LC-MS-Based Phytochemical Profiling of Priyangvadi choorna and its Correlation with Hemostatic Activity

INTRODUCTION

Bleeding is one of the important global health concerns. The commonest causes are injuries, surgeries, obstetrics, and pathological disorders. Traumatic bleeding is the leading cause of preventable death, contributing to nearly 40% of global mortality in early trauma (Watson et al., 2022). Non-cardiac perioperative bleeding accounts for approximately 11.6% of cases within 30 days following surgery (Alderliesten et al., 2024). Globally, bleeding significantly increases the duration of hospital stay and healthcare expenditure and adversely affects clinical outcomes (Leff et al., 2020).

Despite advances in hemostatic management, both local and systemic agents are currently in use with notable limitations. Local hemostatic agents may interfere with wound healing and increase the risk of infection (Achneck et al., 2010; Spotnitz and Burks, 2012). Systemic agents, such as antifibrinolytics, are associated with adverse effects including thromboembolic events, hypersensitivity reactions, and renal complications, particularly in susceptible individuals (Ker et al., 2012; Levi and Hunt, 2015). These challenges emphasize the need for safer, targeted, and biocompatible alternatives, including plant-derived and polyherbal hemostatic formulations.

In Ayurveda, Raktasthambhanopāyas (haemostatic measures) are described in the management of Raktātīpravṛtti (excessive bleeding) and include therapeutic principles such as Sandhāna, Skandhana, Pācana, and Dāhana (Sharma, 2001). Among the formulations employed under Sandhāna therapy, Priyangvadi Choorna is a traditional polyherbal preparation indicated for various bleeding disorders such as epistaxis, oral bleeding, anorectal bleeding, vaginal bleeding, penile bleeding, and wounds caused by sharp instruments, as documented in Yogaratnakara (Shastri, 2010).

The formulation is reported to possess hemostatic, anti-inflammatory, antimicrobial, and wound-healing properties (Sharma et al., 2001; Shastri, 2010). Priyangvadi Choorna is traditionally administered using classical Ayurvedic methods, often with Vasa swarasa. The present work represents the first application of Liquid Chromatography-Mass Spectrometry (LC-MS) for chemical profiling of this formulation. LC-MS analysis revealed the presence of multiple phytoconstituents known for their anti-inflammatory, antioxidant, and cytoprotective activities.

MATERIALS AND METHODS

Priyangvadi Choorna was obtained from GMP Certified Ayurveda pharmacy Khasbag, Belgavi, mentioned in Table 1 (Government of India, Department of AYUSH, 2003) and analysed using LC-MS by standard protocols.

Study conduction

Our study was conducted in Biocyte Research and Development Pvt. Ltd., Sangli, Maharashtra.

Simple Preparation

After purchasing Priyangvadi Choornam in powdered form, 100 mg of the sample was precisely weighed and diluted in 100 mL of methanol. To ensure full extraction of phytochemical contents, the solution underwent sonication for 20 min. After sonication, the solution was filtered to produce an extract clear enough for LC-MS analysis. Analysis of Liquid Chromatography-Mass Spectrometry (LC-MS) was conducted utilizing a Waters Xevo G2-XS QT mass spectrometer alongside a Waters 1525 µ Binary Pump liquid chromatography system. Chromatographic separation was carried out using an Accucore C18 column (50 mm × 4.6 mm, 5 µm particle size; ThermoScientific). The mobile phases consisted of Acetonitrile (solvent B) and 0.1% formic acid in water (solvent A). The gradient elution schedule used was: 95% A and 5% B from 0 to 1 min, transitioning to 50% A and 50% B by 8 min, moving to 5% A and 95% B by 12 min, maintained until 17 min, then returning to 95% A and 5% B by 18 min, which remained until 20 min. Throughout the run, the injection volume stayed at 10 µL, and the flow rate was held steady at 0.5 mL/min.

