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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 13  |  Issue : 2  |  Page : 100-106

Evaluation of antibacterial and antioxidant potential of some homoeopathic mother tinctures


1 Department of BHMS, University College of Conventional Medicine, Faculty of Pharmacy and Alternative Medicine, The Islamia University of Bahahwalpur, Bahwalpur, Punjab, Pakistan
2 Department of Pharmacy, The Islamia University of Bahahwalpur, Bahwalpur, Punjab, Pakistan

Date of Submission03-Aug-2017
Date of Acceptance17-May-2019
Date of Web Publication27-Jun-2019

Correspondence Address:
Dr. Tayyeba Rehman
IUB, Bahawalpur, Punjab
Pakistan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijrh.ijrh_44_17

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  Abstract 


Objectives: In homoeopathic system of medicine, mother tinctures are prescribed for several diseases especially for septic conditions and cure of many illnesses. This study was done to evaluate the antibacterial and antioxidant potential of ten commonly used homoeopathic mother tinctures. Materials and Methods: Ten prepared mother tinctures were tested against five clinically important human pathogenic bacteria (Salmonella typhi [S.T], Escherichia coli [E.C], Bacillus subtilis [B.S], Staphylococcus aureus [S.A], and Pseudomonas aeruginosa [P.A]) by broth micro-dilution method with ciprofloxacin as positive control. Antioxidant activity was estimated by 2,2-diphenyl 1-picryl hydrazyl (DPPH) inhibition assay. Ascorbic acid was taken as positive standard in antioxidant activity. Results: All mother tinctures showed more or less antibacterial activity. Cinchona officinalis had maximum activity (89% inhibition) against Salmonella typhi than all the tested mother tinctures. Pulsatilla nigricans showed the highest inhibition of DPPH (85%) among other tested plant mother tinctures. Conclusion: This study reveals that the above-tested mother tincture has antibacterial and antioxidant potential against the particular microorganism and 2,2- diphenyl 1-picrylhydrazyl (DPHH), respectively.

Keywords: Antibacterial, Antioxidant, Homoeopathic mother tinctures


How to cite this article:
Rehman T, Ahmad S. Evaluation of antibacterial and antioxidant potential of some homoeopathic mother tinctures. Indian J Res Homoeopathy 2019;13:100-6

How to cite this URL:
Rehman T, Ahmad S. Evaluation of antibacterial and antioxidant potential of some homoeopathic mother tinctures. Indian J Res Homoeopathy [serial online] 2019 [cited 2019 Oct 19];13:100-6. Available from: http://www.ijrh.org/text.asp?2019/13/2/100/261637




  Introduction Top


In Homoeopathy, mother tinctures are used for different types of infections including skin infections, wound infections and chronic ulcerative conditions. Some are used as topical antiseptics.[1] Moreover, various homoeopathic mother tinctures have antioxidant potential and are being used for the treatment of degenerative diseases.[2] Homoeopathic mothertinctures of plant origin are the hydroalcoholic extracts of medicinal plants, with some difference of ratio of alcohol and medicinal plants. The extracts of plant source contain secondary metabolites including glycosides, polyphenols, flavonoids, saponins, alkaloids and various others. They can significantly produce antioxidant, antimicrobial, antihypertensive, anti-diabetic, anti-inflammatory, antipyretic and various other therapeutic effects.[3]

Different methods can be used to assess antioxidant activity of a compound. However, the use of free radical 2,2-diphenyl 1-picryl hydrazyl (DPPH) is the preferred method to measure antioxidant activity as it is a simple and rapid method. Moreover, it is not specific for particular antioxidant component, thus measuring the total antioxidant capacity of sample. DPPH-free radical contains an odd electron and is purple. The colour changes from purple to yellow when the odd electron of DPPH radical combines with hydrogen from a free radical scavenging antioxidant to form the reduced DPPH-H.[2]

Different methods can be used to assess the antibacterial activity of a compound. Agar well/disk diffusion assays are the antibacterial methods that estimate antibacterial potential of a sample qualitatively with zone of inhibitions.[4] However, broth micro-dilution assay is the quantitative antibacterial susceptibility testing method that gives the percentage inhibition of bacteria in a micro-well.[5]

