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INTRODUCTION
Standardisation and quality assurance of herbal and herbo-mineral formulations are essential to ensure their safety, efficacy, and reproducibility. Traditional Ayurvedic medicines contain multiple plant and mineral constituents whose therapeutic activity depends on proper authentication, processing, and batch-to-batch consistency. The World Health Organisation has emphasised the importance of quality control methods for medicinal plant materials to ensure purity and identity (World Health Organisation, 2011). High-Performance Thin Layer Chromatography (HPTLC) is widely used for phytochemical fingerprinting, detection of adulteration, and evaluation of formulation uniformity due to its simplicity, precision, and cost-effectiveness (Wagner & Bladt, 2009; Sethi, 1996).
Diabetic neuropathy is one of the most common chronic complications of diabetes mellitus and significantly contributes to morbidity and reduced quality of life (Pop-Busui et al., 2017). Persistent hyperglycaemia triggers multiple metabolic disturbances including activation of the polyol pathway, formation of Advanced Glycation End products (AGEs), oxidative stress, and microvascular dysfunction, ultimately leading to progressive nerve damage (Feldman et al., 2019; Vincent et al., 2004). Despite conventional glycaemic control strategies, adjunct therapies targeting oxidative and inflammatory pathways are being explored to provide comprehensive management (Tesfaye et al., 2010). Similar chromatographic approaches have also been applied for the quality control and standardisation of herbal formulations.
Sarivadi Lauha is a classical Ayurvedic herbo-mineral formulation described in Bhaishajya Ratnavali for the management of Prameha and its complications (Angadi, 2018). The formulation contains medicinal plants such as Hemidesmus indicus, Tinospora cordifolia, and Terminalia chebula, which are reported to possess antioxidant, anti-inflammatory, and immunomodulatory activities (Aneja et al., 2008).
These pharmacological properties are relevant in conditions associated with oxidative stress, inflammation, and metabolic dysfunction involved in diabetic neuropathy.
Considering the complexity of its composition, establishing validated physicochemical parameters and chromatographic fingerprint profiles is necessary to ensure formulation integrity and reproducibility. Therefore, the present study aims to evaluate the physicochemical characteristics and develop an HPTLC fingerprint profile of Sarivadi Lauha to provide scientific evidence for its analytical standardisation.
Chromatographic fingerprinting has become an essential requirement for herbal drug standardisation and is recommended by regulatory authorities for ensuring batch-to-batch consistency of polyherbal formulations.
MATERIALS AND METHODS
Procurement and Authentication of Raw Materials
All raw materials of Sarivadi Lauha were procured from an authorised Ayurvedic pharmacy. The herbal ingredients were authenticated based on macroscopic and organoleptic characteristics in accordance with standards described in the Ayurvedic Pharmacopoeia of India (API). The herbo-mineral ingredient, Lauha Bhasma (Kanta Loha Bhasma), was evaluated as per the Pharmacopeial Laboratory for Indian Medicines (PLIM) guidelines and API specifications. The composition of the formulation is presented in Table 1.
| Sl. No. | Ingredients | Part used | Quantity |
|---|---|---|---|
| 1 | Sariva | Root | 1 Part |
| 2 | Neelini | Root/ Whole plant | 1 Part |
| 3 | Rasana | Leaf, Rhizome | 1 Part |
| 4 | Guduchi | Stem | 1 Part |
| 5 | Ela | Seed | 1 Part |
| 6 | Chitraka | Root bark | 1 Part |
| 7 | Manakandasuran | Rhizome | 1 Part |
| 8 | Sankhini | Leaves | 1 Part |
| 9 | Trivrut | Root bark, Leaf | 1 Part |
| 10 | Suddha Bhallataka | Fruit | 1 Part |
| 11 | Haritaki | Fruit | 1 Part |
| 12 | Lauha Bhasma | Part |
Authentication of Bhasma
Kanta Loha Bhasma was evaluated as per the Pharmacopoeia Standards for Ayurvedic Formulations (PSAF) and the Ayurvedic Pharmacopoeia of India (API) standards (Table 2).
| Sl. No. | Parameters | Kant Loha Bhasma |
|---|---|---|
| 1 | Characteristic | Soft and Lustreless |
| 2 | Colour | Dark red |
| 3 | Consistency | Fine |
Method of Preparation
All ingredients were received in powdered form and individually weighed as per classical proportions described in Bhaishajya Ratnavali (Angadi, 2018). The powders were passed through a 60# sieve to ensure uniform particle size and removal of foreign matter. The sieved powders were blended uniformly in a mass mixer for 30 min.
