International Journal of ISSN: 2381-1803IJCAM

Complementary & Alternative Medicine
Research Article
Volume 5 Issue 1 - 2017
Evaluation of Antimicrobial Activity and Determination of Suitable Method for Antibiogram of Some Commercial Homeopathic Drugs
Foysal Hossen1, Otun Saha1, Sanjoy Kumar Mukharjee1*, Md. Imdadul Huque Khan2 and Mohammad Ruhul Amin1
1Department of Microbiology, Noakhali Science and Technology University, Bangladesh
2Department of Pharmacy, Noakhali Science and Technology University, Bangladesh
Received: August 10, 2016 | Published: January 03, 2017
*Corresponding author: Sanjoy Kumar Mukharjee, Department of Microbiology, Noakhali Science and Technology University, Noakhali-3814, Bangladesh, Tel: +88032162760; Email:
Citation: Hossen F, Saha O, Mukharjee SK, Khan MIH, Amin MR (2017) Evaluation of Antimicrobial Activity and Determination of Suitable Method for Antibiogram of Some Commercial Homeopathic Drugs. Int J Complement Alt Med 5(1): 00139. DOI: 10.15406/ijcam.2016.05.00139

Abstract

Background: Homeopathy is an alternative system of medicine that embraces a holistic, natural approach to treat illness. The aim of this study is to investigate the effectiveness of homeopathic drug against pathogenic organisms as well as to determine the most suitable methods for evaluating the antimicrobial activity of homeopathic drug.

Method: Four common homeopathic drugs which are available in Bangladesh were used in this study as an alternative medicine against various diseases like cholera, typhoid, diarrhea as well as gastrointestinal tract infection. For each drug sample both undiluted and diluted form of drug have been used simultaneously. Four methods (disc diffusion, agar well dilution, turbidimetric and drug inoculation into media) were used to test four common homeopathic drugs for screening antimicrobial activity and find out the inhibitory concentration of drugs. Microorganisms used in this study were clinically isolated Salmonella sp., Vibrio sp. and Escherichia coli.

Result: Among those four testing methods only medium inoculation method showed positive result of susceptibility (300µl/ml) therefore indicates its potentiality to test the homeopathic drugs for antimicrobial activity. In case of minimum inhibitory concentration values of the undiluted form of drugs showed that they are also active even in least concentrations ranged from 5µl/ ml to 200µl /ml but in the case of diluted drugs the ranged from 5µl/ ml to 149µl /ml.

Conclusion: This study has revealed that selected homeopathic drugs have some therapeutic values. Further broad range investigation could lead us to use these drugs as strong medicine in future.

Keywords: Homeopathic drugs; Therapeutic values; Pathogenic organisms; Disc diffusion; Antimicrobial activity

Abbreviations

CAM: Complementary or Alternative Medicine; MHA: Mueller-Hinton agar; TCAM: Traditional medicine/Complementary and Alternative Medicine; WHO: World Health Organization

Introduction

Homeopathic drugs are derived from various substances of plants, minerals, or animals that have been developed as a system of alternative medicine in 1796 by Samuel Hahnemann [1,2]. The word homeopathy is derived from the Greek root “homoios” (means similar or like treatment) and “pathos” (means of disease). Thus, the words together mean “like treatment of disease.” Although this therapeutic method has been practicing for more than 200 years, homeopathy is still among the most debatable of alternative medical therapies. Many scientists doubt about the therapeutic action of homeopathic drugs since homeopathic medicine remedies are so dilute that, in many cases, not a single molecule of the active compound present in the final preparation. Others argue that all curing performances of homeopathic preparations are either a placebo response, or simply a misinterpretation of normal healing that occurs naturally with the transition of time. However, studies involving homeopathic medicine treatment have yielded variable, contradictory results that lead us to nowhere in solving the contradiction.

