Antioxidant effects of Homoeopathic Medicines: Review Based on Preclinical and Clinical Research

Background: Antioxidants are rampantly studied due to their ability to trap the free radicals. Due to excessive stress these free radicals take part as fundamental components in oxidative damage leading to changes in SOD, CT, GSSH, GPx, GSH, LPO, GGT, LDH. High Dilution Medicines (HDM) are also being investigated in deciphering their antioxidant properties. Methodology: The database for the research paper was screened from Google scholar, PubMed, and Web of Science. The search keywords used were Antioxidant, Homoeopathy, Homeopathy, Invitro, In-vivo, Clinical trial in various permutation and combinations. The research article in original full text with English language is included in this manuscript. Score for assessment of biological experiment on homeopathy (SABEH) was referred for screening the research articles. Results: Around 14 research manuscript showed the research conducted on HDM with respect to their antioxidant properties. Four In-vitro, Eight In-vivo studies and two Clinical studies were used in Review Article Patil et al.; JPRI, 33(32B): 8-21, 2021; Article no.JPRI.69721 9 assessing the antioxidant activity. Out of 14, only 12 studies were included in SABEH criteria scores above 5/9. SOD, CT, GSH, GPx, LPO, GSSH, GGT, LDH, 2,2 DPPH were found to be explored with HDM with their antioxidant properties. Conclusion: HDM can further be studied at to what extent they provide preventive role, interception role and repair role pertaining with their antioxidant properties, also comparing the efficacy of antioxidant properties through oral route and injectable in various stages of disease.

assessing the antioxidant activity. Out of 14, only 12 studies were included in SABEH criteria scores above 5/9. SOD, CT, GSH, GPx, LPO, GSSH, GGT, LDH, 2,2 DPPH were found to be explored with HDM with their antioxidant properties. Conclusion: HDM can further be studied at to what extent they provide preventive role, interception role and repair role pertaining with their antioxidant properties, also comparing the efficacy of antioxidant properties through oral route and injectable in various stages of disease.

INTRODUCTION
Antioxidants are rampantly studied due to their ability to trap the free radicals. Highly reactive free radicals formed due to imbalance in the molecular fragments of unpaired electrons in molecular orbit leads to many cascade changes in cellular structures.
[1] These free radicals can cause degenerative diseases by oxidizing proteins, lipids, nucleic acids, and DNA [2] Due to excessive stress these free radicals take part as fundamental components in oxidative damage leading to changes in SOD, CT, GSSH, GPx, GSH, LPO, GGT, LDH. [3] To counteract these changes naturally present antioxidant like enzyme (glutathione) and non-enzymatic antioxidant (Vitamin A, E, C) plays a vital role in bringing the homeostatic changes in cells (Fig.  1). [4] Antioxidants are well known for their various benefits to human health in concerns of improving quality of sleep, preventing neurodegenerative changes, lowering blood pressure and reduced obesity, improves eye vision, protects liver toxification, supports immune system, anti-aging effects and protects renal toxification. Depending upon the stressor's embedding the cellular responses antioxidants have different mechanism of actions like preventive role (which stops the production of Reactive Oxygen Species), interception role (which protects the radical scavenging activity caused by impaired electron of oxygen) and repair role (which brings repair mechanism cellular components caused by free radical molecule). [5][6][7] Homeopathy also known as High dilution medicines (HDM) are used in research for exploring its concealed properties and understanding its pharmacology. There seems to be a paradigm shift in understanding the literature evidence of the HDM and correlating it with preclinical (In vitro & In vivo) and clinical research pertaining to antioxidant activity. This review highlights the role of homeopathic medicines as an antioxidant activity investigated in preclinical and clinical studies.

