With increasing global interest in Ayurvedic traditional medicines, there is a corresponding need to ensure their safety, purity, and therapeutic efficacy. This growing demand places critical responsibility on pharmaceutical manufacturers and regulatory authorities to implement stringent standardisation (Jahanet al., 2018). Drug standardisation encompasses the evaluation of multiple parameters, including phytochemical profiling and physicochemical characterization, which are essential for confirming the identity, quality, and efficacy of Ayurvedic formulations.

Diabetic neuropathy is a prevalent complication of diabetes mellitus and is characterized by peripheral nerve damage due to microvascular dysfunction affecting the vasa nervorum. Among its various forms, distal symmetric polyneuropathy is the most common and involves Type A and Type C nerve fibers. It often remains underdiagnosed, particularly in asymptomatic or painless cases, leading to significant morbidity. The pathological features include distal motor axonal loss, diminished nerve conduction velocity, and degeneration of small and large myelinated as well as unmyelinated sensory fibers. Clinically, it presents with symptoms such as numbness, paresthesia, burning sensations, muscle weakness, reduced reflexes, and impaired coordination-often with nocturnal exacerbation.

Meha Mudgara Rasa is a classical Ayurvedic herbomineral formulation cited in Bhaishajya Ratnavali under Prameha Rogadhikar (Diabetes) (Sivakumaret al., 2022). It contains 14 medicinal herbs and one mineral ingredient. Notably, components such as Dadima (Punica granatum) (Alharthy et al., 2023). Triphala (Ariya et al., 2006), Bilva (Aegle marmelos) (Sayeed et al., 2016), and Gokshura (Tribulus terrestris) (Caporali et al., 2022) have demonstrated antidiabetic, hypolipidemic, and neuroprotective properties in pharmacological studies. These phytotherapeutic actions suggest a potential role for Meha Mudgara Rasa in modulating the pathophysiological mechanisms underlying diabetic neuropathy. Its use may contribute to the management of both peripheral vascular and neuronal complications associated with diabetes.

Raw materials of Meha Mudgara Rasa were procured from an authorized Ayurvedic pharmacy and authenticated by the Dravyaguna department as per API standards (Table 1).

Kanta Loha Bhasma was evaluated as per the Pharmacopoeial Standards for Ayurvedic Formulations (PSAF) and the Ayurvedic Pharmacopoeia of India (API) standards (Table 2).

Amruteekaran is a process adopted to remove residual toxins and enhance the therapeutic action of drugs. First, the Amruteekaran of Kanta Loha Bhasma was performed as classically mentioned by trituration with a decoction of Triphala and subjected to 1 Puta (800ºC for 3 hr) in an electric muffle furnace.

All the fine powders of the herbal drugs are mixed properly in an end-runner machine.

Guggulu is purified and dissolved in Triphala Kashaya, which is used as Bhavana Dravya and mixed with fine powders of herbal drugs.

These mixtures were mixed with Kanta Loha Bhasma processed for 6 hr and dried in a tray-dried machine.

After proper drying, granules are made in the granulator machine, and a binding agent such as starch is added at a ratio of 1:10 to make tablets in a multiple tablet punching machine.

Physiochemical characterization of Meha Mudgara rasa tablets was performed, assessing hardness, friability, pH, ash values, disintegration time, loss on drying, acid-insoluble ash, and extraction values, following the Ayurvedic Pharmacopoeia of India (API) and Indian Pharmacopoeia (IP) standards.

To evaluate its phytochemical profile, the Meha Mudgara rasa methanolic extract was analysed using HPTLC.

The sample (5 g) was weighed, and 100 mL of methanol was added. The mixture was sonicated for 15 min and filtered with simple filter paper. The filtrate was used as a test solution and thus obtained for HPTLC fingerprinting. The details of the HPTLC conditions are outlined in Table 3.

The tablets were black. It has a characteristic taste and aromatic odor.

The organoleptic analysis was performed as follows (Table 4).

The observations of various physicochemical analyses are mentioned below (Table 5).

HPTLC analysis was performed at wavelengths of 254 nm and 366 nm with sample volumes of 10.0 µL, 15.0 µL, and 20.0 µL. Chromatographs at 254 nm are shown in Figures 1–3, while Figure 4 depicts the chromatograph at 366 nm using 20.0 µL. The R_f values, fingerprint profiles, peak heights, and percentage areas were recorded. Chemical substances were identified by comparing these results with PubChem data, with detailed findings and bioactivities presented in Tables 6–9 [Source: PubChem, Google Scholar].

Figure 1:
HPTLC Chromatograph of Meha Mudgara rasa @254 nm and a volume of 5.0 µL (A=Fingerprint, B= Peak height, C= R_f value and area percentage) (A)

Figure 2:
HPTLC Chromatograph of Meha Mudgara rasa @254 nm and 10.0 µL volume (A=Fingerprint, B= Peak height, C= R_f value and area percentage). (A)

Figure 3:
HPTLC Chromatograph of Meha Mudgara rasa @254 nm and 15.0 µL volume (A=Fingerprint, B= Peak height, C= R_f value and area percentage. (A)

Figure 4:
HPTLC Chromatograph of Meha Mudgara rasa @366 nm and 15.0 µL volume (A=Fingerprint, B= Peak height, C= R_f value and area percentage).