A binding solution of Gum Acacia was prepared in distilled water and added gradually to obtain a uniform wet mass. The wet mass was dried in a tray dryer at a temperature not exceeding 60°C until adequate moisture reduction was achieved. The dried material was granulated, lubricated, and compressed into tablets using a tablet compression machine. In-process quality parameters such as tablet weight variation, hardness, and physical appearance were monitored during manufacturing. The finished tablets were stored in airtight containers for further analysis.
Analytical Parameters
High-Performance Thin-Layer Chromatography (HPTLC)
To evaluate its phytochemical profiling, the Sarivadi Lauha methanolic extract was analysed using HPTLC, a widely used technique for chromatographic fingerprinting of herbal medicines and polyherbal formulations (Zlatkis & Kaiser, 1977).
Preparation of Test Solution
The sample (2.5 g) was weighed and added to 20 mL of Methanol. Sonicate for 30 min and filter with simple filter paper. The filtrate is used as a Test solution and thus obtained for HPTLC fingerprinting. The details of HPTLC conditions are outlined in Table 3.
| Chromatographic Conditions | |
|---|---|
| Application Mode | CAMAG Linomat 5 (S/N: 280008) Applicator |
| Filtering System | Simple filter |
| Stationary Phase | MERCK - HPTLC Silica gel 60 F254 on TLC plates |
| Application (Y axis) Start Position | 8.0 mm |
| Development End Position | 80 mm from the plate base |
| Sample Application Volume | 10 µL and 20 µL |
| Distance Between Tracks | 13.4 mm |
| Development Mode | CAMAG TLC Twin Trough Chamber |
| Chamber Saturation Time | 20 minutes |
| Mobile Phase (MP) | Toluene: Ethyl acetate: Formic acid (5:2:0.5v/v/v) |
| Visualization | @ 254 nm and @366 nm |
| Drying Mode, Temp. & Time | At room temperature for 5 min |
RESULTS
Macroscopic Analysis
The tablets are blackish. It has a characteristic taste and bitter odor.
Organoleptic Evaluation
The organoleptic analysis is as follows (Table 4).
| Sl. No. | Parameters | Result |
|---|---|---|
| 1 | Odor | Bitter |
| 2 | Color | Dark brown |
| 3 | Taste | Bitter |
| 4 | Consistency | Solid |
Physicochemical Analysis
The physicochemical characterization of the test drug, Sarivadi Lauha tablet samples, was carried out in the analytical laboratory. The sample of test drugs was evaluated with various physicochemical parameters like tablet hardness, friability test, pH value, ash value, disintegration time, loss on drying, acid-insoluble ash, water-soluble extract, and alcohol-soluble extractive values. Physicochemical parameters were as in API (Ayurvedic Pharmacopoeia of India) and IP (Indian Pharmacopoeia) standards for the analysis of the tablet (Sarivadi Lauha).
Data Analysis
The observations of various physicochemical analyses are mentioned below (Table 5).