Homeopathy is one kind of complementary or alternative medicine (CAM). This means that homeopathy act in different ways from treatments that are part of conventional Western medicine. A central principle of the "treatment" is that "like cures like" which means a substance that causes certain symptoms can also help to eradicate those symptoms. A second central principle is “succession” which is based on a method of dilution and shaking. Practitioners believe that the more a substance is diluted in this way, the better its potential to treat symptoms [3]. Many homeopathic remedies consist of substances that have been diluted many times in water until there is almost none of the original substance left. Homeopaths call this process Potentialisation [4].

To elucidate their mechanisms of action of homeopathic drugs many hypotheses have been postulated. Some hypotheses such as the theory of water memory [5-7], formation of clathrates [8], and epitaxy [9] are hypothetical in nature, while others have not been sufficiently tested, either due to complexity in validating the hypothesis or due to non-reproducible results i. e. those based on the quantum physical aspects of the solutions [10,11]. There is actually a larger body of clinical [12-14] and basic science [15,16] research that has tested various aspects of homeopathic medicines than most people recognize. A recent study claims that, to demonstrate the actual value of homeopathy, it deserves substantial and better research and an open-minded opportunity [17]. Therefore the present study was aimed at evaluating the antimicrobial activity of homeopathy drugs and determining suitable method for antibiotic susceptibility test of some commercial homeopathy drugs.

Materials and Methods

Source of drugs

The drugs used in this study were Podophyllum, China, Nux Vomica, Merc. Sol. Drug samples were collected from various Homeo Hall situated in Bangladesh.

Homoeopathic drugs selection

Those Homoeopathic drugs which are frequently used for gastrointestinal tract infection were selected and with their potency where Q indicates the Mother Tincture of Homeopathy drug and C indicate the Centesimal Scale of dilution. The drugs that are used in mother tincture format are Chaina (D1), Chaina (D2), Chaina (D3), Prodophyllum (D4), NuxVom (D5), Podophyllum (D6) and NuxVom (D7). The drugs that are used in diluted format are Merc. Sol. (D8), Podophyllum (D9), Chaina (D10) and NuxVom (D11).
Determination of antimicrobial efficacy.

Test microorganisms

The bacterial samples used in this experiment were collected from the laboratory of Microbiology department, Noakhali Science and Technology University as preserved sample which are isolated from various clinical samples. In this experiment three organisms were used and these are Escherichia coli, Salmonella sp. and Vibrio sp.

Antibacterial screening

Antimicrobial activities of homeopathic drugs were determined by the disc diffusion assay, agar well diffusion assay, turbidimetric method and drug inoculation into medium method.

Disk diffusion assay

To perform disk diffusion assay [18], previously prepared 3 different bacterial suspensions (Escherichia coli, Salmonella sp. and Vibrio sp.) were inoculated onto three Mueller-Hinton agar (MHA) plates for each (total 9) with a sterile cotton swab. Then the antimicrobial disks were inoculated onto the surface of the inoculated agar plate. Each disk was pressed down individually to ensure complete contact with the agar surface. The disk placed in the agar surface was not closer than 24 mm from center to center. The plates were inverted and placed in an incubator set to 35±2 °C within 15 minutes after the disks were applied. The diameter of inhibition zone was measured after 24 h incubation using a ruler.
Agar well diffusion assay [19].

Small agar wells were prepared with the help of sterilized cork borer with 10 mm diameter. The selected strains of bacteria were inoculated into 10mL of sterile nutrient broth, and incubated at 35±2 °C for 24 hours. The cultures were swabbed on the surface of sterile Mueller Hinton agar plates using a sterile cotton swab. Using a micropipette, 50µl/ml and 100µl/ml of different concentrations of each drug were added to the wells in the plate respectively. The plates were incubated in an upright position at 35±2 ℃ for 24 hours. The diameter of inhibition zones was measured in mm and the results were recorded.
Turbidimetric method [20].

Each drug of 150µl/ml and 200µl/ml concentrations were inoculated in each of the tubes containing 2ml nutrient broth previously seeded with the appropriate test organism. At the same time three control tubes were prepared, one containing the inoculated culture medium (culture control), another identical with the first one but treated immediately with drugs (blank) and a third one containing uninoculated culture medium. All the tubes were placed at randomly distributed manner in an incubator and maintained them at 35±2℃ for 24 hours.

