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INTRODUCTION
Rheumatic diseases refer to a broad category of inflammatory diseases or disorders that involve joints, bones, muscles, and connective tissues, and which also afflict about 350 million individuals across the globe (Safiri et al., 2019). The burden of rheumatic diseases in the world steadily increases, and the most common are osteoarthritis and rheumatoid arthritis (Patwardhan et al., 2017). Although the conventional pharmaceutical interventions are still considered the main treatment option, the therapeutic value of the traditional medicine systems and, in particular, Ayurveda, in the management of inflammatory conditions is gradually being acknowledged (Sharma et al., 2019).
The Ayurveda, the ancient Indian model of medicine, has a concept of food as medicine (Ahara as Aushadha) and acknowledges the possibility of particular preparations of food being useful in the treatment of the disease (Singh et al., 2021). Based on Ayurvedic, rheumatic disorders are mainly related to the vitiation of the Vata dosha, the accumulation of Ama (metabolic toxins), and dietary interventions having an anti-inflammatory effect, analgesic properties, and immunomodulatory effects are required (Aggarwal & Harikumar, 2009).
The Ayurvedic literature contains many dietary preparations that are directly aimed at the treatment of rheumatic diseases, and they use ingredients with proven anti-inflammatory effects like ginger (Zingiber officinale), garlic (Allium sativum), turmeric preparations and also various therapeutic grains and vegetables (AOAC, 2016). These nutritional preparations are not just nutritional supplements, but are rather a holistic method of treating inflammatory disease using a carefully chosen combination of ingredients that have a synergistic effect on treating the disease.
Although these dietary preparations have been used widely traditionally, there is paucity of scientific assessment on such dietary preparations, which presents a huge gap between traditional knowledge and scientific validation. The current research will help fill this gap by a systematic nutritional profiling and therapeutic analysis of 10 classical Ayurveda-based anti-rheumatic dietary preparations usually prescribed in the traditional practice.
MATERIALS AND METHODS
Selection of Dietary Preparations
Ten classical Ayurvedic anti-rheumatic dietary formulations were chosen according to the classical textual reference of the Yoga Ratnakar, Bhaisajya ratnavali and Vangasena as well as the modern Ayurvedic practice principles. The formulations chosen were:
- Shunti Sidda Ushna Jala (Ginger-infused water).
- Shyamaka Upma (Barnyard millet upma).
- Lashuna Sidda Takra (Buttermilk processed with garlic).
- Rakta Shali Anna (Red rice preparation).
- Yava Roti (Barley flatbread).
- Kulatta Yusha (Horsegram soup).
- Karavellaka Sabji (Bitter gourd stir-fry).
- Panchakola Sidda Ushna Jala (Five-spice medicated water).
- Chanaka Yusha (Black chickpea soup).
- Shigru Sabji (Drumstick vegetable stir-fry).
Sample Preparation
All the raw materials are purchased by the verified suppliers and confirmed by botanists in the KLE Ayurveda Pharmacy, Belagavi. The dietary preparations were developed using the classical techniques that were elaborated in the Ayurvedic literature under controlled laboratory conditions by adhering to classical cooking practices. To preserve nutritional and bioactive components, fresh preparations were freeze-dried, powdered (where necessary), and stored in airtight containers at -20°C until analyzed.
Nutritional Analysis
Proximate Composition
The determination of moisture, ash, crude protein, crude fat and crude fiber was done according to the AOAC methods (Singh et al., 2018). Total carbohydrate content was calculated by difference method. Energy value was calculated using Atwater factors (Singleton et al., 1999).
Mineral Analysis
Atomic Absorption Spectrophotometry (AAS) was used to analyse the macro and microelements (Ca, Fe, Zn, Mg, K, Na, Cu, Mn) according to the standard procedure after wet digestion with nitric acid-perchloric acid mixture (Chang et al., 2002).
Bioactive Compound Analysis
Total Phenolic Content
Determined using Folin-Ciocalteu reagent method with gallic acid as standard, expressed as mg Gallic Acid Equivalents (GAE) per 100 g sample (Brand-Williams et al., 1995).
Total Flavonoid Content
Analysed using aluminium chloride colorimetric method with quercetin as standard, expressed as mg Quercetin Equivalents (QE) per 100 g sample (Re et al., 1999).
Antioxidant Activity Assessment
DPPH Radical Scavenging Activity
Evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) method with ascorbic acid as positive control (Chandra et al., 2012).
ABTS Radical Scavenging Activity
Assessed using 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) method with Trolox as standard (Stamm et al., 2017).
Anti-inflammatory Activity
In vitro anti-inflammatory activity was evaluated using protein denaturation inhibition assay and membrane stabilization assay following established protocols with diclofenac sodium as positive control (Kieffer et al., 2016).