METHODOLOGY
The database for the research paper was screened from Google scholar, PubMed, and Web of Science. The search keywords used were Antioxidant, Homoeopathy, Homeopathy, In-vitro, In-vivo, Clinical trial in various permutation and combinations. The research article in original full text with English language is included in this manuscript. Score for assessment of biological experiment on homeopathy (SABEH) was referred for screening the research articles. [8] 2.1 Score for Assessment of Biological Experiment on Homeopathy (SABEH) [8] To identify the quality of research paper for inclusion and exclusion criteria, SABEH was designed. SABEH mentions 9 parameters like a) Defined Objectives b) Control c) Blinding of measurement of outcomes d) Randomization e) Consistency f) Experiment standardization g) Statistical analysis h) Result. Each parameter gives score of 1 mark. Experimental standardization mentions 2 marks. SABEH score is calculated based on presence of abovementioned parameter in selected research paper for review. SABEH score higher than 5 marks are included in for further analysis. Each parameter in SABEH criteria has its relevance when implemented in screening the research paper.
1. Defined Objectives (1 mark) -Research paper defining problems or questions based on relevant theory or empirical theory exploring potentials of hypothesis needs to be taken into consideration. Research paper defining the parameter will be scored as 1 mark, if in case no relevance found will be scored 0.
2. Control (1 mark) -Research paper defining the relevance of control used and its interpretation need to be taken into consideration. Research paper defining the parameter will be scored as 1 mark, if in case no relevance found will be scored 0.
3. Blinding of measurement of outcomes (1 mark) -Research paper defining the blinding measurement as to avoid several biases are present when a study is insufficiently blinded, to avoid confirmation bias (based on existing belief). Research paper defining the parameter will be scored as 1 mark, if in case no relevance found will be scored 0.
4. Randomization (1 mark) -Research paper defining randomization method ensures accidental bias and prevents selection bias. Research paper defining the parameter will be scored as 1 mark, if in case no relevance found will be scored 0.
5. Consistency (1 mark) -Research paper defining consistency for any research work, to which similar findings are reported using similar and different study designs and based on clear chains of inferential reasoning supported and justified by a complete coverage of the relevant literature. Research paper defining the parameter will be scored as 1 mark, if in case no relevance found will be scored 0.
6. Experiment standardization (2 marks) -Research paper defining experiment standardization based on quality and quantity of data interpreted. Quality (1 mark) is scored if the aggregate of quality ratings for individual studies, predicated on the extent to which bias was minimized in the study designs is validated. Quantity (1 mark): the number of studies, the sample size, the study design's statistical power to detect meaningful effects, and magnitude of the effects found or the effect size. Research paper defining the parameter will be scored as 2 marks, if in case no relevance found for either quality or quantity a score of 1 mark will be imposed, while no relevance found for both quality and quantity it will be scored 0.
7. Statistical analysis (1 mark) -Research paper defining correlation of statistical data of text and figures/graph interpreted in research paper need to screen cautiously. Research paper defining the parameter will be scored as 1 mark, if in case no relevance found will be scored 0.
8. Result (1 mark) -Research paper defining correlation of statistical results interpretating with precise term of biological response need to be cross verified. Research paper defining the parameter will be scored as 1 mark, if in case no relevance found will be scored 0.

RESULTS AND DISCUSSION
Around 14 research manuscript showed the research conducted on HDM with respect to their antioxidant properties. Four In-vitro, Eight In-vivo studies and two Clinical studies were used in exploring antioxidant activity highlighted in Table  1. Out of 14, only 12 studies were included in SABEH criteria explained in Table 2. SOD, CT, GSH, GPx, LPO, GSSH, GGT, LDH, 2,2 DPPH were found to be explored with HDM with their antioxidant properties.
The quality of research manuscript was assessed by SABEH criteria modified through previous conducted systematic review. The SABEH criteria include details (Objectives, Controls, Blinding, Randomization, Consistency, Experiment Standardization, Statistical analysis, Results interpretation.), each representing score of 1, which gives sum of 9. Paper's having SABEH score higher than 5 had been included in the review study. [7]

