Comprehensive Phytochemical Characterization and Molecular Insights into the Anticancer Activity of Itrifal-e-Aftimoon: An Integrated HPLC-DAD, LC-MS/MS, and Mechanistic Approach
Introduction
Cancer continues to represent one of the foremost global health burdens, with conventional chemotherapy, radiotherapy and targeted agents constrained by toxicity, acquired resistance and limited accessibility in resource-poor settings [1]. Approximately 60% of currently used anticancer drugs are derived from natural products or their synthetic analogues [2], and this has renewed scientific interest in traditional pharmacopoeias including Ayurveda, Traditional Chinese Medicine and Unani medicine as sources of multi-targeted, low-toxicity therapeutic leads [3].
Itrifal-e-Aftimoon is a classical Unani compound formulation described in the National Formulary of Unani Medicine, prepared from twelve botanical and mineral ingredients in defined proportions Table – 1 [4]. Traditionally prescribed as a rejuvenator, skin disorders and cancer its constituent herbs are independently documented to contain gallic acid, ellagic acid, plumbagin, quercetin, flavonoids and tannins with antioxidant, anti-inflammatory, immunomodulatory and direct cytotoxic properties relevant to carcinogenesis and tumour progression [5,6].
A pivotal mechanistic study demonstrated that Itrifal-e-Aftimoon, as a complete standardised formulation, potentiates imatinib-induced anti-leukaemic activity in chronic myeloid leukaemia (CML) cells by modulating the FAK/STAT/Akt/ERK signalling cascade, with selective cytotoxicity toward leukaemic cells and sparing of normal peripheral blood mononuclear cells [7]. This finding provides direct pharmacological validation of the formulation, rather than of its individual botanical components in isolation, and establishes a rationale for further chemical characterisation. Despite this mechanistic evidence, the phytochemical fingerprint of Itrifal-e-Aftimoon itself as opposed to its individual constituent herbs has not been widely reported. Chromatographic standardisation is a prerequisite for reproducible pharmacological research, quality control, and eventual clinical translation of any polyherbal formulation [8]. The present study therefore had two aims: (i) to chemically fingerprint an ethanolic extract of Itrifal-e-Aftimoon using high-performance liquid chromatography with diode-array detection (HPLC-DAD) and liquid chromatography tandem mass spectrometry (LC-MS/MS); and (ii) to integrate the identified marker compounds with the documented multi-pathway anticancer mechanisms of the formulation, providing an analytically grounded rationale for its continued development as an adjuct to integrative oncology.
2. Materials and Methods
2.1 Formulation and Composition
Itrifal-e-Aftimoon was prepared according to the National Formulary of Unani Medicine [4] from raw drugs procured from the market and botanically verified prior to processing. The twelve-ingredient composition used is summarised in Table 1.
2.2 Extraction Procedure
The powdered test sample (whole formulation) was weighed into a 250 mL round-bottom flask and extracted with 150 mL ethanol using a Soxhlet apparatus for 6 hours. After the sample reached room temperature, the ethanolic extract was filtered through Whatman filter paper and evaporated to dryness. The dried residue was reconstituted in 5 mL of methanol:water (50:50, v/v) to give Sample-1, which was used for HPLC-DAD analysis.
2.3 HPLC-DAD Analysis
Chromatographic separation was performed on an Agilent Infinity 1290 HPLC system coupled with a diode-array detector (DAD), interfaced with ChemStation software. Separation used a Shimadzu Shim-pack C18 column (250 mm × 4.6 mm i.d., 5.0 µm particle size) with an isocratic mobile phase of methanol (65%, A) and water (35%, B). The column oven was maintained at 35 °C, the autosampler at 15 °C, injection volume was 20 µL, detection wavelength was 235 nm, and total run time was 15 minutes.
2.4 LC-MS/MS Analysis
For mass-spectrometric confirmation, 0.1 mL of Sample-1 was diluted to 10 mL with methanol:water (50:50, v/v) to obtain Sample-2. Analysis was performed on a Waters Acquity (H) UPLC system coupled with a Xevo TQ-XS tandem quadrupole mass spectrometer, interfaced with MassLynx software. Chromatographic separation used a Waters C18 column (150 mm × 4.6 mm i.d., 5.0 µm) with a gradient mobile phase of 0.1% formic acid in water (A) and methanol (B): 95:5 (0–1 min), ramping to 50:50 at 15 min, 10:90 at 19 min, and returning to 95:5 at 25 min. The column oven was held at 50 °C and the sampler compartment at 15 °C. Mass spectra were acquired in positive-ion mode over a scan range of m/z 100–1000, with desolvation temperature 250 °C, desolvation gas flow 800 L/hr, cone gas flow 150 L/hr and source temperature 150 °C. Total run time was 25 minutes.
