and safety guidelines.

The efficacy of WS is also significantly influenced by extraction techniques and plant part selection (Kushwaha et al. 2012). Different extraction techniques, such as aqueous, hydro‐alcoholic, and alcoholic methods, yield varying profiles of bioactive compounds. Hydroalcoholic extracts are often preferred, as they capture a broader spectrum of bioactivity by extracting both polar and non‐polar constituents (Dar et al. 2015; Sahoo et al. 2024). Furthermore, the roots, leaves, and seeds of WS possess distinct phytochemical compositions (Shinde et al. 2023). The roots are particularly rich in withanolides, such as Withanolide A and Withaferin A, which are responsible for its immunomodulatory and adaptogenic effects (Shinde et al. 2023). In contrast, the leaves contain higher concentrations of flavonoids and phenolic acids, known for their potent antioxidant properties. These differences highlight the importance of standardizing extraction processes and systematically characterizing the bioactivity of specific plant parts, with detailed mechanistic insights to optimize therapeutic outcomes.

WS demonstrates a multifaceted capacity for immune regulation by modulating pro‐ and anti‐inflammatory cytokines, enhancing phagocytic activity, and supporting immune cell survival. While substantial progress has been made in understanding its effects on innate immune cells such as macrophages and neutrophils, its impact on other immune populations, including microglia, Kupffer cells, dendritic cells, and natural killer cells, remains insufficiently explored. Research on eosinophils and basophils is particularly limited. There is a critical need for integrative transcriptomic and proteomic studies to identify unbiased cytokine networks and signaling pathways underlying WS‐mediated immunomodulation. Furthermore, the specific contributions of individual WS constituents to these immune effects require deeper investigation.

Although preclinical and clinical studies support the efficacy of WS in modulating innate immunity, further targeted research is essential to elucidate its molecular mechanisms. Comprehensive and well‐designed clinical trials are needed to validate its therapeutic potential, establish standardized treatment regimens, and assess long‐term safety across diverse populations and clinical conditions. With continued focused research and development, WS holds considerable promise as a key component of integrative medicine for managing immune‐related disorders.

7. Conclusions

Taken together, WS exhibits significant immunomodulatory potential, encompassing both immunostimulatory and immunosuppressive activities. Its bioactive components, particularly withanolides and withaferin A, mediate diverse immune responses, including enhanced macrophage function, stimulation of natural killer cell activity, and anti‐inflammatory effects through the reduction of pro‐inflammatory cytokine production under various pathological conditions. Though studies on Kupffer cells, dendritic cells, eosinophils, and basophils remain limited, thus requiring further investigations to unravel WS effects on key immune players in more detail. Preclinical and clinical studies substantiate the medical efficacy of this herb in treating a wide range of conditions, such as cancer, autoimmune disorders, infections, and stress‐induced immune dysfunctions. However, significant knowledge gaps remain regarding the molecular mechanisms underlying its immune modulation and the roles of individual phytochemicals. Further studies should focus on elucidating the cellular and molecular interactions influenced by WS, as well as exploring its pharmacokinetics and optimal dosages. Consistent with Ayurvedic uses, WS roots are found to be safe and well‐tolerated in multiple clinical trials up to 12 months. There is still a need for universal standards and safety guidelines for WS parts, extraction methods, therapeutic formulations, and uses to fully realize the potential of WS in integrative medicine.

Author Contributions

Rana Yukta: investigation, methodology, data curation, writing – original draft. Sonalika Gupta: methodology, data curation, formal analysis, investigation, writing – original draft. Gagandeep Kaur: methodology, data curation, investigation. Partha Sarathi Mondal: methodology, data curation, investigation. Milirani Das: methodology, data curation, investigation. Anagha Ranade: investigation, methodology, formal analysis. Alok Kumar Srivastava: conceptualization, formal analysis. Sachin Kumar: conceptualization, methodology, data curation, formal analysis, supervision, funding acquisition, writing – original draft, writing – original draft, writing – review and editing, validation. Manoj Kumar Barthwal: conceptualization, methodology, supervision, project administration, visualization, funding acquisition, writing – review and editing. Madhu Dikshit: conceptualization, methodology, data curation, supervision, resources, project administration, formal analysis, funding acquisition, writing – review and editing.

Funding

This work was supported by CCRAS, HQ‐Proj011/20/2022‐Proj and Science and Engineering Research Board, NSC/2025/000020 and Ministry of Ayush, Government of India, CoES‐12011/5/2022‐SCHEME.

Disclosure

During the finalization of this work, the authors used the Grammarly generative AI tool to improve language and readability. After using this tool, the authors re‐viewed and edited the content as needed and take full responsibility for the content of the publication.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.