Perspective on Pharmacology of Antioxidants of Homeopathic Medicines Based on Biological Studies (Preclinical)
Saeed Ahmad et.al explored the antioxidant activity of Mother Tinctures (Q) through 2,2 Diphenyl 1 picrylhydrazyl free radical scavenging Assay and further demonstrated the phenolic content in the Q's. The medicines used were Syzygium jambolanum, Damiana, Cinchona officinalis, Chelidonium majus, Convallaria majalis and Coca (All Q's had 90% alcohol as a solvent base). The comparator used was Quercitin and the phenolic content was compared with gallic acid as to explore the antioxidant activity through 2,2DPPH. S. jambolanum (5 µl) showed 87.2% inhibition and S. jambolanum (1.25 µl) showed 39.9% inhibition of DPPH. Damiana, C. officinalis, C. majus, C. majalis, and Coca inhibited DPPH 81.6%, 69%, 68.5%, 65.1%, and 61%, respectively, at 5 µl volume. There was decreased in percent inhibition with the decrease of volume against DPPH. [9] Celso Fernandes Batello studied antioxidant activity of Homeopathic medicines through Lipid peroxidation inhibition assay in-vitro in Rat Brain. The medicines used were Arsenic album, Cuprum metallicum, Zinc metallicum and Magnum in 6C, 12C & 30C potencies respectively.

10]
Vasavan Jyothilakshmi et.al investigated the antioxidant activity of Homeopathic preparations of Berberis Vulgaris (HPBV) against the oxidative stress induced in renal tissues of experimental rats. Group I (G1) rats were untreated control. Group II (G2) received ethylene glycol (EG 0.75% in drinking water) for induction of a chronic low-grade hyperoxaluria and further to generate CaOx deposition in kidneys. Group III (G3) rats received oral administration of HPBV, (20 ul/ day/100 mg body weight) concomitant with EG administration as in group II. Group IV (G4) rats served as drug controls and were given HPBV (20 ul/day/100 mg body weight, oral gavage) for 28 days. Berberis vulgaris 200C (20ul/100mg bw) and Ethylene glycol (EG 0.75% in drinking water) as comparator was investigated through assay of Enzymatic   Nat sulph (17%), Kali carb (19%), Ferrum Phos (14%) and Ammonium carb (13%) were prescribed to the patients. 11 homeopathic medications most prescribed for 23 patients whose follow up blood sample was taken. Ars alb, Puls nigrican, Nat sulph, Ferrum phos and Antim tart was observed to be prescribed for the patients for treatment. LP had decreased in the erythrocyte which showed that homeopathic treatment had some effect in reducing oxidative stress. Further it was confirmed with plasma vitamin C and erythrocyte SOD levels which interpreted to be normal, but oxidant stress had not resolved completely within the period of study as plasma AOA had still not returned to be in its normal control levels. Pre-treatment levels of GSH µmol/g Hb), SOD (units/g Hb), Catalase (Units/g Hb) & GR (Units/g Hb) were 6.02+0.

Future Perspective
Homeopathy also known as HDM was discovered by German physician Samuel Hahnemann through a process named as drug proving (DP) which is performed on healthy volunteers.
[23] DP comprises of interpretation of drug action of homeopathic medicines in the form of subjective analysis or perception explained by the volunteer to whom the homeopathic medicine was administered.
[24] As per the empirical sources homeopathy is safe for human consumptions. Recent appraisal of presences of physical entity in this HDM had made a remarkable change in perspective of understanding its pharmacology, as the HDM earlier was questioned about its efficacy (Safety & Mechanism of action) and controversy of Avogadro's equation (pertaining to presence of any physical entity in HDM preferably above 12C as it crosses the limit of Avogadro constant i.e 6.024 x 10 23 ) researched and now explored in a domain of nanoscience. [25-27] From the above cited research these HDM from various preclinical studies have shown antioxidant properties which might provide an essential guide in predicting dose for first in human or initial phase studies or in drug proving trial, which might be used as standard protocol in chronic disease conditions wherein these free radicals are targeted to be researched. Allium cepa 200C (NS) had also shown antioxidant activity. HDM can further be studied at to what extent they provide preventive role, interception role and repair role pertaining with their antioxidant properties, also comparing the efficacy of antioxidant properties through oral route and injectable in various stages of disease. This review also confirms that the HDM's have no toxicity and provides antioxidant properties when compared with standard control and vehicle control.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.