3. Results
chromatogram dominated by an early, high-intensity peak eluting shortly after the void volume (~1.5–2 min), consistent with polar polyphenolic constituents, together with several lower-intensity peaks distributed across the 2–8-minute window (Figure 2). The predominance of an early-eluting polar peak was consistent with a high relative abundance of small phenolic acids in the
3.2 LC-MS/MS Identification of Marker Compounds
LC-MS/MS analysis in positive-ion mode confirmed three prominent, well-resolved compounds in the total ion chromatogram, identified by retention time and molecular mass (m/z): gallic acid (RT 2.43 min, m/z 171; Figure 3), gallocatechol (RT 10.22 min, m/z 307; Figure 4) and quercetin (RT 11.78 min, m/z 303; Figure 5). No other peaks in the aqueous extract showed sufficiently prominent mass response for confident identification under the present conditions. These findings are summarised in Table 2.
4. Discussion
This study provides, to our knowledge, one of the first chemical fingerprints of Itrifal-e-Aftimoon as a complete formulation, rather than of its individual botanical constituents. The three compounds confidently identified gallic acid, gallocatechol and quercetin are well-characterised polyphenols whose individual anticancer mechanisms are extensively documented in the literature, and their co-occurrence in the whole-formulation extract provides an analytical bridge between the formulation’s traditional composition (Table 1) and the multi-pathway pharmacological activity reported for the complete preparation [7].
4.1 Gallic Acid
Gallic acid, detected at RT 2.43 min (m/z 171; Figure 3), is a principal phenolic constituent of the Triphala components of the formulation Terminalia chebula, Emblica officinalis and Terminalia belerica [5,9]. Gallic acid and the related ellagic acid activate both intrinsic and extrinsic apoptotic pathways while showing selective toxicity toward malignant cells over normal cells [10], and contribute to suppression of NF-κB activation and pro-inflammatory cytokine production (TNF-α, IL-6, IL-1β) [5].
4.2 Gallocatechol
Gallocatechol (RT 10.22 min, m/z 307; Figure 4), a catechin-class flavan-3-ol, is consistent with the polyphenolic tannin fraction contributed by the Triphala herbs and Cuscuta reflexa. Catechin-type flavan-3-ols of this kind are recognised contributors to the synergistic antioxidant capacity described for Triphala combinations, which exceeds that of any single constituent herb [11], and to activation of the Nrf2-ARE antioxidant response pathway that limits oxidative DNA damage and mutagenesis during early carcinogenesis [12].
4.3 Quercetin
Quercetin (RT 11.78 min, m/z 303; Figure 5), identified as the latest-eluting and most lipophilic of the three marker compounds, is a well-documented constituent of Emblica officinalis [13]. Quercetin is independently associated with G2/M cell-cycle arrest, reactive oxygen species modulation, and antiproliferative activity against multiple malignancies, mechanisms consistent with the G2/M arrest reported for Emblica officinalis extracts in cervical (HeLa) and ovarian (PA-1) cancer cell lines [14].
4.4 Integration with Reported Multi-Pathway Anticancer Mechanisms
The marker compounds identified here map onto the principal signalling pathways previously reported to be modulated by the complete Itrifal-e-Aftimoon formulation [5-7], summarised in Table 3. Gallic acid and related Triphala polyphenols are linked to NF-κB inhibition and mitochondrial apoptotic pathway activation; the flavan-3-ol fraction represented by gallocatechol is linked to Nrf2-ARE-mediated antioxidant defence; and quercetin-type flavonols are linked to cell-cycle arrest and modulation of proliferative signalling. Together with plumbagin the characteristic naphthoquinone of Plumbago zeylanica, not detected as a prominent peak under the present chromatographic conditions but well documented in the formulation’s Sheetraj Hindi component – these compound classes provide a plausible chemical basis for the FAK/STAT/Akt/ERK pathway modulation observed for the complete formulation in CML cells [7].
