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Pre-clinical study on ethanolic preparations of Ruta graveolens and Thlaspi bursa pastoris
*Corresponding author: Dr. Rumsha Tamkeen, Department of Organon of Medicine, National Institute of Homoeopathy, Kolkata, West Bengal, India. dr.rumshat@outlook.com
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Received: ,
Accepted: ,
How to cite this article: Tamkeen R, Chourasia P, Paul S, Das KK, Vel VK. Pre-clinical study on ethanolic preparations of Ruta graveolens and Thlaspi bursa pastoris. J Integr Stand Homoeopath. doi: 10.25259/ JISH_100_2024
Abstract
Objectives:
The investigation of plant-based medicines has garnered significant attention globally due to their antioxidant properties. Various plant compounds, particularly phenols and flavonoids, contribute to this antioxidant activity. This study aimed to evaluate the antioxidant capacity and the total phenolic and flavonoid contents of ethanolic extracts of Ruta graveolens (RG) and Thlaspi bursa pastoris (TBP).
Material and Methods:
RG Mother tincture (Q) and TBP Q, along with their potencies (6C, 12C and 30C), were analysed using 2,2-Diphenyl-1-picrylhydrazyl (DPPH)-free radical scavenging and ferric ion reducing antioxidant power (FRAP) assays to determine the antioxidant power of the substances. In addition, the total phenolic content (TPC) and total flavonoid content (TFC) of both RG Q and TBP Q were assessed.
Results:
The findings indicated that RG Q inhibits the DPPH-free radical at 128.89 µg/mL of ascorbic acid equivalents (AAE) and the FRAP-free radical at 97.16 µg/mL of AAE. TBP Q demonstrated inhibition of the DPPH-free radical at 111.96 µg/mL of AAE and FRAP-free radical at 97.74 µg/mL of AAE. The TPC of RG Q at 100 µg/mL was 359.6 µg/mL and for TBP Q at 100 µg/mL, it was 210.8 µg/mL of gallic acid equivalents. The TFC of RG Q at 100 µg/mL was 313.37 µg/mL, while for TBP Q at 100 µg/mL, it was 89.6 µg/mL of quercetin equivalents.
Conclusion:
The study concluded that ethanolic extracts of RG and TBP exhibit antioxidant potential and contain substantial concentrations of phenols and flavonoids.
Keywords
Antioxidant
Flavonoids
Phenols
Ruta graveolens
Thlaspi bursa pastoris
INTRODUCTION
For centuries, plant-based medicines have been integral to various traditional systems for treating many kinds of disorders. Plant-derived substances containing various bio compounds such as phenols, flavonoids, alkaloids and other phytochemicals are used as medicines to promote health and combat illness. The World Health Organization has acknowledged the significance of medicinal plants and their role in healthcare, especially due to the antioxidant, anti-inflammatory and antimicrobial properties of many of these compounds.[1,2] Amongst these phytochemicals, phenols and flavonoids are more important bioactive molecules due to their potent antioxidant potential, which has demonstrated a significant role in biomedical research in conditions such as diabetes, Alzheimer’s disease and various forms of cancer.[3-5] Although several synthetic drugs and their derivatives are available at present, side effects and increasing resistance to conventional therapies have led to the search for natural plant compounds.[6]
Homoeopathy, one of the complementary systems of medicine, which has many plant based medicines, is prepared by the process of serial dilution and succussion (vigorous shaking).[7,8] Despite the high dilutions employed, often beyond Avogadro’s limit (e.g., 12 centesimal potency [12C] or 30C), practitioners report that these remedies possess therapeutic effects. However, the scientific understanding of the mechanisms has not been explored properly till now. Few studies have demonstrated the presence of nanoparticles in homeopathic higher potencies and antioxidant properties. Ruta graveolens (RG) and Thlaspi bursa pastoris (TBP), both traditionally utilised in various medical systems.[4,9-11] While ethanolic extracts of these plants have been examined for their antioxidant potential, there is a paucity of data regarding their homoeopathic preparations, especially across various potencies.
The use of antioxidant assays such as 2,2-Diphenyl-1-picrylhydrazyl (DPPH), ferric ion reducing antioxidant power (FRAP), total phenolic content (TPC) and total flavonoid content (TFC) for testing homoeopathic potencies is required to explore the chemical and biological properties of homoeopathic remedies. These assays are commonly employed to identify the antioxidant potential and phytochemical composition (such as phenolics and flavonoids) of plant-based extracts and preparations. RG and TBP are prepared from plant sources and are commonly used for various kinds of cancer and haemorrhagic properties. However, the scientific basis for evaluating whether these potencies retain measurable antioxidant activity or phytochemical content, which could contribute to their therapeutic effects, has not been adequately addressed until now. The present study addresses this knowledge gap.
This study, therefore, aimed to investigate the TPC, TFC and antioxidant activity of homoeopathic preparations of RG and TBP at different potencies - Mother Tincture (Q), 6C, 12C and 30C.
MATERIAL AND METHODS
Materials and reagents
All the homoeopathic preparations of RG and TBP (Q, 6C, 12C and 30C) and ethanol were procured from a good manufacturing practice pharmaceutical company in Kolkata, India. Aluminium chloride, sodium acetate, DPPH, Folin–Ciocalteu reagent (FCR), sodium phosphate, 1% ferrocyanide, 10% trichloroacetic acid, 0.1% ferric chloride, gallic acid (GA), quercetin (QC) and ascorbic acid (AA) were procured from Hi Media, Mumbai, India.