Drug inoculation into media method [21]

In drug inoculation into media method, 300µl of each drug solution was added to nutrient agars that have previously been autoclaved to 121 °C for 15 minutes. The agar and drug solution were mixed thoroughly and poured into Petri dishes on a level surface to result in an agar depth of 3mm to 4mm. After proper mixing the plates were poured quickly to prevent cooling and partial solidification in the mixing container. Then the plates were kept at room temperature to solidify and then one loop full of desired organisms were transferred in these plates through streaking method. All the plates were incubated at 35±2 °C for 24 hours.

Results and Discussion

In this study 3 clinically isolated Microbes, 11 homeopathic drugs were used and anti-microbial test were performed by four methods. All the isolates were showing resistance against all forms of homeopathy drugs in disc diffusion method and Agar well diffusion methods. On the other hand, in turbidimetric methods isolates were showing resistance against all forms of homeopathy drugs except D8, D9, D10 and D11 (diluted form). Finally isolates were showing sensitivity against all forms of homeopathy drugs in drug inoculation into media method. The results are shown in Table 1-3.

Tested Microorganisms (Salmonella Sp.)

70% Alcohol (Control) In Every Case

Disk Diffusion Test (5µl)

Agar Well Diffusion (50µl)

Agar Well Diffusion (100µl)

Turbidimetric (150µl)

Turbidimetric (200µl)

Drug Inoculation Into Media (300 µl)

D1-Chaina

S

R

R

R

R

R

S

D2-Chaina

S

R

R

R

R

R

S

D3-Chaina

S

R

R

R

R

R

S

D4-Podophyllum

S

R

R

R

R

R

S

D5-NuxVom

S

R

R

R

R

R

S

D6-Podophyllum

S

R

R

R

R

R

S

D7-NuxVom

S

R

R

R

R

R

S

D8-Merc Sol

S

R

R

R

S

S

S

D9-Podophyllum

S

R

R

R

S

S

S

D10-Chaina

S

R

R

R

S

S

S

D11-NuxVom

S

R

R

R

S

S

S

Table 1: Effect of different conc. of selected homeopathic drug on clinically isolated Salmonella sp. through different methodology.

Tested Microorganisms
(Vibrio sp.)

70% Alcohol (Control) In Every Case

Disk Diffusion Test (5µl)

Agar Well Diffusion (50µl)

Agar Well Diffusion (100µl)

Turbidimetric (150µl)

Turbidimetric (200µl)

Drug Inoculation Into Media (300 µl)

D1-Chaina

S

R

R

R

R

R

S

D2-Chaina

S

R

R

R

R

R

S

D3-Chaina

S

R

R

R

R

R

S

D4-Podophyllum

S

R

R

R

R

R

S

D5-NuxVom

S

R

R

R

R

R

S

D6-Podophyllum

S

R

R

R

R

R

S

D7-NuxVom

S

R

R

R

R

R

S

D8-Merc Sol

S

R

R

R

S

S

S

D9-Podophyllum

S

R

R

R

S

S

S

D10-Chaina

S

R

R

R

S

S

S

D11-NuxVom

S

R

R

R

S

S

S

Table 2: Effect of different conc. of selected homeopathic drug on clinically isolated Vibrio Sp. through different methodology.

Tested Microorganisms
(E. coli)

70% Alcohol (Control) In Every Case

Disk Diffusion Test (5µl)

Agar Well Diffusion (50µl)

Agar Well Diffusion (100µl)

Turbidimetric (150µl)

Turbidimetric (200µl)

Drug Inoculation Into Media (300 µl)

D1-Chaina

S

R

R

R

R

R

S

D2-Chaina

S

R

R

R

R

R

S

D3-Chaina

S

R

R

R

R

R

S

D4-Podophyllum

S

R

R

R

R

R

S

D5-NuxVom

S

R

R

R

R

R

S

D6-Podophyllum

S

R

R

R

R

R

S

D7-NuxVom

S

R

R

R

R

R

S

D8-Merc Sol

S

R

R

R

S

S

S

D9-Podophyllum

S

R

R

R

S

S

S

D10-Chaina

S

R

R

R

S

S

S

D11-NuxVom

S

R

R

R

S

S

S

Table 3: Effect of different conc. of selected homeopathic drug on clinically isolated E. coli through different methodology.