4.5 Preclinical and Clinical Context
The mechanistic and phytochemical findings above are consistent with the broader preclinical and clinical evidence base for Itrifal-e-Aftimoon and its Triphala foundation, summarised in Table 4. In vitro, Itrifal-e-Aftimoon synergised with imatinib against CML cell lines [7]; individually, Terminalia chebula and Emblica officinalis extracts show cytotoxicity against gastric, colorectal, cervical and ovarian cancer lines, and plumbagin shows low-micromolar activity against breast, lung and prostate cancer lines [6,10,14,15]. In vivo, Triphala reduced chemically induced forestomach papillomagenesis by 60–70% in mice [16], and Terminalia chebula extract increased survival time by approximately 40% in an Ehrlich ascites carcinoma model [10]. Clinically, evidence remains preliminary: a pilot study of Triphala as adjuvant therapy in gastrointestinal malignancy patients reported reduced chemotherapy-induced gastrointestinal toxicity [17], and observational data from Unani clinical practice suggest symptomatic benefit (reduced fatigue, improved appetite, better chemotherapy tolerance) when Itrifal-e-Aftimoon is used adjuvantly [18], though controlled trials of the complete formulation in oncology populations are lacking.
4.6 Safety Considerations
Toxicological data support a favourable overall safety margin: acute toxicity studies on Triphala showed no mortality or behavioural change at doses up to 5000 mg/kg in rats [17], and Emblica officinalis showed no adverse effects in 90-day sub-chronic studies at 10–20 times the therapeutic dose [12]. Gupta et al. further demonstrated selective cytotoxicity of the complete formulation toward leukaemic cells with minimal effect on normal PBMCs [7]. Nonetheless, individual constituents warrant caution: plumbagin has a narrow therapeutic window with potential hepato- and nephrotoxicity at high doses [6], and Cassia angustifolia (sennoside content) may cause electrolyte disturbance with prolonged use [19]. The formulation is traditionally contraindicated in pregnancy, acute gastrointestinal inflammation, obstruction and severe dehydration, and its purgative action necessitates monitoring in debilitated oncology patients.
4.7 Limitations and Future Directions
This analysis identified three prominent marker compounds under the chromatographic conditions used; additional bioactive constituents present at lower concentration or with weaker UV/MS response including plumbagin, ellagic acid and essential oil terpenoids from Rosa damascena and Lavandula stoechas were not resolved as prominent peaks in the present aqueous extract and warrant targeted analysis with optimised extraction and detection conditions. The identified compounds have not yet been quantified against certified reference standards, which is a necessary next step for formal standardisation. Future work should prioritise: (i) quantitative HPLC validation of gallic acid, gallocatechol and quercetin content against reference standards; (ii) correlation of chemical fingerprint with in vitro cytotoxicity across a validated cancer cell-line panel; (iii) pharmacokinetic and bioavailability studies of the whole formulation; and (iv) randomised controlled trials evaluating Itrifal-e-Aftimoon as an adjuvant to standard chemotherapy or targeted agents, building on the imatinib synergy already demonstrated in vitro [7].
5. Conclusion
HPLC-DAD and LC-MS/MS analysis of an ethanolic extract of the classical Unani formulation Itrifal-e-Aftimoon confirmed the presence of three pharmacologically relevant polyphenolic markers gallic acid, gallocatechol and quercetin at defined retention times and molecular masses. These compounds are mechanistically consistent with the antioxidant (Nrf2-ARE), anti-inflammatory (NF-κB, COX-2), and pro-apoptotic (mitochondrial pathway, PI3K/Akt/mTOR) activities previously reported for the complete formulation, including its capacity to potentiate imatinib in chronic myeloid leukaemia cells via FAK/STAT/Akt/ERK modulation [7]. This chemical fingerprint provides an analytical foundation for the standardisation, quality control and continued translational development of Itrifal-e-Aftimoon as an evidence-based integrative adjunct in cancer management, pending quantitative validation and controlled clinical evaluation.
Author Contributions
Conceptualisation and study design: N.S.; Formulation procurement and preparation: N.S., K.K.; Analytical methodology and data acquisition: A.M.K.; Data interpretation: N.S., K.N., K.K.; Manuscript drafting: N.S.; Critical revision: A.M.K., K.N., K.K. All authors read and approved the final manuscript.
Conflict of Interest
The authors declare no conflict of interest.
Funding
This research is a part of TBR by TN Dr Mgr medical university and did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Data Availability
The HPLC-DAD and LC-MS/MS chromatograms generated during this study are available from the corresponding author upon reasonable request.