Apparatus used
Trivitron LDx R1 is the name of a specific model of SemiAutomated ELISA Microplate Reader manufactured by Trivitron Healthcare (LDxR1)-enzyme-linked immunosorbent assay (ELISA) reader, flat-bottomed 96-well plates of maximum capacity 200 µL of each well (Tarson - 39233090), micropipette (Thermo Fisher Scientific) and microtips (Tarson).
DPPH-free radical scavenging assay
The antioxidant activity of the homoeopathic preparations of RG and TBP was evaluated with the DPPH radical with slight modifications. A solution of 0.1 mM of DPPH was prepared in a glass bottle by mixing 20 mg of DPPH with 500 mL of 91% ethanol and kept in the dark for 20 min after wrapping it with an aluminium foil. AA was used as the standard. The stock solution of AA was prepared by adding 0.5 g of ascorbic acid to 10 mL of ethanol, and a total volume of 100 mL was obtained by adding distilled water. Different concentrations (50, 100, 150, 200, 250, 300, 350, 400, 450, 500 mg/L) of AA were taken as working standard solutions, added to distilled water in 20 mL test tubes with a cap, and made to a total volume of 500 µL. To 100 µL of AA, 90 µL of DPPH solution was added in the 96-well plate. The 96-well plates were then incubated for 30 min at room temperature in the dark. Absorbance was measured, and a graph was plotted based on the mean values of AA. A calibration curve of ascorbic acid was prepared [Figure 1a]. The process was repeated with 50 µL each of the ethanolic preparations of RG and TBP (Q, 6C, 12C and 30C) and different concentrations of ethanol (56%, 70% and 91%). The concentration of antioxidants in the ethanolic preparations of RG and TBP was obtained as µg/mL of ascorbic acid equivalents (AAE). The entire process was performed in triplicate. Absorbance measured at 492 nm using the Trivitron LDxR1-ELISA reader was calculated as follows,
Free radical scavenging activity % = ([A0 – Am]/A0) × 100
Where, A0 = Absorbance of negative control, Am = Absorbance of mother tincture.
- Calibration curves (a) ascorbic acid, (b) gallic acid, (c) quercetin.
FRAP assay
The FRAP assay was performed as per the literature with slight modification, by a short procedure. AA was used as the standard. 100 µL each of Q, 6C, 12C and 30C of RG and TBP were taken in eight 20 mL test tubes with caps. 1 mL of ethanol, 2.5 mL of phosphate buffer (pH 7.4) and 2.5 mL of 1% ferrocyanide were added to each test tube. The test tubes were then incubated at 50°C for 20 min, wrapped with aluminium foil. Trichloroacetic acid (2.5 mL) was added, followed by centrifuging at 3000 rpm for 5 min and separation of 2.5 mL supernatant fluid. 2.5 mL of distilled water and 0.5 mL of ferric chloride were added to the supernatant fluid. Formation of bluish colour and its intensity indicated the antioxidant power. Finally, 96-well plates were used to find the absorbance at 630 nm. The concentration of antioxidants in the ethanolic preparations of RG and TBP was obtained as µg/mL of AAE. The entire process was performed in triplicate for greater accuracy.
TPC
The TPC of RG and TBP, each at Q, 6C, 12C and 30C, was estimated by FCR using GA as the standard. A stock solution of GA was made by adding 0.5 g of GA to 10 mL of ethanol, and a final volume of 100 mL was obtained by adding distilled water. Different concentrations of GA ranging from 50 mg/L to 500 mg/L were prepared. The various concentrations of working standard solutions (50, 100, 150, 200, 250, 300, 350, 400, 450, 500 mg/L) of GA were prepared with distilled water to get a final volume of 100 mL. To 100 µL of the above concentrations of working standard solutions of GA, 400 µL of distilled water, followed by 150 µL of FCR were added in test tubes. The solutions were incubated at room temperature for 5 min in the dark. 500 µL of 7.5% sodium carbonate was added, and the test tubes were incubated at room temperature in the dark for 45 min. They were transferred to a 96-well plate, and absorbance was checked at 630 nm. A graph was plotted based on the absorbance values of GA. A calibration curve was prepared taking GA as the standard [Figure 1b]. 100 µL each of RG (Q, 6C, 12C, 30C) and TBP (Q, 6C, 12C, 30C) was taken in different test tubes and the above procedure was repeated. The phenolic contents of RG and TBP were expressed in µg/mL of GA equivalents (GAE). The entire process was performed in triplicate for greater accuracy.
TFC
Aluminium chloride assay was used to determine the TFC of RG (Q, 6C, 12C, 30C) and TBP (Q, 6C, 12C, 30C). QC was the standard phenolic substance used for this study. The stock solution of QC was prepared by adding 10 mg of QC to 10 mL of ethanol and obtaining a volume of 100 mL by adding distilled water. The various concentrations of working standard solutions (50, 100, 150, 200, 250, 300, 350, 400, 450, 500 mg/L) of QC were prepared with distilled water to get a final volume of 1000 ml. To 100 µL of the above working standard solutions of QC were added 400 µL of ethanol, followed by 100 µL of aluminium chloride and 100 µL of 1M sodium acetate. The test tubes were incubated for 45 min in a dark room at room temperature. This solution was transferred to a 96-well plate and absorbance was measured at 450 nm using the Trivitron LDxR1-ELISA reader. A graph was plotted based on the absorbance values of QC. A calibration curve was prepared, taking QC as the standard [Figure 1c]. The above procedure was repeated with 100 µL of RG (Q, 6C, 12C, 30C) and TBP (Q, 6C, 12C, 30C) taken in different test tubes. The TPCs of RG (Q, 6C, 12C, 30C) and TBP (Q, 6C, 12C, 30C) were expressed in µg/mL of QC equivalents (QE). The entire process was performed in triplicate for greater accuracy.