All the selected mother tinctures are conventionally used for the treatment of various types of diseases including infections, e.g., Pulsatilla nigricans belongs to Ranunculaceae family and it is well-known remedy for the treatment of anxiety, melancholy, mild restlessness and mental disturbance. Ovaritis, ovarialgia, pain due to acute inflammation, epididymitis, orchitis, uterine affections, indigestion, coryza, otitis, rhinitis, conjunctivitis, cough, cutaneous affections and acute meningitis are treated by Pulsatilla nigricans. Its roots are reported as antibacterial, anti-amoebic, antitumor, blood-cooling and detoxifier.[6] Thuja occidentalis belongs to Cupressaceae family. It helps avoid surgical intervention in cases of papilloma, epithelioma, polyps, pustules, ulcers and skin eruptions. In different research studies, Thuja occidentalis has been proved as antimicrobial against multiple pathogens.[7] Atropa belladonna belongs to Solanaceae family and is used for teething complaints, fever and different types of inflammatory conditions. Nux vomica belongs to Loganiaceae family and is used for various types of gastrointestinal diseases due to sedentary life style. Achillea millefolium belongs to the family Asteraceae and is useful for haematuria, dentition, chlorosis and leucorrhoea. Cinchona officinalis belongs to Rubiaceae family and is used for parasitic infections, diarrhoea and bloating conditions. Allium sativum belongs to Amaryllidaceae family and is used for colitis and various types of cardiovascular diseases.[8] Hamamelis virginiana belongs to Hamamelidaceae family and is useful for the treatment of inflammatory disorders and tumours.[9] Rhus toxicodendron belongs to Anacardiaceae family and treats skin ailments such as fever blisters, chicken pox and shingles hives. Berberis vulgaris belongs to Berberidaceae family and is used for the treatment of fever, cough, liver diseases and kidney diseases. Matricaria chamomilla belongs to Asteraceae family and is useful for the treatment of to cure digestive disorders, inflammatory disorders and anger related problems.[8]

The aim of the current study was to evaluate ten above-mentioned homoeopathic mother tinctures for their antioxidant and antibacterial properties. In literature review, no study has reported antioxidant and quantitative antibacterial potential of these homoeopathic mother tinctures.


  Materials and Methods Top


Chemicals and bacterial strains

DPPH, ascorbic acid and ciprofloxacin (Sigma-Aldrich, Germany) were used. Staphylococcus aureus (S.A) ATCC-6538 and Pseudomonas aeruginosa (P.A) ATCC-9027 were purchased from Microbiologics Inc. Bacillus subtilis (B.S),  Salmonella More Details typhi (S.T) and  Escherichia More Details coli (E.C) were purchased from first Fungal Culture Bank of Pakistan, Institute of Agricultural Sciences, University of Punjab, Lahore, Pakistan. Accession numbers were 12, 14 and 72, respectively.

Plant material

Plant parts were purchased from herbalist in local market. Ten plants were tested: (1) Cinchona officinalis bark, (2) Allium sativum fruit, (3) Strychnos nux-vomica seeds, (4) Pulsatilla nigricans whole plant, (5) Atropa belladonna, (6) Hamamelis virginiana, (7) Rhus toxicodendron leaves and twigs, (8) Berberis vulgaris, (9) Matricaria chamomilla, (10) Thuja occidentalis leaves and twigs and (11) Achillea millefolium whole fresh plant. The plants were identified by the botanist, Dr Sarwar, Lecturer, The Islamia University, Bahawalpur (Voucher No. 2201/L.S–2203/L.S, 2206/L.S–2212/L.S, respectively) and were deposited to IUB Herbarium.

Preparation of mother tinctures

100 g of each plant material in the powder form was placed in a flask of 2 L capacity for soaking in 900 mL of 70% ethanol for 15 days. The flask was kept in dark cool place after sealing and shaken for 10 min daily. After that, the material was filtered by coarse filtration through multiple layers of muslin cloth and then filtered by a filter paper. The filtrate was taken in glass bottles, sealed and stored in dark cool place.[10],[11]

Antioxidant activity

DPPH-free radical scavenging test was used for assessing antioxidant activity of homoeopathic mother tinctures.