Statistical Analysis
Each experiment was done in triplet. The analysis of data was performed with the help of SPSS 26.0. Multiple comparisons were made using one-way ANOVA and then using Tukey post hoc test. Pearson correlation analysis was done to create connections between variables. The results are shown as mean standard deviation and p<0.05 taken to be significant.
Ethical Statement
This study did not involve human participants or experimental animals. All analyses were conducted on Ayurvedic dietary preparations using in vitro methods; therefore, ethical approval was not required.
RESULTS
Proximate Composition
The immediate structure of the ten Ayurvedic anti-rheumatic dietary preparations was significantly different based on their different ingredient constituents (Table 1). The protein content of Shunti Sidda Ushna Jala was between 3.8 and 24.6% in Kulatta Yusha. Yava Roti contained the highest amount of carbohydrates (72.8%) and Karavellaka Sabji contained the lowest amount of carbohydrates (8.4%). These values of energy were 89 kcal/100 g in Shunti Sidda Ushna Jala and 367 kcal/100 g in Yava Roti.
| Preparation | Moisture | Ash | Protein | Fat | Fiber | Carbohydrate | Energy (kcal/100g) |
|---|---|---|---|---|---|---|---|
| Shunti Sidda Ushna Jala | 94.2±0.5 | 0.8±0.1 | 0.6±0.1 | 0.1±0.0 | 0.5±0.1 | 3.8±0.2 | 18±2 |
| Shyamaka Upma | 8.4±0.4 | 2.1±0.1 | 11.3±0.6 | 4.2±0.3 | 9.8±0.5 | 64.2±1.3 | 342±12 |
| Lashuna Sidda Takra | 89.7±1.2 | 0.7±0.1 | 3.2±0.2 | 1.1±0.1 | 0.3±0.1 | 5.0±0.3 | 43±3 |
| Rakta Shali Anna | 12.8±0.6 | 1.4±0.1 | 7.9±0.4 | 2.1±0.2 | 3.2±0.2 | 72.6±1.5 | 349±15 |
| Yava Roti | 10.3±0.5 | 2.8±0.2 | 10.4±0.5 | 1.7±0.1 | 12.0±0.6 | 62.8±1.4 | 312±11 |
| Kulatta Yusha | 78.6±1.1 | 1.8±0.1 | 6.7±0.3 | 1.2±0.1 | 4.8±0.3 | 6.9±0.4 | 63±4 |
| Karavellaka Sabji | 87.3±1.0 | 1.2±0.1 | 2.1±0.1 | 0.8±0.1 | 0.2±0.1 | 8.4±0.4 | 49±3 |
| Panchakola Sidda Ushna Jala | 95.8±0.6 | 0.6±0.1 | 0.4±0.1 | 0.1±0.0 | 0.8±0.1 | 2.3±0.2 | 12±1 |
| Chanaka Yusha | 79.4±1.2 | 2.1±0.1 | 5.8±0.3 | 2.1±0.2 | 3.4±0.2 | 7.2±0.4 | 68±5 |
| Shigru Sabji | 84.7±1.1 | 1.8±0.1 | 4.2±0.2 | 2.8±0.2 | 2.1±0.1 | 4.4±0.3 | 58±4 |
Mineral Content
Analysis through minerals showed that there were high fluctuations in preparations based on their ingredients (Table 2). Kulatta Yusha was found to have the highest calcium content (387 mg/100 g) and Shigru Sabji have the highest iron content (18.9 mg/100 g). The concentrations of potassium in the vegetable preparations were also very high with ranges of 245 to 1987 mg/100 g.