Statistical analysis
Mean and standard deviation of the absorbance values of DPPH, FRAP, TPC and TFC were calculated. The DPPH and FRAP absorbance values of Q, 6C, 12C and 30C of both RG and TBP were analysed using the calibration curve of AA, and the concentration was expressed in µg/ml of AAE. TPC of the preparations of RG and TBP was calculated using the calibration curve of GA and the concentration was expressed in µg/ml of GAE. TFC of preparations of RG and TBP was calculated using the calibration curve of QC and the concentration was expressed in µg/ml of QE. The absorbance values were analysed using Microsoft Excel, with a oneway ANOVA conducted to assess the significance amongst Q, 6C, 12C and 30C potencies for each drug. Tukey’s post hoc test was subsequently applied to identify specific differences between the potencies. Statistical significance was determined using a p-value threshold of 0.05 and the results were reported accordingly.
RESULTS
The DPPH-free radical scavenging assay was performed to identify the antioxidant activity of RG and TBP (Q, 6C, 12C and 30C), and it was validated by FRAP. RG Q inhibits the DPPH-free radical at a higher concentration of 128.89 µg/mL and the FRAP-free radical at a higher concentration of 97.16 µg/mL of AAE, respectively. TBP Q inhibits the DPPH free radical at a higher concentration of 111.96 µg/mL and FRAP-free radical at a higher concentration of 97.74 µg/mL of AAE. RG Q and TBP Q showed higher antioxidant concentrations than their potencies [Table 1, Figures 2 and 3]. Further assays of RG Q , TBP Q, and their potencies were carried out for the identification of their TPC and TFC. The yellow coloured FCR became bluish coloured and the intensity of the blue colour indicated the percentage of its phenolic compounds. Dark blue colour shows more phenolic contents than light blue colour. TPC of RG Q at 100 µg/mL is 428.62 µg/mL and of TBP Q at 100 µg/mL is 158.06 µg/mL, respectively, of GAE; the rest of the potencies of both drugs demonstrated a concentration of phenols ranging from 63.47 µg/mL (TBP 6C) to 68.36 µg/mL (RG 6C) [Table 2 and Figure 4]. The colourless aluminium chloride becomes golden yellow in colour, and the intensity of the golden yellow colour indicates the percentage of its flavonoid content. The TFC of RG Q at 100 µg/mL is 313.37 µg/mL, and that of TBP Q at 100 µg/mL is 89.6 µg/mL, respectively, of QE; the rest of the potencies of both drugs did not demonstrate any flavonoid contents [Table 3 and Figure 5].
- 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay. (a) DPPH assay of Ruta graveolens (RG) and Thlaspi bursa pastoris (TBP). (b) Concentration of ascorbic acid equivalents (AAE) in DPPH assay of RG Q, TBP Q and their potencies (6C, 12C and 30C). (c) Mean absorption values of RG and TBP. Standard error of mean is represented by error bars.
- Ferric ion reducing antioxidant power (FRAP) assay (a) Ruta graveolens (RG) Q and its potencies. (a1) RG Q and its potencies - 96-well plate. (b) Thlaspi bursa pastoris (TBP) Q and its potencies. (b1) TBP Q and its potencies - 96-well plate. (c) Concentration of ascorbic acid equivalents (AAE) of RG Q and TBP Q and their potencies (6C, 12C and 30C). (d) Mean absorption values of RG and TBP. Standard error of mean is represented by error bars.
- Total phenolic content (TPC) analysis. (a) Ruta graveolens (RG) Q comparison with control. (b) Thlaspi bursa pastoris (TBP) Q comparison with control. (c) TPC analysis of RG Q, TBP Q and their potencies (6C, 12C and 30C). (d) Concentration of gallic acid equivalents (GAE) of RG Q, TBP Q and their potencies (6C, 12C and 30C). (e) Mean absorption values of RG and TBP. Standard error of mean is represented by error bars.
- Total flavonoid content (TFC) analysis. (a) Ruta graveolens (RG) Q comparison with control. (b) Thlaspi bursa pastoris (TBP) Q. comparison with control. (c) TFC analysis of RG Q, TBP Q and their potencies (6C, 12C and 30C). (d) Concentration of Quercetin equivalents (QE) of RG Q and TBP Q. (e) Mean absorption values of RG and TBP. Standard error of mean is represented by error bars.