2,2-diphenyl 1-picryl hydrazyl-free radical scavenging test

There are multiple methods for the evaluation of antioxidant activity, but DPPH-free radical scavenging method was the preferred one because it could conduct easily as it is stable, rapid and less expensive.[12]

Method of 2,2-diphenyl 1-picryl hydrazyl inhibition assay

DPPH inhibition assay was performed as mentioned by Mel et al.[13] with some adjustment. The concentration of DPPH 100 μM in methanol was used. Total assay volume was 100 μL containing 10 μL of the test solution and 90 μL of DPPH solution in a 96-well plate. The contents were mixed and incubated at 37°C for 30 min. Synergy HT BioTek® USA micro-plate reader was used to determine the diminution in absorbance at 517 nm. Standard antioxidant was ascorbic acid (0.1 mM). Seventy per cent alcohol was taken as negative control. Triplicate method was used for carrying out this experiment. The value of IC50 was calculated by Ez-fit-5 Perrella Scientific Inc., Amherst, USA software. Reduction in absorbance indicated greater radical scavenging activity which was determined by the following formula.

% Inhibition of free radicals = 100 − [(Absorbance of test solution/Absorbance of control)] × 100

Where

Absorbance of control = Total radical activity with solvent.

Absorbance of test = Activity in presence of test compound.

Antibacterial activity

Ten homoeopathic mother tinctures were evaluated for their antibacterial activity through broth micro-dilution method.

Method for inoculum preparation

For the preparation of bacterial inoculum, few colonies of each bacterium were taken from 24-h-old cultures and shifted into nutrient broth. Then, solution was set for the concentration of 1 × 108 CFU/mL. At this concentration, turbidity of the solution was nearly equivalent to 0.5 McFarland standard. Bacterial inoculums were stored at 4°C.[4]

Method for McFarland standard preparation

For preparation of 0.5 McFarland standard, 0.5 ml of 1.175% w/v barium chloride was added to 85 mL of 1% sulphuric acid (H2 SO4) and mixed them thoroughly. After mixing, 1% H2 SO4 was further added to make volume of 100 mL. Prepared solutions were checked for its optical density at 540 nm range which gives absorbance ranging from 0.12 to 0.19.

Broth micro-dilution method

The assay of antibacterial activity was described by Andrews.[14] In this method, the antibacterial activity was performed in sterile 96-well micro-plates. Total mixture volume in each well was 150 μL. It contained 75 μL of extract solution (5 mg/mL) and 75 μL of bacterial inoculum. Absorbance was checked at 540 nm using an ELISA micro-plate reader and it was taken as pre-read. After-read was taken at 540 nm after plates were incubated for 16–24 h at 37°C. The difference between pre-read and after-read was taken as an index of bacterial growth. All readings were taken in triplicate. Ciprofloxacin was taken as positive control (2 mg/mL) and distilled water was taken as negative control. Percentage inhibition was measured by following formula.

Inhibition (%) = O.D of sample/O.D of control × 100

Statistical analysis

The results were analysed by SPSS (IBM Statistical Package of the Social Sciences version 20.0) software. One-way analysis of variance (ANOVA) for repeated measures followed by least significant difference post hoc test was applied for checking statistical significance of results. Significance level was set at P ≤ 0.05.


  Results and Discussion Top


In the current study, ten homoeopathic mother tinctures were investigated for their antibacterial and antioxidant potential. Antibacterial activity was checked against two Gram-positive bacteria (S.A and B.S) and three Gram-negative bacteria (S.T, E.C and P.A). These bacterial strains are the commonly infection-causing bacteria in humans as B.S causes allergic reactions, food poisoning and eye infections.[15] S.A is responsible for various diseases including skin (boils, itch), soft tissue, bone, joint, food poisoning, cardiovascular, respiratory and wound infections. S.T is responsible for typhoid fever. P.A can cause infections such as urinary tract infections, pneumonia, ear and eye infections and traumatic wound infections. E.C is an opportunistic organism causing pneumonia and sepsis in immunocompromised host and meningitis.[16]

Flavonoids and phenolic compounds present in tinctures and extracts possess strong antioxidant potential.[2] Several homoeopathic mother tinctures investigated in this study contain these active constituents.