Mass Spectrometry Parameters

The mass spectrometer functioned in positive and negative Electrospray Ionization (ESI) modes to detect each component present. The collision energy was configured to 20 V, and the capillary voltage was adjusted to 3.0 kV. The collision energy could range from 30 to 90 V. The temperatures for the source and desolvation were held constant at 150°C and 450°C, respectively. The gas flow rate for desolvation was 800 L/h, whereas the cone gas flow rate stood at 50 L/h. Data Gathering Information was obtained using MassLynx software (version 4.1). For analyzing the chemical composition of the plant sample, both positive and negative ionization modes can be used to produce Total Ion Chromatograms (TIC) and Base Peak Intensity (BPI) chromatograms.

Ethical Statement

Not applicable, as this study involved an in vitro phytochemical analysis of a commercial product. No human or animal subjects were used.

Statistical Analysis

Because the study concentrated on analytical profiling without assessing differences between groups, inferential statistical approaches were not applicable.

RESULTS

The LC-MS analysis of Priyangvadi Choorna was performed in both negative and positive ionization modes. Figure 1 shows the negative mode LC-TIC and BPI chromatograms, while Figure 2 depicts the positive mode LC-TIC and BPI chromatograms. The analysis revealed the presence of multiple bioactive phytochemicals, including flavonoids (Tectochrysin, Kaempferol, Liquiritigenin, Hesperetin, Ononin), alkaloids (Sanguinarine), phenolic acids (Ellagic acid, 1,3-Benzenedicarboxylic acid), and other compounds such as Thalidomide and 4-Methylumbelliferone.

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Several of these constituents, including Ellagic acid, Hesperetin, Sanguinarine, and Tectochrysin, have been reported to modulate platelet activation, thrombus formation, and vascular health. Ellagic acid acts as a haemostatic agent by activating Factor XII in the intrinsic coagulation pathway. Sanguinarine modulates the GPVI signaling pathway and regulates thromboxane, thereby controlling platelet activation. Hesperetin and Tectochrysin inhibit platelet aggregation and inflammation, helping maintain endothelial stability and preventing thrombosis. Additionally, 4-Methylumbelliferone (4-MU) and Biochanin A exert antioxidant and anti-inflammatory effects, contributing to vascular protection and maintaining haemostatic balance.

These findings support the traditional use of Priyangvadi Choorna in wound healing and bleeding management. Table 2 summarizes the phytocomponents detected in the acetone-extracted Priyangvadi Choorna by LC-MS, highlighting their potential role in modulating platelet function, stabilizing the endothelium, and maintaining haemostatic equilibrium.