| S. No. | DPPH | Ethanolic preparations | FRAP | ||
|---|---|---|---|---|---|
| Concentration (μg/ml) of AAE | Absorbance±SDM | Concentration (μg/ml) of AAE | Absorbance±SDM | ||
| 1. | 97.16 | 0.16±0.06 | RG Q | 128.89 | 0.39±0.04 |
| 2. | 92.83 | 0.13±0.01 | RG 6C | 94.63 | 0.14±0.02 |
| 3. | 91.95 | 0.12±0.004 | RG 12C | 102.76 | 0.19±0.03 |
| 4. | 103.09 | 0.20±0.07 | RG 30C | 102.49 | 0.19±0.02 |
| 5. | 97.74 | 0.16±0.03 | TBP Q | 111.96 | 0.26±0.02 |
| 6. | 92.47 | 0.12±0.01 | TBP 6C | 98.6 | 0.16±0.01 |
| 7. | 91.99 | 0.12±0.003 | TBP 12C | 96.09 | 0.15±0.01 |
| 8. | 92.47 | 0.12±0.002 | TBP 30C | 87.43 | 0.08±0.004 |
DPPH: 2,2-Diphenyl-1-picrylhydrazyl, FRAP: Ferric ion reducing antioxidant power, SDM: Standard deviation of mean, RG: Ruta graveolens, TBP: Thlaspi bursa pastoris, Q: Mother tincture, C: Centesimal potency, AAE: Ascorbic acid equivalents
| S. No. | Medicine | Concentration (μg/ml) of QE | Absorbance |
|---|---|---|---|
| 1. | RG Q | 428.62 | 2.829 |
| 2. | RG 6C | 68.36 | 0.091 |
| 3. | RG 12C | 64.68 | 0.063 |
| 4. | RG 30C | 65.16 | 0.066 |
| 5. | TBP Q | 158.06 | 0.772 |
| 6. | TBP 6C | 63.47 | 0.054 |
| 7. | TBP 12C | 65.87 | 0.072 |
| 8. | TBP 30C | 64.11 | 0.058 |
RG: Ruta graveolens, TBP: Thlaspi bursa pastoris, Q: Mother tincture, C: Centesimal potency, TPC: Total phenol content, QE: Quercetin equivalents
| S. No. | Medicine | Concentration (μg/ml) of QE | Absorbance |
|---|---|---|---|
| 1. | RG Q | 313.37 | 1.05 |
| 2. | RG 6C | Nil | 0.059 |
| 3. | RG 12C | Nil | 0.066 |
| 4. | RG 30C | Nil | 0.14 |
| 5. | TBP Q | 89.6 | 0.45 |
| 6. | TBP 6C | Nil | 0.071 |
| 7. | TBP 12C | Nil | 0.060 |
| 8. | TBP 30C | Nil | 0.054 |
RG: Ruta graveolens, TBP: Thlaspi bursa pastoris, Q: Mother tincture, C: Centesimal potency, TFC: Total flavonoid content, QE: Quercetin equivalents
The DPPH radical scavenging activity of RG preparations (Q, 6C, 12C, 30C) was analysed using one-way analysis of variance (ANOVA) and Tukey’s Honestly Significant Difference (HSD) post hoc test. The ANOVA revealed no statistically significant differences amongst the groups (F = 2.02, p = 0.1902), and none of the pairwise comparisons reached significance at α = 0.05. The DPPH radical-scavenging activity of TBP extracts demonstrated significant differences amongst the tested groups (one-way ANOVA: F (3,8) = 6.18, P = 0.0177, η2 = 0.699) and post hoc Tukey’s HSD analysis revealed that TBP Q exhibited significantly higher antioxidant activity than TBP 6C, TBP 12C and TBP 30C extracts (P < 0.05). However, no significant differences were observed amongst the three extract concentrations (TBP 6C, TBP 12C and TBP 30C), suggesting comparable antioxidant potentials within the tested range [Table 4].
| (a) One-way ANOVA summary | |||||||||
| Drug | Source | SS | df | MS | F | P-value | η2 | RMSSE | ω2 |
| RG | Between Groups | 0.01317 | 3 | 0.00439 | 2.02 | 0.1902 | 0.431 | 0.820 | 0.203 |
| Within Groups | 0.01741 | 8 | 0.00218 | ||||||
| Total | 0.03058 | 11 | |||||||
| TBP | Between Groups | 0.00378 | 3 | 0.001259 | 6.18 | 0.0177 | 0.699 | 1.435 | 0.564 |
| Within Groups | 0.00163 | 8 | 0.000204 | ||||||
| Total | 0.00541 | 11 | |||||||
| (b) Tukey HSD post hoc pairwise comparisons (α=0.05) for DPPH radical scavenging activity of RG and TBP | |||||||||
| Drug | Comparison | Mean difference | Standard error | Q-statistic | 95% confidence interval | P-value | HSD threshold | Cohen’s d | Significant (α=0.05) |
| RG | RG Q versus RG 6C | 0.0328 | 0.02694 | 1.22 | −0.0892, 0.1548 | 0.825 | 0.122 | 0.70 | No |
| RG Q versus RG 12C | 0.0393 | 0.02694 | 1.46 | −0.0827, 0.1613 | 0.737 | 0.122 | 0.84 | No | |
| RG Q versus RG 30C | 0.0443 | 0.02694 | 1.65 | −0.0777, 0.1663 | 0.664 | 0.122 | 0.95 | No | |
| RG 6C versus RG 12C | 0.0065 | 0.02694 | 0.24 | −0.1155, 0.1285 | 0.998 | 0.122 | 0.14 | No | |
| RG 6C versus RG 30C | 0.0771 | 0.02694 | 2.86 | −0.0449, 0.1991 | 0.256 | 0.122 | 1.65 | No | |
| RG 12C versus RG 30C | 0.0836 | 0.02694 | 3.10 | −0.0384, 0.2056 | 0.204 | 0.122 | 1.79 | No | |
| TBP | TBP Q versus TBP 6C | 0.0396 | 0.00824 | 4.81 | 0.0023, 0.0769 | 0.0380 | 0.0373 | 2.77 | Yes |
| TBP Q versus TBP 12C | 0.0432 | 0.00824 | 5.24 | 0.0059, 0.0805 | 0.0248 | 0.0373 | 3.03 | Yes | |
| TBP Q versus TBP 30C | 0.0397 | 0.00824 | 4.82 | 0.0024, 0.0770 | 0.0376 | 0.0373 | 2.78 | Yes | |
| TBP 6C versus TBP 12C | 0.0036 | 0.00824 | 0.44 | −0.0337, 0.0409 | 0.989 | 0.0373 | 0.25 | No | |
| TBP 6C versus TBP 30C | 0.00008 | 0.00824 | 0.01 | −0.0372, 0.0374 | 1.000 | 0.0373 | ~0 | No | |