Over the last few decades there has been a substantial consciousness worldwide in Traditional medicine/ Complementary and Alternative Medicine (TCAM) particularly in herbal products [22] and virtually half of the population currently often customize some form of complementary and alternative medicine [23]. Homeopathy can be the best choice among them. Today homeopathy is widely practiced in all over the world: North America, India, Mexico, South America, Germany, France, Great Britain, Russia, and other European countries [24]. In recent years, there have been a number of calls on World Health Organization (WHO) to support efforts to regulate the safety of homeopathic medicines [25]. Several clinical trials have evidenced the effectiveness of homeopathic medicines for treatment of serious illnesses. A study showed a substantial improvement with homeopathic treatment, where conventional treatment had been unsatisfactory or contraindicated [26].

In Bangladesh, homeopathy dug treatment is very popular with other forms of treatment. Though, educated person and peoples of urban area commonly deny homeopathy but it’s a reliable cure system in village. Still now there was no significant in vitro research about the acceptability of this drug treatment. Many people are used to take this treatment from village homeopathic doctors when they suffer some gastrointestinal dieses like as typhoid, cholera as well as diarrhea. In this research, we tried to determine the antimicrobial activity of some selected homeopathic drugs by in vitro method as well as to evaluate the most effective methods for determining antibacterial activity. The study reveals that, the inhibitory concentration range for the selected drug against clinically isolated microorganisms is within 201µl/ml to 300µl/ml. This finding claims the antibacterial potentiality of homeopathic drugs.

At present the concern about the homeopathy is that, most of the homeopathy doctors don’t know or concern about the dose as well as concentration of drugs which is really active against pathogens. Again, they are not well trained to make a dilution from pure or mother drugs. That way, due to improper dilution, the ratio of active ingredient would be down and ultimately the microbes will be resistant.

From this study, we have seen that diluted drugs of different drug samples were active against all pathogenic microorganisms using 150µl/ml and 200µl/ml where mother drugs remain resistant. So, here, the active ingredient was not mother component, it was dilutant. Commonly, most of the doctors use pure alcohol as a dilutant and it is well know that alcohol has the antimicrobial activity.

The Disc diffusion method is not totally suitable for screening the antimicrobial activity of homeopathic drugs. This is due to the fact that discs don’t absorb huge amount of drugs. It is not possible to apply more than 5µl/disc. Again, agar well diffusion method is better than disc diffusion method but it has also limitations in regards of amount. It is contradictory to use more than 100µl of amounts because the well doesn’t carry this huge amount.

Turbidimetric is the more acceptable method if sample amount is more than other. We can observe a clear growth of microorganisms visually as well as determining the concentration by spectrophotometric method. But using homeopathic drugs, more drugs can change the color of media if we use more than 200µl. So, it is very difficult to determine the growth of microorganisms by observing turbidity using naked eye as the only way. But it is very much effective using any amount of drugs if drugs are incorporated into the media directly.

It is clear that homeopathic drugs have the antibacterial activity but in high amount of dose. Although the determination and maintenance of concentration in homeopathic drugs is not mainstream practice methods, maintaining the appropriate concentration might help to identify more potent drug preparation. In our country the actual concentration of homeopathy drugs is not also maintained by the doctors. For commercial purposes and to earn high profit, they make more dilution of drugs which might not be adequate enough to kill the disease causing microorganisms. Again, they are not so much literate about the origin, specification and doses of the homeopathic drugs. So, to evaluate the actual efficiency of those drugs, a lot of research is needed in future.

Conclusion

The present investigation can conclude that the homeopathic drugs showed antimicrobial activity at very concentrated form. However whether such drugs will act as an effective therapeutic agent remain to be investigated and to identify the actual effectiveness of those drugs in antimicrobial activity testing need to use mother tincture form of those drugs from actual manufacturer industry.

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