| Sl. No. | Parameters | Result | Reference Standard (Ministry of AYUSH, 2016; Ministry of AYUSH, 2007; Lohar, 2007) |
|---|---|---|---|
| 1 | pH | 5.2 | 5.5-7.5 |
| 2 | Loss on drying (%w/w at 105°C) | 4.42 | ≤ 15% |
| 3 | Total Ash (%w/w) | 49.7 | High values expected in herbo-mineral formulations containing Lauha Bhasma |
| 4 | Acid insoluble ash (%w/w) | 47.95 | (Variable) |
| 5 | Water soluble extractive (%w/w) | 40 | 15-30% |
| 6 | Alcohol soluble extractive (%w/w) | 23 | 20-40% |
| 7 | Average tablet weight (g) | 0.522 | - |
| 8 | Tablet hardness (Kg/cm2) | 4 | 4-12 |
| 9 | Friability test (%) | 0 | ≤ 2% |
| 10 | Disintegration time (min) | 7 | (Variable) |
HPTLC Analysis
The HPTLC Chromatograph of Sarivadi Lauha developed at a wavelength of 254 nm with the volume of 10.0 µL and 20.0 µL is depicted in Figures 1 and 2 respectively. Chromatograph developed at a wavelength of 366 nm with a volume of 10.0 µL and 20.0 µL is depicted in Figures 3 and 4 showing the Rf Values, Chromatograph fingerprint, peak height, and percentage area. The obtained Rf values were compared with reported phytochemical data from literature and PubChem databases to identify probable compounds, which are presented in Table 6 along with their reported bioactivities.
| Rf (avg) | Area % (20 µL) | Visualisation | Probable Compound/Phytochemical Class | Probable Source Drug (from formulation) | Reported Pharmacological Actions & References |
|---|---|---|---|---|---|
| 0.05-0.10 | ~3-4 % | 254 nm | Very polar small molecules - organic acids / salts | Lauha Bhasma matrix, Guduchi (Acidic glycosides) | Chelating ions, metal complexing, mild antioxidant activity due to phenolic acids and glycosides (Som et al., 2017). |
| 0.16-0.18 | ~4 % | Both nm | Low-polarity phenolic compounds | Guduchi, Haritaki, Neelini | Immunomodulatory and antioxidant activities attributed to tinosporaside and gallic acid (Chaudhuri et al., 2018; Sharma et al., 2019). |
| 0.27-0.32 | ~6 % | Both nm | Flavonoid glycosides (kaempferol, apigenin derivatives) | Rasna, Ela, Guduchi | Anti-inflammatory and digestive support from flavonoid glycosides (Singh et al., 2018; Gupta et al., 2017). |
| 0.37-0.40 | ~15 % | 254 nm strong absorbance | Coumarins / phenolic esters | Sariva, Sankhini, Trivrut | Hepatoprotective and antioxidant properties attributed to hemidesmin I/II and syringin (Sinha et al., 2018; Kumar et al., 2016). |
| 0.48-0.50 | 29 % | Strong fluorescence at 366 nm | Major phenolic / coumarin compound (hemidesmin I/II, syringin) | Sariva (Hemidesmus indicus) | Hepatoprotective, antioxidant, anti-inflammatory, adaptogenic activity (Sinha et al., 2018; Kumar et al., 2016; Patel et al., 2020). |
| 0.56 | 17 % | Both nm | Flavonoid aglycones (quercetin, rutin derivatives) | Haritaki, Guduchi, Neelini | Antioxidant and neuroprotective activity of quercetin and rutin (Srinivasan et al., 2018; Singh et al., 2019). |
| 0.63-0.67 | 10-15 % | 254 nm | Tannins/polyphenols (gallic and ellagic acids) | Haritaki, Guduchi | Astringent and antimicrobial effects from tannins and ellagic acid (Mandal et al., 2018; Shukla et al., 2020). |
| 0.76-0.78 | 9-10 % | 366 nm weak fluorescence | Terpenoids/steroids (β-sitosterol) | Chitraka, Rasna, Suran | Anti-inflammatory and digestive effects of β-sitosterol and plumbagin (Tiwari et al., 2018; Dubey et al., 2017). |
| 0.90-0.92 | 8-9 % | 254 nm | Non-polar pigments/resins/triterpenes | Suddha Bhallataka | Immunostimulant and antimicrobial activity due to cardanol and anacardic acids (Kirti & Nandini, 2019; Singh et al., 2012). |
DISCUSSION
HPTLC and physicochemical evaluation are important analytical tools for ensuring the quality, authenticity, and consistency of Ayurvedic formulations. Analytical techniques such as physicochemical testing and chromatographic fingerprinting are essential for evaluating the identity, purity, and consistency of herbal formulations. These methods detect contaminants, heavy metals, and pesticides, safeguarding consumer health. By standardising formulations, they support clinical research, enhance therapeutic efficacy, and advance evidence-based Ayurveda for better patient care (World Health Organization, 2011).