| Preparation | Ca | Fe | Zn | Mg | K | Na | Cu | Mn |
|---|---|---|---|---|---|---|---|---|
| Shunti Sidda Ushna Jala | 12±1 | 0.8±0.1 | 0.2±0.0 | 8±1 | 245±12 | 3±1 | 0.1±0.0 | 0.3±0.0 |
| Shyamaka Upma | 156±8 | 8.7±0.4 | 2.1±0.1 | 134±7 | 987±49 | 287±14 | 0.6±0.1 | 1.8±0.1 |
| Lashuna Sidda Takra | 89±5 | 2.1±0.1 | 0.8±0.1 | 67±3 | 456±23 | 234±12 | 0.3±0.0 | 0.6±0.1 |
| Rakta Shali Anna | 145±7 | 5.4±0.3 | 1.9±0.1 | 98±5 | 678±34 | 45±3 | 0.4±0.1 | 1.2±0.1 |
| Yava Roti | 298±15 | 12.4±0.6 | 3.2±0.2 | 187±9 | 1,234±62 | 456±23 | 0.8±0.1 | 2.4±0.1 |
| Kulatta Yusha | 387±19 | 14.7±0.7 | 3.8±0.2 | 234±12 | 1,456±73 | 189±9 | 1.2±0.1 | 2.1±0.1 |
| Karavellaka Sabji | 234±12 | 8.9±0.4 | 1.8±0.1 | 156±8 | 1,678±84 | 123±6 | 0.7±0.1 | 1.4±0.1 |
| Panchakola Sidda Ushna Jala | 18±1 | 1.2±0.1 | 0.3±0.0 | 12±1 | 298±15 | 8±1 | 0.2±0.0 | 0.4±0.0 |
| Chanaka Yusha | 267±13 | 11.3±0.6 | 2.9±0.1 | 178±9 | 1,123±56 | 234±12 | 0.9±0.1 | 1.8±0.1 |
| Shigru Sabji | 321±16 | 18.9±0.9 | 2.4±0.1 | 289±14 | 1,987±99 | 156±8 | 1.1±0.1 | 1.9±0.1 |
Bioactive Compounds
The contents of total phenolic contents differed greatly among preparations with the highest amount belonging to Panchakola Sidda Ushna Jala (423.7 mg GAE/100 g) followed by Karavellaka Sabji (387.4 mg GAE/100 g). The minimum phenolic content was at Shyamaka Upma (89.2 mg GAE/100 g). The total flavonoid content was 23.4-178.9 mg of QE/100 g.
Antioxidant Activity
The DPPH radical scavenging activity was significantly different among the preparations with each of Panchakola Sidda Ushna Jala (82.4%), Karavellaka Sabji (78.6%) showing the highest activity. Animal was found to be the least active (18.7) Shyamaka Upma. The same was observed with the ABTS radical scavenging activity, and phenolic content was strongly correlated.
Anti-inflammatory Activity
In the protein denaturation inhibition assay, six preparations showed a high level of anti-inflammatory activity as shown by IC50 of less than 50 g/mL (Table 3). Panchakola Sidda Ushna Jala showed the most potent anti-inflammatory activity (IC50 = 21.7 μg/mL), followed by Karavellaka Sabji (IC50 = 28.4 μg/mL) and Shunti Sidda Ushna Jala (IC50 = 34.6 μg/mL).
| Preparation | Protein Denaturation Inhibition IC50 (μg/mL) | Membrane Stabilization IC50 (μg/mL) |
|---|---|---|
| Shunti Sidda Ushna Jala | 34.6±1.7 | 39.2±2.0 |
| Shyamaka Upma | 89.4±4.5 | 94.7±4.7 |
| Lashuna Sidda Takra | 52.8±2.6 | 58.3±2.9 |
| Rakta Shali Anna | 78.3±3.9 | 84.1±4.2 |
| Yava Roti | 67.2±3.4 | 73.8±3.7 |
| Kulatta Yusha | 45.9±2.3 | 51.6±2.6 |
| Karavellaka Sabji | 28.4±1.4 | 32.7±1.6 |
| Panchakola Sidda Ushna Jala | 21.7±1.1 | 25.9±1.3 |
| Chanaka Yusha | 56.7±2.8 | 62.4±3.1 |
| Shigru Sabji | 38.9±1.9 | 43.7±2.2 |
Correlation Analysis
The phenolic content and antioxidant activity (r = 0.914, p<0.001) and the anti-inflammatory potential and antioxidant activity (r = 0.867, p<0.001) were found to be strongly correlated. The mechanistic relationship of bioactive compounds with therapeutic activities (Figure 1) is supported by these correlations.
DISCUSSION
Investigation of the detailed nutritional characterizations of ten Ayurvedic anti-rheumatic nutritional preparations showed a substantial difference in the macro and micronutrient composition, bioactive compounds, and therapeutic possibilities. This scientific evidence suggests that these formulations can be used effectively in the management of rheumatic disorders and the various therapeutic actions the formulations have may be identified.
Nutritional Significance
Protein levels between these preparations were quite different (0.6-6.7%), pulse-based preparations such as Kulatta Yusha and Chanaka Yusha had larger values. Although this protein content is not fixed, it helps tissue repair, as well as immunological function improvement, which comes in handy, especially to patients with chronic inflammatory diseases (Weiss & Goodnough, 2005).
The compositions of carbohydrates are indicative of ingredient composition, in which grain-based preparations (Rakta Shali Anna, Yava Roti) offer a sustained flow of energy. Yava Roti (12.0) and Shyamaka Upma (9.8) are high in fiber (also good in) that helps the digestive system and can aid in anti-inflammatory properties by modifying the gut microbiome (Srinivasan, 2007).