| TBP 12C versus TBP 30C | 0.0035 | 0.00824 | 0.43 | −0.0338, 0.0409 | 0.990 | 0.0373 | 0.25 | No | |
RG: Ruta graveolens, TBP: Thlaspi bursa pastoris, Q: Mother tincture, C: Centesimal potency, HSD: Honestly significant difference, SS: Sum of squares, df: Degree of freedom, MS: Mean of squares, RMSSE: Root means square error, ANOVA: Analysis of variance. The significance value of ANOVA is p=0.05 and Post hoc test P=0.016. F: F-Statistic (F-Ratio), P: Probability Value, ɳ2: Effect Size (Eta Squared), RMSSE: Root mean square standard error, ω2: Effect size (Omega squared)
The FRAP assay of RG revealed highly significant differences in antioxidant activity amongst the tested groups (oneway ANOVA: F(3,8) = 53.28, P < 0.00001) and post hoc Tukey’s HSD analysis showed that the RG Q demonstrated significantly greater ferric reducing antioxidant power compared to RG 6C, RG 12C and RG 30C (P < 0.0001). The FRAP assay of TBP demonstrated significant differences in antioxidant capacity amongst the tested groups (F(3,8) = 127.65, P < 0.000001, η2 = 0.979), and Tukey’s HSD post hoc analysis revealed that the TBP Q exhibited significantly higher FRAP compared to both TBP 6C and TBP 12C extracts (P < 0.00001). However, no significant difference was observed between TBP 6C and TBP 12C (P = 0.310), suggesting similar antioxidant potential between these extract concentrations [Table 5].
| (a) One-way ANOVA summary | |||||||||
| Drug | Source | SS | df | MS | F | P-value | η2 | RMSSE | ω2 |
| RG | Between Groups | 0.112691 | 3 | 0.037564 | 53.28 | <0.000013 | 0.952 | 4.214 | 0.929 |
| Within Groups | 0.00564 | 8 | 0.000705 | ||||||
| Total | 0.118331 | 11 | 0.010757 | ||||||
| TBP | Between Groups | 0.054433 | 3 | 0.018144 | 127.65 | <0.000001 | 0.979 | 6.523 | 0.969 |
| Within Groups | 0.001137 | 8 | 0.000142 | ||||||
| Total | 0.05557 | 11 | 0.005052 | ||||||
| (b) Tukey HSD post hoc pairwise comparisons (α =0.05) for FRAP assay of RG and TBP | |||||||||
| Drug | Group comparison | Mean difference | Standard error | Q-statistic | 95% confidence interval | P-value | Mean-Crit | Cohen’s d | Significant (α=0.05) |
| RG | RG Q versus RG 6C | 0.2565 | 0.01533 | 16.73 | 0.1871, 0.3260 | <0.0000012 | 0.0694 | 9.66 | Yes |
| RG Q versus RG 12C | 0.1959 | 0.01533 | 12.78 | 0.1264, 0.2653 | <0.00009 | 0.0694 | 7.38 | Yes | |
| RG Q versus RG 30C | 0.1972 | 0.01533 | 12.86 | 0.1277, 0.2666 | <0.00008 | 0.0694 | 7.43 | Yes | |
| RG 6C versus RG 12C | 0.0607 | 0.01533 | 3.96 | −0.0088, 0.1301 | 0.0884 | 0.0694 | 2.28 | No | |
| RG 6C versus RG 30C | 0.0594 | 0.01533 | 3.87 | −0.0101, 0.1288 | 0.0962 | 0.0694 | 2.24 | No | |
| RG 12C versus RG 30C | 0.0013 | 0.01533 | 0.085 | −0.0681, 0.0707 | 0.9999 | 0.0694 | 0.05 | No | |
| TBP | TBP Q versus TBP 6C | 0.100633 | 0.006883 | 14.61973 | 0.069458, 0.131808 | <0.000031 | 0.031175 | 8.440707 | Yes |
| TBP Q versus TBP 12C | 0.118867 | 0.006883 | 17.26862 | 0.087692, 0.150042 | <0.000009 | 0.031175 | 9.970043 | Yes | |
| TBP Q versus TBP 30C | 0.188336 | 0.006883 | 27.36098 | 0.157161, 0.219511 | <0.000001 | 0.031175 | 15.79687 | Yes | |
| TBP 6C versus TBP 12C | 0.018233 | 0.006883 | 2.648888 | −0.01294, 0.049408 | 0.310346 | 0.031175 | 1.529336 | No | |
| TBP 6C versus TBP 30C | 0.087703 | 0.006883 | 12.74125 | 0.056528, 0.118878 | <0.00009 | 0.031175 | 7.356164 | Yes | |
| TBP 12C versus TBP 30C | 0.06947 | 0.006883 | 10.09236 | 0.038295, 0.100645 | 0.00045 | 0.031175 | 5.826828 | Yes | |
RG: Ruta graveolens, TBP: Thlaspi bursa pastoris, Q: Mother tincture, C: Centesimal potency, SS: Sum of squares, df: Degree of freedom, MS: Mean of squares, RMSSE: Root means square error, ANOVA: Analysis of variance. The significance value of ANOVA is p=0.05 and Post hoc test P=0.016. F: F-Statistic (F-Ratio), P: Probability Value, ɳ2: Effect Size (Eta Squared), RMSSE: Root mean square standard error, ω2: Effect size (Omega squared)
The TPC assay of RG showed highly significant differences in the phenolic content amongst the groups (one-way ANOVA: F(3,8) = 2364.859, P < 10−14, η2 = 0.998) and Tukey’s HSD analysis revealed that RG Q had significantly higher phenolic content than all extract groups (RG 6C, RG 12C, RG 30C; P < 10−11 for each comparison). The TPC assay of TBP showed highly significant differences in the phenolic content amongst the groups (one-way ANOVA: F(3,8) = 14140.55, P < 10−14, η2 = 0.998) and Tukey’s HSD analysis revealed that comparison of only TBP 6C versus TBP 30C was not significant (P = 0.6801) [Table 6].