[Table 1] describes antibacterial activity of different mother tinctures against five bacterial strains while [Figure 1] describes antioxidant potential of homoeopathic mother tinctures. In the current study, homoeopathic mother tincture Thuja occidentalis showed percentage inhibition of 62.62 ± 1.33, 62.75 ± 1.15, 43.88 ± 1.15, 14.09 ± 0.44 and 37.25 ± 0.49 against S.T, E.C, B.S, S.A and P.A, respectively. In a previous study, Thuja occidentalis homoeopathic mother tincture was evaluated for its antibacterial potential against S.A and P.A. Disk diffusion assay showed 20 mm zone of inhibition against S.A while 24 mm zone of inhibition against P.A.[4] The results of the study were comparable to the current study in which mother tincture of Thuja occidentalis showed sufficient inhibition against all the tested strains. Hence, antibacterial activity of mother tincture of Thuja occidentalis is verified by broth micro-dilution assay that estimates the quantitative inhibition of bacteria. In the current study, homoeopathic mother tincture of Thuja occidentalis showed percentage inhibition of 82.34 ± 2.08 against DPPH at highest concentration tested (10 μL). In a previous study, extracts from cones of Thuja occidentalis were tested for their antioxidant potential using DPPH-free radical scavenging assay. This study expressed significant antioxidant results of tested extracts.[17]
Table 1: Antibacterial activity of mother tinctures

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Figure 1: Antioxidant potential of various mother tinctures against 2,2-diphenyl 1-picryl hydrazyl-free radical in descending order

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Homoeopathic mother tincture Pulsatilla showed that the percentage inhibition against S.T, E.C, B.S, S.A and P.A was 80.64 ± 0.89, 12.99 ± 2.85, 79.28 ± 0.59, 58.49 ± 2.66, 82.28 ± 2.96, respectively. Pulsatilla showed good percentage inhibition against S.T and P. Aand showed moderate-to-medium activity against B.S.Pulsatilla showed poor activity against E.C. Pulsatilla contains flavonoids, tannins, carbohydrates, glucoside pulsatoside, triterpene saponins and steroids.[18],[19] Flavonoids and triterpenoids have marked antimicrobial activity.[20] Antibacterial activity of Pulsatilla nigricans may be due to the presence of flavonoids and triterpenoids. Moreover, a previous study showed that protoanemonin from Pulsatilla nigricans is the main constituent responsible for its antibacterial activity.[21] Pulsatilla nigricans showed the highest inhibition of DPPH (85%) among other tested plant mother tinctures. It contains high content of flavonoids[18],[19] that might be responsible for its antioxidant activity.

Homoeopathic mother tincture Matricaria chamomilla showed the percentage inhibition against S.T, E.C, B.S, S.A and P.A 85.46 ± 2.24, 76.91 ± 3.40, 77.80 ± 1.60, 74.58 ± 0.04 and 79.18 ± 2.27, respectively. Chamomilla showed the highest percentage inhibition against S.T. Chamomilla showed medium percentage inhibition against E.C, B.S, S.A and P.A. Homoeopathic mother tincture Chamomilla showed 71.17 ± 2.80% inhibition of DPPH. In a previous study, Matricaria chamomilla essential oil showed decreased oxidation rate of soybean oil under accelerated conditions at 60°C.[22]

Homoeopathic mother tincture Hamamelis showed that the percentage inhibition against S.T, E.C, B.S, S.A, Pseudomonas is 80.64 ± 0.89, 12.99 ± 2.85, 79.28 ± 0.59, 58.49 ± 2.66 and 82.28 ± 2.96, respectively. Hamamelis mother tincture showed excellent antibacterial activity against S.T, B.S and P. Aand moderate-to-medium activity against S.A.Hamamelis showed medium antioxidant activity which is 68.10 ± 3.66. Polyphenolic flavonoids, anthocyanins, auxin and cytokinin are present in it[23] that might be responsible for its antioxidant and antibacterial potential.

Homoeopathic mother tincture Atropa belladonna showed that the percentage inhibition against S.T, E.C, B.S, S.A, P.A is 85.99 ± 0.22, 74.21 ± 2.56, 81.02 ± 0.38, 69.03 ± 1.55, 81.49 ± 2.25, respectively. Belladonna has excellent percentage inhibition against S.T, E.C and P.A and moderate activity against E.C and S.A. In a previous study, Atropa belladonna was evaluated for the antibacterial activity against certain pathogenic bacteria, namely B.S and Staphylococcus epidermidis antibacterial activity. Both the extracts, i.e., ethanolic and methanolic extract, of the therapeutic plant Atropa belladonna has revealed antibacterial activity against the tested bacterial species.[24] Belladonna has poor percentage inhibition of free radical which is 15.02 ± 3.24.