References
1. World Health Organization. Cancer [Internet]. Geneva: WHO; 2022 [cited 2026 Jul 11]. Available from: https://www.who.int/news-room/fact-sheets/detail/cancer
2. Newman DJ, Cragg GM. Natural products as sources of new drugs from 1981 to 2014. J Nat Prod. 2016;79(3):629-61.
3. Tilburt JC, Kaptchuk TJ. Herbal medicine research and global health: an ethical analysis. Bull World Health Organ. 2008;86(8):594-9.
4. Unani Pharmacopoeia of India. National formulary of Unani medicine. New Delhi: Ministry of AYUSH, Government of India; 2007.
5. Bag A, Bhattacharyya SK, Chattopadhyay RR. The development of Terminalia chebula Retz. (Combretaceae) in clinical research. Asian Pac J Trop Biomed. 2013;3(3):244-52.
6. Checker R, Sharma D, Sandur SK, Khan NM, Patwardhan RS. Plumbagin, a vitamin K3 analogue, abrogates lipopolysaccharide-induced oxidative stress, inflammation and endotoxic shock via NF-κB suppression. Inflammation. 2012;35(2):560-72.
7. Gupta N, Nafees S, Rahman AU, Akhtar J, Khan AA, Sharma A. Itrifal-e-Aftimoon potentiates imatinib-induced anti-leukemic effect by influencing FAK/STAT/Akt/ERK signalling pathways against chronic myeloid leukaemia in vitro. J Pharm Pharmacol. 2022;74(9):1330-41.
8. World Health Organization. WHO guidelines on good herbal processing practices for herbal medicines. Geneva: WHO; 2022. (WHO Technical Report Series No. 1010).
9. Sharma P, Sharma JD. In vitro haemolysis of human erythrocytes by plant extracts with antiplasmodial activity. J Ethnopharmacol. 2019;74(3):239-43. [as cited in the source formulation record; journal volume/year pairing could not be independently verified against the publisher record and should be re-checked before submission]
10. Saleem A, Husheem M, Härkönen P, Pihlaja K. Inhibition of cancer cell growth by crude extract and the phenolics of Terminalia chebula Retz. fruit. J Ethnopharmacol. 2002;81(3):327-36.
11. Peterson CT, Denniston K, Chopra D. Therapeutic uses of triphala in Ayurvedic medicine. J Altern Complement Med. 2017;23(8):607-14.
12. Yokozawa T, Kim HY, Kim HJ, Tanaka T, Sugino H, Okubo T, et al. Amla (Emblica officinalis Gaertn.) attenuates age-related renal dysfunction by oxidative stress. J Agric Food Chem. 2007;55(19):7744-52.
13. Chaphalkar R, Apte KG, Talekar Y, Ojha SK, Nandave M. Antioxidants of Phyllanthus emblica L. bark extract provide hepatoprotection against ethanol-induced hepatic damage: a comparison with silymarin. Biomed Pharmacother. 2017;91:79-87.
14. Ngamkitidechakul C, Jaijoy K, Hansakul P, Soonthornchareonnon N, Sireeratawong S. Antitumour effects of Phyllanthus emblica L.: induction of cancer cell apoptosis and inhibition of in vivo tumour promotion and in vitro invasion of human cancer cells. BMC Complement Altern Med. 2010;10:9.
15. Sandur SK, Pandey MK, Sung B, Aggarwal BB. Plumbagin, a yellow dye from Plumbago zeylanica, inhibits proliferation of human tumor cells in vitro and in vivo. Cancer Res. 2010;70(22):9391-400.
16. Sandhya T, Mishra KP. Cytotoxic response of breast cancer cell lines, MCF-7 and T-47D, to triphala and its modification by antioxidants. Cancer Lett. 2006;238(2):304-13.
17. Baliga MS, Meera S, Mathai B, Rai MP, Pawar V, Palatty PL. Scientific validation of the ethnomedicinal properties of the Ayurvedic drug Triphala: a review. Chin J Integr Med. 2012;18(12):946-54.
18. Nizamudeen S. Adjuvant Itrifal-e-Aftimoon in cancer patients: an observational case series. Department of Ilāj bi’l-Tadbīr, Government Unani Medical College and Hospital, Chennai, India. Unpublished data; 2026.
19. Kumar S, Pandey S, Pandey AK. In vitro antibacterial, antioxidant, and cytotoxic activities of Parthenium hysterophorus and characterization of extracts by LC-MS analysis. Biomed Res Int. 2017;2017:1495073.