Inclusion criteria.

Participants of either gender between 25–60 years of age, diagnosed with moderate to high ASCVD risk (10-year ASCVD risk ≥7.5%) dyslipidemia in accordance with the 2019 ACC/AHA Guidelines for the Primary Prevention of Cardiovascular Disease and indicated for statin therapy, will be considered eligible for inclusion. Eligible individuals must have a Body Mass Index (BMI) ranging from 18.5 to 34.9 kg/m², provide written informed consent after adequate understanding of study details, and express willingness to adhere to the intervention protocol for a 12-week duration.

Exclusion criteria.

Exclusion criteria include the use of lipid-modifying agents (other than statins), including Ayurvedic or nutritional supplements, within four weeks prior to screening. Individuals with low ASCVD risk not requiring statin therapy, those on systemic corticosteroids or hormone therapies within six weeks of screening, and pregnant or lactating women will be excluded. Smokers, individuals with poorly controlled diabetes mellitus (HbA1c > 9%), significant hepatic or renal impairment (including total bilirubin, AST, ALT, or alkaline phosphatase levels >2 × the upper normal limit, or serum creatinine >1.2 mg%), and those with a history of acute myocardial infarction or cardiac arrest within the previous 24 months are ineligible. Participants with musculoskeletal conditions limiting exercise, uncontrolled hypertension (systolic ≥160 mmHg or diastolic ≥100 mmHg), and those with co-morbidities potentially affecting study participation; such as myopathies, chronic pulmonary or endocrine diseases, systemic infections, arrhythmias, neurological or psychiatric illnesses—will be excluded. Active alcohol or substance abuse, or history thereof in the last six months, and any other medical or psychosocial condition deemed by the investigator to interfere with study integrity or participant safety, will also constitute grounds for exclusion.

Statin Therapy.

The statin prescribed in both groups will be Atorvastatin, administered once daily 30 minutes before dinner. The dosage—20 mg for moderate ASCVD risk and 40 mg for high ASCVD risk—will be determined based on baseline ASCVD risk assessment and clinical judgement by the consulting modern medicine physician on the research team, in accordance with the ACC/AHA 2019 guidelines and presence of comorbidities such as cardiovascular disease or diabetes mellitus.

Study Drug and Quality Control.

The trial intervention, Trikatu, has been procured from Good Manufacturing Practice (GMP) -certified Ayurvedic manufacturing pharmacies, following standard procedures for manufacturing of Churna (powder), prescribed in the Ayurvedic formulary of India (AFI). [9] All raw materials are botanically authenticated prior to processing, and quality assessment is performed in accordance with Ayurvedic pharmacopeial standards, including evaluation of relevant physicochemical parameters. Batch-to-batch consistency is ensured through standardized manufacturing processes and quality control protocols, thereby maintaining uniformity of the intervention throughout the study.

The phytochemical composition of Trikatu and its constituent drugs has been well documented in previous studies. Piperine, derived from Piper nigrum and Piper longum, is reported to be one of the principal bioactive constituents and is commonly used as a marker compound for quality evaluation of Trikatu. [10]

The dosage of Trikatu used in the present study was selected based on classical Ayurvedic dosage recommendations and previously published preclinical safety studies. Experimental toxicity evaluations have reported that Trikatu is well tolerated at therapeutic and higher dose ranges in animal models. [7,11]

Criteria for discontinuing or modifying the allocated intervention or comparator will include the occurrence of serious adverse events, clinically significant laboratory abnormalities, participant request for withdrawal, evidence of disease progression or intolerance to the study medication, or the investigator’s clinical judgment that continuation may pose undue risk to the participant’s health.

The use of any other lipid-lowering agents—including Ayurvedic or modern medicines, herbal supplements, or nutraceuticals such as omega-3 fatty acids—is prohibited during the trial to avoid confounding effects on lipid levels and gut microbiota composition. Routine medications for stable co-morbid conditions such as diabetes and hypertension, as well as short-term treatments for emergent conditions like flu or infections, may be continued if deemed necessary and approved by the study investigator, provided they are not known to significantly alter gut microbial ecology.