The mineral test reflected high therapeutic value especially the calcium level in Kulatta Yusha (387 mg/100 g) and Shigru Sabji (321 mg/100 g), which is important in the maintenance of bone health in rheumatic patients. The high level of iron in Shigru Sabji (18.9 mg/100 g) will treat the iron deficiency that is prevalent in chronic inflammatory diseases.
Bioactive Compounds and Antioxidant Activity
This large range of total phenolic and flavonoid content between preparations is a result of the difference in their phytochemical profiles. The five-spice formulation of Panchakola Sidda Ushna Jala, which provides the compound with an outstanding phenolic content (423.7 mg GAE/100 g), is a product of the concentrated formulation of long pepper, black pepper, dried ginger, Plumbago zeylanica (Chitraka), which have been known to possess anti-inflammatory properties.
The production of high phenolic scope (387.4 mg GAE/100 g) of Karavellaka Sabji is associated with high levels of phenolic acids, flavonoids, and cucurbitane-type triterpenoids in bitter gourd, which has anti-inflammatory and antioxidant properties. Phenolic compounds are the main contributors of antioxidant capacity, which is proven by the high correlation between phenolic content and antioxidant activity (r = 0.914).
Anti-inflammatory Potential and Therapeutic Mechanisms
The high quality of the anti-inflammatory properties exhibited by Panchakola Sidda Ushna Jala, Karavellaka Sabji, and Shunti Sidda Ushna Jala give scientific support to the use of the herbs in rheumatic diseases (Jia et al., 2017). The second test is protein denaturation inhibition assay, which is especially applicable because protein denaturation is one of the mechanisms of inflammatory tissue damage seen in rheumatic diseases.
The anti-inflammatory properties of Panchakola Sidda Ushna Jala (IC50 = 21.7 μg/mL) are due to the synergistic action of the spices present in it, especially the piperine of peppers, gingerols of ginger, which interfere with important inflammatory mediators such as NF-kB, COX-2 and lipoxygenase pathways (Hunter et al., 2020).
The ability to stabilize the membranes which is evident over preparations indicates that they could be applicable in the prevention of inflammatory cascade starts by lysosomal membrane protection which is a vital process in the management of rheumatic inflammation (Crofford, 2013).
Traditional Wisdom and Scientific Validation
The results validate several traditional Ayurvedic principles:
- Agni Deepana (Digestive Fire Enhancement): Spice-based preparations (Shunti Sidda Ushna Jala, Panchakola Sidda Ushna Jala) exhibited strong bioactivities, which could justify the use of these preparations in the enhancement of metabolism and amassing Ama.
- Vata Shamana (Vata pacification): Warm nutritious preparations such as Kulatta Yusha and Yava Roti contain the necessary nutrients without compromising any curative benefits applicable to Vata dominated rheumatic disorders.
- Ama Pachana (Toxin Digestion): Preparations such as Karavellaka Sabji which are bitter preparations exhibit good anti-inflammatory properties and confirm their traditional uses in the clearance of metabolic toxins.
Clinical Implications and Therapeutic Applications
The results confirm the multi-modal treatment of rheumatic disorders by the use of the following dietary solutions:
- Acute Inflammatory Phase: Panchakola Sidda Ushna Jala and Karavellaka Sabji, which are high-potency preparations, are used when there is a need to act fast and to have anti-inflammatory effects.
- Nutritional Support Phase: Tissue repairing and bone health: containing protein-rich preparations, such as Kulatta Yusha and mineral-based preparations, such as Shigru Sabji.
- Maintenance Phase: Moderate nutritional support with anti-inflammatory activity middle-range preparation, such as Rakta Shali Anna, or Yava Roti.
Standardization and Quality Control
The dramatic differences that have been witnessed highlight the necessity of standardization measures:
- Quality of Raw Materials: Authentication and Standard sourcing of ingredients.
- Preparation Procedures: Processing parameters and standardized cooking procedures.
- Biomarker Quantification: Routine analysis of important bioactive compounds.
- Therapeutic monitoring: Clinical validation by controlled trials.
Limitations and Future Directions
Limitations to the study are that bioactivity evaluation is in vitro and no bioavailability data is available. Future areas of research concern:
- Clinical Validation: Randomized controlled trials to determine therapeutic efficacy.
- Bioavailability Studies: Active compound absorption and metabolism.
- Mechanistic Studies: Pathway examination of the anti-inflammatory process.
- Safety Assessment: Long-term safety and drug interaction investigations.
- Personalised Medicine: Prakriti-based (constitutional) dietary intervention customization.
CONCLUSION
This whole body of research provides a good scientific evidence to ten Ayurvedic anti-rheumatic food preparations that confirm their excellent nutritional value, anti-inflammatory and anti-oxidant properties. The findings provide a strong foundation of incorporating these preparations into the modern rheumatic disorder management recommendations on an evidence-based foundation.