Homoeopathic mother tincture Nux vomica showed that the percentage inhibition against S.T, E.C, B.S, S.A and P.A is 88.34 ± 1.40, 71.41 ± 1.20, 85.95 ± 1.40, 83.20 ± 0.55 and 88.90 ± 0.90, respectively. Nux vomica has excellent percentage inhibition against S.T, B.S, S.A and P.A and moderate activity against E.C. In a previous study, the antibacterial activity of the Strychnos nux-vomica extract was performed by disc diffusion method. The extract was tested against four pathogenic bacterial strains of Gram-negative and Gram-positive bacteria. Ethyl acetate extract of Strychnos nux-vomica showed a significant antimicrobial activity.[25] Nux vomica Q has poor antioxidant activity which is 47.64 ± 4.49. Strychnine, the major component in the alcoholic extract of the seeds of Strychnos nux vomica, has no pro-oxidant property.[26]

Homoeopathic mother tincture Allium sativum showed percentage inhibition against S.T, E.C, B.S, S.A and Pseudomonas is 88.49 ± 0.52, 83.29 ± 0.18, 84.68 ± 0.12, 86.90 ± 1.66 and 81.14 ± 2.20, respectively. Allium sativum Q has excellent percentage inhibition against S.T, E.C, B.S, S.A and P.A. In a previous study, antibacterial activity of Allium sativum extract was apparent within 1 h of incubation and 93% killing of Staphylococcus epidermidis and S.T was achieved within 3 h of incubation.[27],[28] Radical scavenging and anti-oxidative effects of garlic powder have proved to be an effective antioxidant when tested against at hepatic micro some stressed by ascorbic acid/Fe”. Allicin has a very good hydroxyl radical. However, Allium sativum has poor percentage inhibition of DPPH in current study, i.e., 14.61.

Homoeopathic mother tincture Cinchona officinalis showed that the percentage inhibition against S.T, E.C, B.S, S.A and P.A is 89.23 ± 0.22, 85.29 ± 0.51, 75.33 ± 1.34, 80.79 ± 1.69 and 71.73 ± 2.49, respectively. Cinchona has excellent percentage inhibition against S.T, E.C and S.A. Medium percentage inhibition was showed by B.S. In previous studies, Cinchona showed antibacterial activity against several bacteria. Cinchona is highly recommended for the formulation of cosmetic products to protect the skin and hair against harmful microorganisms. Due to the presence of caffeic acid, flavonoids and the extract itself, Cinchona is useful in treating skin affections.[29] Homoeopathic mother tincture Cinchona officinalis has excellent percentage inhibition, which is 84.61 ± 3.98.

Homoeopathic mother tincture Achillea millefolium showed percentage inhibition against S.T, E.C, B.S, S.A and P.A is 29.03 ± 2.8, 59.50 ± 0.88, 65.73 ± 0.08, 68.03 ± 1.86 and 74.85 ± 1.24, respectively. Millefolium Q has moderate to medium percentage inhibition against E.C, B.S, S.A and P.A. In a previous study, Achillea millefolium was found to be mildly active against Gram-positive and Gram-negative bacteria.[30] Millefolium mother tincture has good antioxidant activity which is 77.81 ± 3.98. Results are in line to previous studies that showed Achillea millefolium possess a significant free radical scavenging activity which is due to the presence of phenolic compounds. The extract of Achillea millefolium has been investigated scavenging effects against DPPH radical. The antioxidant and total phenolic constituent levels are also positively correlated.[31]

Homoeopathic mother tincture Berberis vulgaris showed that the percentage inhibition against S.T, E.C, B.S, S.A and P.A was 60.75 ± 2.14, 87.83 ± 0.12, 69.69 ± 2.04, 71.14 ± 0.09 and 64.69 ± 0.09, respectively. Berberis vulgaris has excellent percentage inhibition against E.C and moderate-to-medium percentage inhibition against S.T, B.S, S.A and P.A. In a previous study, the ethanolic and aquatic extracts of Berberis vulgaris, at a concentration from 35 to 40 μg/mL, showed an antibacterial effect against both Gram-positive and Gram-negative bacteria. In particular, P.A and E.C were the most inhibited.[32] Berberis Q has good antioxidant activity which is 98.43%. Immunomodulatory and antioxidant effects of Berberis vulgaris fruits have been reported previously.[33]

Homoeopathic mother tinctures are available in market in ready for use form and this study provides their antioxidant and antibacterial potential. Hence, despite the reported antibacterial and antioxidant potential of some of these extracts, reporting antioxidant and antibacterial activities of mother tinctures is important and valuable.


  Conclusion Top


This study reveals that the above-tested mother tinctures have antibacterial and antioxidant potential against the particular microorganism and DPHH, respectively.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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