Primary outcome.

The primary efficacy endpoint is the change in fasting low-density lipoprotein cholesterol (LDL-C) from baseline to 12 weeks, assessed as both percent change and absolute (mean) change, in participants receiving Trikatu compared with placebo, when administered as an add-on to standard statin therapy.

Secondary outcomes.

Secondary efficacy endpoints include the absolute change from baseline in fasting LDL-C at 12 weeks; the percent change from baseline in total cholesterol, high-density lipoprotein cholesterol, and triglyceride levels at 12 weeks; and the proportion of participants achieving fasting total cholesterol levels below 200 mg/dL at the end of the intervention period.

Secondary endpoints also include changes from baseline in gut microbiota composition at week 12, assessed using shotgun metagenomic sequencing of fecal samples. Total microbial DNA will be extracted using validated commercial kits following manufacturer-recommended protocols. Libraries will be prepared and sequenced on a next-generation sequencing platform with a target sequencing depth sufficient to ensure robust taxonomic and functional profiling.

Bioinformatic analysis will be conducted using standard, validated pipelines for quality control, host DNA removal, taxonomic profiling, and functional annotation. Microbial diversity will be evaluated using α-diversity indices (Shannon and Simpson indices) and β-diversity measures, while differential relative abundance of key bacterial taxa will be assessed. Functional metabolic pathway analysis relevant to lipid metabolism and inflammation will be predicted using curated reference databases.

Changes in gut microbiota–derived short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, will be quantified from fecal samples using gas chromatography–mass spectrometry (GC–MS) following standard derivatization procedures. SCFA concentrations will be expressed as μmol/g of fecal matter and analyzed to evaluate shifts in microbial metabolic activity associated with the intervention.

Changes in metabolic biomarkers such as adiponectin, ghrelin, apolipoprotein A1, and apolipoprotein B; and changes in inflammatory markers including high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) are also included as secondary outcome measures. Role of intervention on Glycemic parameters will be evaluated through changes in fasting blood glucose, HbA1c, and fasting insulin levels, with insulin resistance estimated using the homeostatic model assessment for insulin resistance (HOMA-IR). Resting blood pressure (measured in triplicate) will be assessed at baseline, week 6, and week 12. Safety and adherence will be monitored throughout the study using structured compliance forms completed at each visit, along with systematic documentation of all participant-reported adverse events (AEs) or adverse drug reactions (ADRs).

Sequence generation.

Data collection methods.

All trial data will be systematically collected using standardized Case Report Forms (CRFs) and electronic Case Report Forms (e-CRFs) specifically designed for this study. The CRFs capture detailed information on participant demographics, medical history, intervention adherence, outcome measures, adverse events, and laboratory findings.

Study investigators, co-investigators, and designated research personnel underwent comprehensive training sessions on the study protocol, data collection procedures, and source documentation requirements prior to initiation of participant recruitment. This training was aimed at ensuring consistency in data capture, adherence to protocol-specific timelines, and compliance with Good Clinical Practice (GCP) standards. All laboratory investigations, including lipid profile, inflammatory markers, glycemic indices, and gut microbiome analysis, will be conducted in NABL-accredited laboratories using validated instruments and standardized protocols. The gut microbiota analysis will utilize metagenomic sequencing and SCFA profiling, conducted under quality-controlled conditions.

To promote participant retention and ensure complete follow-up, regular reminders via phone calls or messages will be used to maintain engagement and schedule visits. Study staff will maintain a supportive and responsive communication channel to address concerns, encourage adherence, and reinforce the importance of follow-up assessments. For participants who discontinue or deviate from the intervention protocol, efforts will be made to collect outcome data at all subsequent scheduled visits, including primary and secondary endpoints such as lipid profile, inflammatory markers, glycemic indices, and gut microbiota composition. Reasons for withdrawal or deviation will be documented in detail, and participants will be encouraged to complete end-of-study assessments, even if they do not continue the assigned treatment.