| (a) One-way ANOVA summary | |||||||||
| Drug | Source | SS | df | MS | F | P-value | η2 | RMSSE | ω2 |
| RG | Between groups | 17.0839 | 3 | 5.6946 | 2364.859 | <1×10−14 | 0.998 | 28.08 | 0.998 |
| 0.01926 | 8 | 0.002408 | |||||||
| 17.1032 | 11 | 1.5548 | |||||||
| TBP | Between groups | 1.1385 | 3 | 0.3795 | 14140.55 | <1×10−14 | 0.998 | 68.66 | 0.999 |
| 0.0002 | 8 | 0.0000268 | |||||||
| 1.1387 | 11 | 0.103516 | |||||||
| (b) Tukey HSD post hoc pairwise comparisons (α =0.05) for TPC assay of RG and TBP | |||||||||
| Drug | Comparison | Mean difference | Std error | Q-statistic | 95% confidence interval | P-value | Mean- Crit | Cohen’s d | Significant (α=0.05) |
| RG | RG Q versus RG 6 | 2.7379 | 0.028332 | 96.64 | 2.6097,2.8662 | <10−11 | 0.1283 | 55.80 | Yes |
| RG Q versus RG 12 | 2.7659 | 0.028332 | 97.63 | 2.6376,2.8942 | <10−11 | 0.1283 | 56.37 | Yes | |
| RG Q versus RG 30 | 2.7623 | 0.028332 | 97.49 | 2.6339,2.8906 | <10−11 | 0.1283 | 56.29 | Yes | |
| RG 6 versus RG 12 | 0.0279 | 0.028332 | 0.99 | −0.1003,0.1562 | 0.8950 | 0.1283 | 0.57 | No | |
| RG 6 versus RG 30 | 0.0243 | 0.028332 | 0.86 | −0.1039,0.1526 | 0.9269 | 0.1283 | 0.50 | No | |
| RG 12 versus RG 30 | 0.0036 | 0.028332 | 0.13 | −0.1247,0.1319 | 0.9997 | 0.1283 | 0.074 | No | |
| TBP | TBP Q versus TBP 6 | 0.7188 | 0.00299 | 240.34 | 0.7053, 0.7324 | <10−11 | 0.0135 | 138.76 | Yes |
| TBP Q versus TBP 12 | 0.7006 | 0.00299 | 234.24 | 0.6871, 0.7141 | <10−11 | 0.0135 | 135.24 | Yes | |
| TBP Q versus TBP 30 | 0.7140 | 0.00299 | 238.73 | 0.7005, 0.7276 | <10−11 | 0.0135 | 137.83 | Yes | |
| TBP 6 versus TBP 12 | 0.0182 | 0.00299 | 6.10 | 0.0047, 0.0318 | 0.0110 | 0.0135 | 3.52 | Yes | |
| TBP 6 versus TBP 30 | 0.0048 | 0.00299 | 1.60 | −0.0088, 0.0183 | 0.6801 | 0.0135 | 0.93 | No | |
| TBP 12 versus TBP 30 | 0.0134 | 0.00299 | 4.49 | −0.0001, 0.0269 | 0.0519 | 0.0135 | 2.59 | Yes | |
RG: Ruta graveolens, TBP: Thlaspi bursa pastoris, Q: Mother tincture, C: Centesimal potency, TPC: Total phenolic content, SS: Sum of squares, df: Degree of freedom, MS: Mean of squares, RMSSE: Root means square error, ANOVA: Analysis of variance. The significance value of ANOVA is p=0.05 and Post hoc test P=0.016. F: F-Statistic (F-Ratio), P: Probability value, ɳ2: Effect Size (Eta squared), RMSSE: Root mean square standard error, ω2: Effect size (Omega squared)
The TFC assay of RG showed highly significant differences in flavonoid content amongst the groups (one-way ANOVA: F(3,8) = 1225.33, P < 10−10, η2 = 0.998) and Tukey’s HSD analysis revealed that RG Q had significantly higher flavonoid content than all extract groups (RG 6C, RG 12C, RG 30C; P < 0.000001 for each comparison). The TFC assay of TBP revealed highly significant differences in flavonoid content amongst the groups (one-way ANOVA: F(3,8) = 2190.33, P < 10−11, η2 = 0.999) and Tukey’s HSD post hoc analysis showed TBP Q had significantly higher flavonoid content compared to TBP 6C, TBP 12C and TBP 30C (P < 0.000001) [Table 7].