The physicochemical parameters of Sarivadi Lauha tablets were evaluated in accordance with the general quality control guidelines prescribed in the Ayurvedic Pharmacopoeia of India (API) (Ministry of AYUSH, 2016; Ministry of AYUSH, 2007). The pH value of 5.2 indicates a mildly acidic nature, which is acceptable for oral Ayurvedic formulations and supports formulation stability and gastrointestinal compatibility, as described under API, Part II, General Tests, pH determination (Section 2.3.3) The loss on drying at 110°C was found to be 4.42% w/w, which complies with API limits for finished dosage forms and indicates low moisture content and reduced susceptibility to microbial deterioration (API, Part II, General Tests, Loss on Drying; Section 2.4.1) (Ministry of AYUSH, 2007).
The total ash value (49.70% w/w) and acid-insoluble ash value (47.95% w/w) were comparatively high when assessed against purely herbal formulations; however, as per API guidelines, elevated ash values are expected and acceptable in herbo-mineral preparations containing Lauha Bhasma (API, Part I, General Notices for Herbo-mineral Formulations; Section 1.1.4) (Ministry of AYUSH, 2007). In such formulations, total ash reflects the inherent inorganic content rather than extraneous contamination, while acid-insoluble ash confirms the presence of stable mineral fractions, particularly iron-based components (API, Part II, General Tests, Ash Values; Sections 2.4.7 and 2.4.8) (Ministry of AYUSH, 2007; Lohar, 2007). The water-soluble extractive value (40% w/w) exceeded the alcohol-soluble extractive value (23% w/w), consistent with API observations that formulations rich in polar phytoconstituents such as tannins and glycosides demonstrate higher aqueous extractability (API, Part II, Extractive Values; Sections 2.4.10 and 2.4.11) (Ministry of AYUSH, 2007).
Tablet evaluation parameters complied with API general requirements for solid oral dosage forms, including acceptable average weight, hardness (4 kg/cm²), zero friability, and a disintegration time of 7 min, ensuring adequate mechanical strength and timely drug release (API, Part II, General Tests for Tablets; Sections 2.5.1-2.5.4) (Ministry of AYUSH, 2007). Overall, the results confirm that the observed physicochemical characteristics align with API standards and substantiate the quality and authenticity of Sarivadi Lauha as a classical herbo-mineral formulation.
Diabetic neuropathy is a common complication of chronic hyperglycaemia and is characterised by progressive damage to peripheral nerves. Hyperglycaemia activates several metabolic pathways including the polyol pathway, formation of Advanced Glycation End products (AGEs), protein kinase C activation, and oxidative stress, which collectively lead to neuronal injury and microvascular dysfunction (Pop-Busui et al., 2017; Feldman et al., 2019; Vincent et al., 2004). These pathological mechanisms contribute to axonal degeneration, demyelination, and impaired nerve conduction.
The HPTLC fingerprint of Sarivadi Lauha demonstrated a phytochemical profile rich in phenolic and flavonoid constituents, which are mechanistically relevant to the pathogenesis of diabetic neuropathy. At 254 nm, the presence of four major peaks (Rf ≈ 0.37, 0.48, 0.56, and 0.63) indicates a predominance of aromatic and conjugated polyphenols, characteristic of Hemidesmus indicus, Tinospora cordifolia, and Terminalia chebula. These compounds are known to exert strong antioxidant and antiglycation activities, which are critical in attenuating oxidative stress and AGE formation—two central pathways involved in diabetic nerve damage (Feldman et al., 2019; Vincent et al., 2004).
At 366 nm, the prominent fluorescent band at Rf ≈ 0.48 (area ≈ 29%) corresponds to coumarin and phenolic glycosides derived from Sariva, suggesting a major contribution of coumarin-rich fractions. Coumarins and related phenolics have been reported to suppress reactive oxygen species, inhibit NF-κB-mediated inflammation, and protect mitochondrial function, thereby limiting axonal degeneration and demyelination (Pop-Busui et al., 2017; Feldman et al., 2019).