Data accuracy will be further supported by regular monitoring, cross-verification of source documents with CRFs, and automated validation checks embedded in the e-CRF platform.

Data management.

Data will be coded, de-identified, and stored securely with restricted access. Regular data audits will be conducted to maintain quality.

Statistical methods.

A comprehensive Statistical Analysis Plan (SAP) will be finalized prior to unblinding the randomization code. All analyses will be conducted following the modified intention-to-treat (mITT) principle, including all randomized participants who completed the intervention period and had evaluable data for gut microbiota, metabolic and inflammatory markers, and lipid profile assessments at the 84th day. Per-protocol (PP) analyses will also be performed as supportive. Continuous outcomes will be summarized using means, standard deviations, medians, interquartile ranges, and minimum/maximum values. Categorical variables will be presented as frequencies and percentages. No imputation will be applied for missing data; all analyses will be based on observed data.

For the primary outcome (change in fasting LDL-C from baseline to 12 weeks, expressed as percent and absolute change), between-group comparisons will be conducted using analysis of variance (ANOVA) when baseline LDL-C values are comparable between groups. In the presence of baseline variability, analysis of covariance (ANCOVA) will be employed, with treatment group as the fixed effect and baseline LDL-C included as a covariate to adjust for baseline differences.

Similarly, for secondary continuous outcomes measured at baseline and 12 weeks (total cholesterol, HDL-C, triglycerides, metabolic biomarkers, inflammatory markers, glycemic parameters), ANOVA will be applied when baseline values are balanced, while ANCOVA adjusted for baseline values will be used if baseline variability is observed.

For outcomes assessed at multiple time points, repeated-measures analysis of variance (r-ANOVA) will be used when assumptions of sphericity and normality are satisfied. If r-ANOVA assumptions are not met, linear mixed-effects models with fixed effects for treatment group, time, and group-by-time interaction, will be employed as an alternative.

Prespecified subgroup analyses will explore differential effects by baseline cardiovascular risk (e.g., Framingham Risk Score), comorbidity status, and concomitant medications. Safety assessments, including AEs and SAEs, will be summarized descriptively by treatment group, with frequencies and proportions reported. All statistical analyses will be performed using statistical software SPSS 29.0, and a significance level of 5% (two-sided) will be considered for hypothesis testing unless otherwise specified.

Data monitoring committee.

A formal independent Data Monitoring Committee has not been established for this study, as the interventions involved are considered low risk and the study interventions are classical Ayurveda formulations in use. However, to ensure oversight and quality assurance, CCRAS will nominate a Clinical Monitoring Committee responsible for site monitoring and review of protocol adherence.

This committee, composed of designated CCRAS representatives and monitors, will conduct periodic visits to the study site to inspect facilities, verify source documents, review CRFs, and assess compliance with the protocol and GCP guidelines. The committee will work in coordination with the investigators to resolve any discrepancies or issues identified during monitoring. All data will be reviewed centrally at CCRAS to ensure consistency, completeness, and accuracy of the data. No conflicts of interest are declared by the monitoring team.

Research ethics approval.

The study protocol, case report forms (CRF), and participant information sheet (PIS) were reviewed and approved (Ref no. T/EMF/Biochem/22/15 by the Institutional Ethics Committee on 9^th June 2022. The study protocol is enclosed as a S1 File. The protocol was registered with the Clinical Trials Registry of India (CTRI) (registration number CTRI/2023/04/051942) Registered on 25/04/2023. The CTRI registration is enclosed as a S2 File. The trial will be conducted in accordance with the National Ethical Guidelines for Biomedical and Health Research Involving Human Participants (ICMR, 2017) and the Good Clinical Practice Guidelines for Clinical Trials in Ayurveda, Siddha, and Unani Medicine (GCP-ASU), 2013. Written informed consent will be obtained from all eligible participants prior to enrollment. In cases where a participant is temporarily unable to provide consent, consent will be sought as soon as capacity is regained. Participants and their legally authorized representatives will be informed of their right to withdraw from the study at any time and to withhold the use of their data.