| (a) One-way ANOVA summary | |||||||||
| Drug | Source | SS | df | MS | F | P-value | η2 | RMSSE | ω2 |
| RG | Between Groups | 2.1091 | 3 | 0.7030 | 1225.33 | <1×10−10 | 0.998 | 20.21 | 0.997 |
| Within Groups | 0.0046 | 8 | 0.000574 | ||||||
| Total | 2.1137 | 11 | |||||||
| TBP | Between Groups | 0.3400 | 3 | 0.1133 | 2190.33 | <1×10−11 | 0.999 | 27.02 | 0.998 |
| Within Groups | 0.0004 | 8 | 0.0000517 | ||||||
| Total | 0.3405 | 11 | |||||||
| (b) Tukey HSD post hoc pairwise comparisons (α =0.05) for TFC assay of RG and TBP | |||||||||
| Drug | Comparison | Mean difference | Std error | q statistic | 95% confidence interval | P-value | Mean-Crit | Cohen’s d | Significant (α=0.05) |
| RG | RG Q versus RG 6 | 0.9942 | 0.01383 | 71.89 | 0.9315, 1.0568 | <0.000001 | 0.0626 | 41.50 | Yes |
| RG Q versus RG 12 | 0.9883 | 0.01383 | 71.47 | 0.9257, 1.0510 | <0.000001 | 0.0626 | 41.26 | Yes | |
| RG Q versus RG 30 | 0.9138 | 0.01383 | 66.07 | 0.8511, 0.9764 | <0.000001 | 0.0626 | 38.15 | Yes | |
| RG 6 versus RG 12 | 0.0058 | 0.01383 | 0.42 | −0.0568, 0.0685 | 0.9901 | 0.0626 | 0.24 | No | |
| RG 6 versus RG 30 | 0.0804 | 0.01383 | 5.81 | 0.0178, 0.1430 | 0.0144 | 0.0626 | 3.36 | Yes | |
| RG 12 versus RG 30 | 0.0746 | 0.01383 | 5.39 | 0.0119, 0.1372 | 0.0215 | 0.0626 | 3.11 | Yes | |
| TBP | TBP Q versus TBP 6 | 0.3787 | 0.00415 | 91.18 | 0.360, 0.398 | <0.000001 | 0.0188 | 52.64 | Yes |
| TBP Q versus TBP 12 | 0.3905 | 0.00415 | 94.03 | 0.372, 0.409 | <0.000001 | 0.0188 | 54.29 | Yes | |
| TBP Q versus TBP 30 | 0.3962 | 0.00415 | 95.40 | 0.377, 0.415 | <0.000001 | 0.0188 | 55.08 | Yes | |
| TBP 6 versus TBP 12 | 0.0118 | 0.00415 | 2.85 | −0.007, 0.031 | 0.2591 | 0.0188 | 1.64 | No | |
| TBP 6 versus TBP 30 | 0.0175 | 0.00415 | 4.21 | −0.0013, 0.0363 | 0.0685 | 0.0188 | 2.43 | No | |
| TBP 12 versus TBP 30 | 0.0057 | 0.00415 | 1.36 | −0.0131, 0.0245 | 0.7724 | 0.0188 | 0.79 | No | |
RG: Ruta graveolens, TBP: Thlaspi bursa pastoris, Q: Mother tincture, C: Centesimal potency, TFC: Total flavonoid content, SS: Sum of squares, df: Degree of freedom, MS: Mean of squares, RMSSE: Root means square error, ANOVA: Analysis of variance. The significance value of ANOVA is p=0.05 and Post hoc test P=0.016. F: F-Statistic (F-Ratio), P: Probability Value, ɳ2: Effect Size (Eta Squared), RMSSE: Root Mean Square Standard Error, ω2: Effect Size (Omega Squared)
DISCUSSION
The present study evaluated the antioxidant potential of Thlaspi bursa-pastoris and RG using two antioxidant assays (DPPH and FRAP) along with TPC and TFC analyses [Supplementary Data]. The mother tinctures of both plants exhibited significant antioxidant activity, with RG Q demonstrating more antioxidant activity compared to TBP Q. The TPC and TFC were also higher in RG Q. The antioxidant capacity of these mother tinctures supports the previous reports on the medicinal properties of these plants, particularly in traditional and homoeopathic medicine. Phenolic compounds and flavonoids are well-documented for their ability to scavenge reactive oxygen species (ROS), thereby helping to alleviate oxidative stress disorders such as diabetes, neurodegeneration and cancer.[12-17] However, these findings are based solely on in vitro chemical assays and cannot be directly extrapolated to clinical outcomes. Interestingly, lower potencies (Q and 6C) exhibited measurable antioxidant activity; the higher potencies (12C and 30C), particularly those beyond Avogadro’s number, exhibit antioxidant properties and show small amounts of phenolic content, but not flavonoids, despite the potencies being widely used are widely used in clinical homoeopathic practice. RG 30C exhibits higher antioxidant activity than TBP 30C, but evaluating its TPC and TFC provided more insight. While small amounts of phenols were found in 6C, 12C and 30C of both drugs, no flavonoid content was found in these potencies of either drug. This study does not provide evidence supporting the biological or therapeutic activity of higher potencies, as DPPH, FRAP, TPC and TFC assays showed results with Q and not with potencies. Hence, these assays may not be suitable for highly diluted homoeopathic potencies, as their sensitivity is limited to low concentrations. This represents a methodological limitation of the study. Therefore, higher potencies in homoeopathy require more preclinical investigations such as dynamic light scattering, Zeta potential analysis, ultraviolet-visible spectroscopy, Raman spectroscopy, thermal scanning and Fourier-Transform infrared spectroscopy, to identify and characterise nanoparticles, especially with higher potencies (30C).