DISCUSSION

LC-MS (liquid chromatography-mass spectrometry) analysis identified several compounds with potential haemostatic properties, including flavonoids, alkaloids, and phenolic acids such as tectochrysin, hesperetin, kaempferol, biochanin A, 4-methylumbelliferone, ononin, columbianetin, and ellagic acid. Ellagic acid enhances coagulation by activating Hageman factor (factor XII) in the intrinsic pathway, resulting in increased serine protease activity. It is a naturally occurring polyphenolic compound and is also known for its antiproliferative, wound-healing, and antioxidant properties (Gopalakrishnan et al., 2014; Umesalma and Sudhandiran, 2011). Sanguinarine, an alkaloid belonging to the benzophenanthridine class, exhibits anti-inflammatory, antioxidant, and anticancer activities. It attenuates collagen-induced platelet activation and thrombus formation by modulating the GPVI pathway, reducing intracellular calcium mobilization and integrin αIIbβ3-mediated outside-in signaling (Shu et al., 2021). Biochanin A, an isoflavone, enhances vasorelaxation in coronary arteries and may indirectly support haemostasis by maintaining vascular tone. It also exhibits neuroprotective, anti-inflammatory, anticancer, and antioxidant properties through modulation of NF-κB, AP-1, and JAK/STAT signaling pathways (Feng and Lai, 2023; Sobotková et al., 2009). Tectochrysin, a methylated flavonoid, demonstrates antithrombotic activity by inhibiting NF-κB signaling, thereby reducing platelet aggregation and pro-inflammatory cytokine expression. Additionally, it exhibits anti-inflammatory, antibacterial, hepatoprotective, neuroprotective, and antioxidant effects (Lee et al., 2003; Lu et al., 2020). Liquiritigenin reduces oxidative stress and extracellular matrix accumulation under hyperglycaemic conditions by suppressing NF-κB and NLRP3 inflammasome activation, thereby promoting wound healing (Zhu et al., 2018). 4-Methylumbelliferone exhibits anti-inflammatory effects by regulating MAPK, SYK, and NF-κB signaling pathways. Although it does not directly influence coagulation, it helps maintain endothelial integrity, which is essential for vascular homeostasis (Wang et al., 2022). Kaempferol prevents platelet activation and thrombus formation while offering vascular protection. It also possesses anti-inflammatory, antioxidant, and anticancer activities (Lee et al., 2018; Liu et al., 2020). Ononin reduces inflammatory cytokine production by downregulating inflammatory mediator mRNA expression via inhibition of the NOD1/RIP2/NF-κB signaling pathway (Yu et al., 2023). Columbianetin exhibits significant anti-inflammatory and antioxidant effects in immune cells (Lu et al., 2018). Hesperetin, a bioflavonoid, exhibits antioxidant, anti-inflammatory, antihypertensive, antiatherogenic, and anticancer properties. It reduces oxidative stress, DNA damage, and cellular apoptosis by modulating p38 MAPK, NF-κB, and caspase-3 signaling pathways, thereby reducing doxorubicin-induced cardiotoxicity (Rehman et al., 2024). Thalidomide exhibits procoagulant effects under specific conditions by increasing tissue factor expression and thrombin generation in endothelial cells and also possesses anti-inflammatory and anti-angiogenic properties (Akter et al., 2022; Chen et al., 2009; Qiao et al., 2017). Isophthalic acid (1,3-benzenedicarboxylic acid) exhibits antibacterial and antifungal activities (Hamsalakshmi et al., 2021). Coumarins are naturally occurring compounds with diverse biological properties, including antibacterial, anti-inflammatory, and anticancer activities (Garrard, 2014; Lake, 1999; Önder, 2020). Laxapur, a flavonoid compound, acts as a potent free-radical scavenger and exerts anti-inflammatory effects by inhibiting NF-κB signaling and reducing pro-inflammatory mediators such as iNOS and COX-2 (Egbuna et al., 2020; Lin et al., 2009). Khellin is primarily known for its vasodilatory and smooth muscle relaxant properties (National Center for Biotechnology Information [NCBI], 2025a). Constictic acid, a lichen-derived depsidone, exhibits antimicrobial and antioxidant activities (NCBI, 2025b). Antipyrine, a pyrazolone derivative, exhibits analgesic, antipyretic, anti-inflammatory, and platelet-inhibitory effects through inhibition of cyclooxygenase-mediated prostaglandin synthesis (NCBI, 2025c). Tryptophan, an essential amino acid, serves as a precursor for bioactive molecules such as melatonin and kynurenine, contributing to antioxidant and immunomodulatory effects (Ghazaghi et al., 2024). 5-Methoxytryptamine, a serotonin derivative, exhibits free-radical scavenging activity and suppresses inflammation via cytokine modulation (NCBI, 2025d; Tan et al., 1993).

CONCLUSION

A detailed phytochemical investigation of Priyangvadi Choorna through LC-MS analysis revealed the presence of 454 compounds, among them 223 are unknown molecules which may exhibit coagulative properties. The known compounds, particularly those associated with haemostasis, inflammatory control, and wound healing. Principal ingredients, including Ellagic acid, Sanguinarine, Hesperetin, Tectochrysin, and Liquiritigenin, indicated established functions in augmenting coagulation pathways and maintaining endothelial integrity. No sufficient study has been conducted for particularly for haemostatic activity in many compounds found in Priyangvadi Choorna. These results illustrate that the traditional usage of Priyangvadi Choorna is a treatment for bleeding disorders, inflammatory diseases, and wound healing issues. The findings further underscore its promise as a natural, plant-derived treatment for the development of innovative haemostatic medicines.