HPTLC and physicochemical tests ensure Ayurvedic medicine quality by verifying authenticity and purity, detecting contaminants, and standardizing formulations, supporting clinical research and advancing evidence-based Ayurveda for improved patient care.

This study analyzed the physicochemical parameters of Meha Mudgara Rasa tablets. The pH was 6.8, the water- and alcohol-soluble extraction values were 41.7% and 4.5%, respectively, and the moisture content was 8.88%, all within acceptable limits. The tablet hardness was 6.8 kg/m². The total ash (34.19%) and acid-insoluble ash (28.6%) contents were greater due to Loha Bhasma but may suggest potential contamination if they exceed standard limits, which are common in herbo-mineral drugs.

The pathophysiology of diabetic neuropathy involves four pathways: the Protein Kinase C signaling (PKC) pathway, Advanced Glycation end Products (AGEs), increased free radical formation and the polyol pathway. In addition, Hypoinsulinemia also contributes to damage to nerves. The mechanism that causes sensory and motor axonal neuron degeneration and the development of neuropathy, along with its types, is outlined in Figure 5.

Figure 5:
Pathophysiology of diabetic neuropathy.

HPTLC analysis of the methanolic extract of Meha Mudgara Rasa revealed multiple phytoconstituents at varying concentrations (Figures 1–4, Tables 6–9). Chromatograms scanned at 245 nm and 366 nm presented diverse peaks and R_f values, which were compared to standards to identify key phytochemicals. The significant compounds detected include quercetin (Sousaet al., 2018), terpenes (Jainet al., 2013), flavonoids (World Health Organization, 2004), and coumarin (Maharaj and Rohan, 2013). These bioactive compounds are known for their anti-inflammatory, antioxidant, and analgesic properties, potentially contributing to reversing the pathophysiology of diabetic neuropathy.

The PKC pathway, which is influenced by vascular ischemia, inflammation, and oxidative stress, plays a key role in diabetic complications. Quercetin and terpenes enhance blood circulation, reducing ischemia, while quercetin also regulates AGE production. Inflammation is mitigated by quercetin and flavonoids through procytokine and IL inhibition. Additionally, quercetin improves mitochondrial function and reduces ROS, whereas flavonoids act as antioxidants, scavenging ROS to combat oxidative stress. These combined actions help reverse PKC pathway activation, positioning Meha Mudgara Rasa as a promising therapeutic for diabetic neuropathy (Figure 6).

Figure 6:
Possible effects of Meha Mudgara Rasa on the Protein Kinase C signaling (PKC) pathway.

The second pathway known to cause DN is endothelial dysfunction and AGE production. Coumarin improves endothelial function and reduces blood viscosity, whereas quercetin and flavonoids combat oxidative stress by scavenging Reactive Oxygen Species (ROS). Additionally, coumarin alleviates vasculitis by inhibiting inflammatory markers, and terpenes aid in reducing ROS formation. Together, these compounds mitigate endothelial dysfunction, oxidative stress, and inflammation and promote vascular health and slow aging processes (Figure 7).

Figure 7:
Possible effects of Meha Mudgara Rasa on endothelial dysfunction and advanced glycation end products (AGEs).

Hypoinsulinemia leads to a disruption in nerve growth factors such as BDNF, IGF, VEGF, and NGF, which are essential for nerve regeneration and maintenance. This imbalance results in reduced nerve repair function, ultimately contributing to neuropathy. Flavonoids counteract this process by increasing nerve growth factor levels, thereby assisting in nerve repair and reducing neuropathic damage. Thus, flavonoids serve as crucial protective agents against nerve degeneration, promoting neuroprotection and functional recovery (Figure 8).

Figure 8:
Possible effects of Meha Mudgara Rasa on Hypoinsulinemia.

In addition, HPTLC also contains many other phytochemicals, such as umbelliferone (Ginwalaet al., 2019), myristicin (Gautam and Ramanathan, 2019). and stigmasterol (Wuet al., 2016). in relatively small quantities. These findings can help in the management of diabetes because of their antidiabetic and insulin-stimulating effects. It can mitigate hyperglycemia, which is a major cause of nerve damage in diabetic neuropathy. They are also reported to correct dyslipidemia, which can improve blood circulation and decrease the formation of atherosclerotic plaques in the arteries supplying the nerves, ultimately supporting better nerve function. These additional effects can help in conditions that are contributory but not directly associated with diabetic neuropathy.

Hence, the primary phytochemicals observed through HPTLC analysis of Meha Mudgara Rasa could be useful in the management of diabetes, peripheral neuronal disorders, peripheral vascular disorders, and inflammatory disorders. Further analyses, such as gas chromatography, liquid chromatography, and experimental and clinical studies, are necessary to substantiate the therapeutic potential of Meha Mudgara Rasa in diabetic neuropathy.