In the DPPH assay, RG did not exhibit significant variation in DPPH scavenging across its potencies (Q, 6C, 12C, 30C). Although effect size measures (η2 = 0.431; η2 = 0.203) suggested a moderate proportion of variance explained by treatment, Tukey’s post hoc comparisons did not reveal statistically significant differences. Conversely, the potencies of TBP demonstrated measurable radical scavenging activity, albeit significantly lower than that of TBP Q. No significant variation was observed amongst TBP 6C, TBP 12C and TBP 30C, indicating similar activity across these potencies. The large effect size (η2 = 0.699) and strong Cohen’s d values (>2.7) between TBP Q and its potencies underscore the biological relevance of the observed differences. These findings are consistent with previous reports suggesting that TBP possesses antioxidant properties attributable to its phenolic and flavonoid constituents. The FRAP assay further strengthens these observations. RG Q has measurable reducing ability, and no significant differences were found amongst RG 6C, RG 12C and RG 30C, suggesting that increasing concentration within the tested range did not enhance antioxidant potential. These findings are consistent with DPPH assay outcomes, where RG Q exhibited antioxidant activity, but its potencies showed no significant enhancement. Similarly, TBP potencies demonstrated ferric reducing power, though considerably lower than TBP Q. The very high effect size (η2 = 0.979) and strong Cohen’s d values (8.44–15.80) confirmed the substantial difference in antioxidant activity between the standard and the potencies. The similar values of concentrations of AAE in the FRAP assay of TBP 6C (98.6) and TBP 12C (96.09) are corroborated in the Tukey HSD Post Hoc comparison between TBP 6C and TBP 12C (p = 0.31). The phytochemical analysis corroborated these results. TPC measurements confirmed that both TBP Q and RG Q contained higher levels of phenolic compounds compared to their potencies. Since phenols are well-established contributors to antioxidant activity, these results support the phenolic-mediated antioxidant capacity observed in the DPPH and FRAP assays. In RG, TFC confirmed the presence of measurable flavonoids, though RG 30C showed slightly lower content compared to RG 6C and RG 12C, based on Tukey HSD post hoc pairwise comparisons. This reduction at higher concentrations may be attributed to assay interference, reduced solubility, or flavonoid degradation/ complexation. Similarly, the Tukey HSD post hoc pairwise comparisons for TFC analysis of TBP Q demonstrated that it contained flavonoids, with no significant differences amongst TBP 6C, TBP 12C and TBP 30C.
These study findings showed that RG and TBP possess antioxidant properties that are largely dependent on their phenolic and flavonoid contents. The Q potencies consistently exhibited higher antioxidant potential compared to higher dilutions, while no substantial differences were observed amongst 6C, 12C and 30C. This suggests that the antioxidant activity of these plants is concentration-dependent, and higher dilutions may not significantly contribute additional effects. This study contributes to the small but growing body of research examining homoeopathic preparations through modern scientific methodologies. Although therapeutic efficacy cannot be claimed, this study instead highlights the presence of antioxidant activity in various potencies under experimental conditions. Further research, including in vivo models, pharmacokinetic profiling and clinical studies, is essential to understand the relevance and mechanisms better better.
Highlights
Mother tinctures (Q) of both plants showed significantly higher antioxidant activity compared to potencies (6C, 12C, 30C).
Ruta graveolens Q exhibited stronger antioxidant activity than Thlaspi bursa pastoris Q.
Total phenolic content (TPC) and total flavonoid content (TFC) were higher in mother tinctures than in potencies.
These findings provide preliminary in vitro evidence of antioxidant potential in mother tinctures (Q), but no direct clinical claims can be made.
CONCLUSION
This study demonstrates that RG and TBP possess measurable antioxidant activity, primarily mediated through their phenolic and flavonoid contents. The mother tinctures of both plants exhibited significantly higher antioxidant capacity compared to their respective potencies (6C, 12C, 30C), as shown in DPPH and FRAP assays. RG Q displayed higher antioxidant activity, phenolic and flavonoid contents than TBP Q. These findings confirm the phytochemical basis of antioxidant activity in mother tinctures, but they also highlight the need for further preclinical investigations, including advanced physicochemical characterisation and in vivo studies, to clarify the mechanisms and potential therapeutic relevance of higher homoeopathic dilutions.
Acknowledgement:
The authors would like to acknowledge the National Institute of Homoeopathy, where the study was conducted.
Ethical approval:
This is an article of an original, preclinical study where no patients and/or experimental animals were involved. Hence, institutional ethical approval is not required.
Declaration of patient consent:
Patient’s consent not required as there are no patients in this study.
Conflicts of interest:
There are no conflicts of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation:
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
Financial support and sponsorship: Nil.
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