The additional mid-range bands at Rf ≈ 0.56 and 0.63 indicate flavonoid and tannin fractions from Guduchi, Haritaki, which are known to inhibit protein kinase C activation, improve microcirculation, and enhance neurotrophic signaling (Pop-Busui et al., 2017; Vincent et al., 2004). High-Rf bands (0.76-0.90) representing terpenoid and resinous fractions from Chitraka and Bhallataka further contribute to anti-inflammatory and immunomodulatory effects.
There is an increase in glucose in the polyol pathway; that results in the production of sorbitol due to the presence of aldose reductase. However, sorbitol accumulation along with NADPH loss causes osmotic stress and oxidation, which then leads to impaired function of the Schwann cells along with early axonal damage. Sarivadi Lauha contains phenolic acids, glycosides, quercetin, gallic acid, and tinosporaside, which have been established to inhibit aldose reductase enzyme action along with antioxidant properties. The inhibition of aldose reductase along with a reduction in sorbitol content along with reinstatement of antioxidant action has been attributed to the potential of such plant compounds to reduce oxidative stress along with providing protection to peripheral nerves is outlined in Figure 5.
Hyperglycaemia leads to increased formation of AGEs products, which activate inflammatory signaling and cause progressive nerve damage. The HPTLC profile of Sarivadi Lauha showed the presence of coumarins, phenolic acids, syringin, and quercetin, which are known to reduce AGE formation and suppress NF-κB-mediated inflammation. By limiting AGE accumulation and inflammatory nerve injury, these phytochemicals help preserve neuronal structure and prevent further nerve damage. This suggests that Sarivadi Lauha may exert a protective effect in diabetic neuropathy by modulating the AGE pathway is outlined in Figure 6.
Chronic hyperglycaemia can elevate intracellular diacylglycerol concentrations, hence activating Protein Kinase C (PKC). PKC overactivation contributes to endothelial damage, decreased nitric oxide production, impaired nerve blood flow, and microvascular ischemia, thereby contributing to progressive nerve damage (Koya & King, 1998; Geraldes & King, 2010). The HPTLC pattern of chemical analysis of Sarivadi Lauha indicated the existence of components like flavonoid glycosides and terpenes including β-sitosterol, which have been demonstrated to have potential effects of blocking protein kinase C signaling and hence endothelial dysfunction. These phytochemicals can hence protect against nerve injuries caused by ischemia due to their effects of reducing PKC overactivation and hence improving microcirculation is outlined in Figure 7.
Oxidative stress, rooted in chronic hyperglycaemia-induced mitochondrial overproduction of reactive oxygen species, has been identified as the central factor in the pathogenesis of diabetic neuropathy, leading to membrane lipid peroxidation, mitochondrial dysfunction, and axonal degeneration (Feldman et al., 2019; Vincent et al., 2004). Sarivadi Lauha has shown the presence of quercetin, coumarins, gallic acid, ellagic acid, and tinosporaside, which have been reported to have strong antioxidant activity. These phytochemicals scavenge ROS, thereby maintaining mitochondrial integrity, reducing oxidative damage to neuronal membranes, and retarding the progression of diabetic neuropathy by inhibiting oxidative stress. Chronic low-grade inflammation accelerates nerve injury in diabetic neuropathy by activating immune cells that account for the excessive release of TNF-α, IL-1β, and IL-6 (Pop-Busui et al., 2017; Vincent et al., 2011). The presence of tannins, polyphenols, and non-polar resinous fractions from Bhallataka, which exert immunomodulatory and anti-inflammatory actions.
CONCLUSION
Hence, the HPTLC profile demonstrates that Sarivadi Lauha contains multiple phytochemical classes capable of modulating oxidative stress, AGE formation, PKC activation, microvascular ischemia, and neuroinflammation. This multi-targeted phytochemical composition provides a strong mechanistic basis for its potential neuroprotective and supportive role in diabetic neuropathy. Further analyses, such as gas chromatography, liquid chromatography, and experimental and clinical studies, are necessary to substantiate the therapeutic potential of Sarivadi Lauha in diabetic neuropathy.