Prophylactic potential of a Panchgavya formulation against certain pathogenic bacteria

A Panchgavya preparation was evaluated for its prophylactic efficacy against bacterial infection, employing the nematode worm Caenorhabditis elegans as a model host. Worms fed with the Panchgavya preparation prior to being challenged with pathogenic bacteria had a better survival rate against four out of five test bacterial pathogens, as compared to the control worms. Panchgavya feeding prior to bacterial challenge was found to be most effective against Staphylococcus aureus, resulting in 27% (p=0.0001) better worm survival. To the best of our awareness, this is the first report demonstrating in vivo prophylactic efficacy of Panchgavya mixture against pathogenic bacteria.

Introduction 'Panchgavya' is a term used to describe a combination of five major substances obtained from cow, including cow's urine, milk, ghee (clarified butter), curd and dung. Dhanvantari, referred to as the God of Indian Medicine, is said to have offered to mankind this wonder medicine called Panchgavya. In Sanskrit, all its five ingredients are individually called 'Gavya' and collectively termed as Panchgavya (panch means five). Panchgavya products have been claimed to be beneficial in curing several human ailments, enhancing immunity and providing resistance to fight infections (Dhama et al., 2005). Panchgavya therapy (cowpathy) has been indicated as an alternate prophylactic and therapeutic modality for sound livestock and poultry health along with human health (Dhama et al., 2014). Panchgavya Prashan is a common tradition followed by certain communities (e.g. Telugu Brahmins) in India, wherein a Panchgavya dose is taken once every year during monsoon season. The potential applications of Panchgavya as antimicrobials, immune boosters, antidiabetics, anticancer, anticonvulsant, aphrodisiac, blood purifiers, and as a suitable medium to deliver medicines, have caught the attention of scientists and medical professionals (Dhama et al., 2014). In this context, we undertook an investigation on the prophylactic potential of a Panchgavya preparation against bacterial infections in the nematode host Caenorhabditis elegans.

Panchgavya preparation
The Panchgavya formulation used in this study was prepared using a method that was different from the one practiced traditionally (which yields a fermented preparation). Fresh cow dung and urine, sourced from a cow fed on cottonseed and sugarcane grass, were mixed thoroughly in a glass beaker. This mix was allowed to stand for 10 min and subjected to filtration through a muslin cloth (the traditional method does not involve filtration). To this filtrate, fresh cow's milk and fresh curd was added, and mixed until a uniform mixture was formed. Finally, cow ghee was added to this mixture and mixed thoroughly.
Dung, urine, and milk were all sourced from a single cow. From the same batch of milk, curd and ghee were prepared. Cream of this milk was boiled for 30-40 min and filtered; the filtrate was taken as ghee. For curd preparation, one part of this milk was inoculated with previous batch of curd (prepared using milk from the same cow by adding few drops of lemon juice to the milk) followed by overnight incubation at room temperature.
The ratio of dung:urine:milk:curd:ghee in this preparation was 1:2:3:3:1. This Panchagavya mixture was then transferred to a copper vessel (covered with a muslin cloth) and allowed to rest for 30 min. This was followed by freeze-drying at -20 °C to convert the preparation in powder form, which was stored under refrigeration (4-8°C) until used for the microbiological experiments. When required for use, the Panchgavya powder was suspended in sterile distilled water to attain OD 625 = 0.10±0.01.
In vivo assay C. elegans worms (received gift from the Biology Division, Sophia College, Mumbai) maintained on NGM (Nematode Growing Medium; 3 g/L NaCl, 2.5 g/L peptone, 1 M CaCl 2 , 1 M MgSO 4 , 5 mg/mL cholesterol, 1 M phosphate buffer of pH 6, 17 g/L agar-agar; this medium was prepared by us using the listed ingredients purchased from Merck, Mumbai or HiMedia, Mumbai) agar plate with E. coli OP50 (LabTIE B.V., JR Rosmalen, the Netherlands) as food, were kept unfed 24h prior to being used for experiments.
These worms were fed with Panchgavya by mixing this formulation (100 µL) with M9 medium (800 µL) and placed in a 24-well plate (sterile, non-treated polystyrene plates; HiMe-diaTPG24) containing 10 worms per well. Duration of exposure of worms to Panchgavya was kept 24, 48, 72 or 96 h, followed by addition of pathogenic bacteria (100 µL of bacterial suspension with OD 764 = 1.50). Appropriate controls i.e. worms previously not exposed to Panchgavya, but exposed to pathogenic bacteria; worms exposed neither to Panchgavya nor bacteria; and worms exposed to Panchgavya, but not to bacterial pathogens, were also included in the experiment. Incubation was carried out at 22°C.
Number of live vs. dead worms were counted every day for 5 days by putting the plate (with lid) under a light microscope (4X). Straight worms were considered to be dead. Plates were gently tapped to confirm lack of movement in the dead-looking worms. On the last day of the experiment, when plates could be opened, their death was also confirmed by touching them with a straight wire, wherein no movement was taken as confirmation of death.

Statistical analysis
Values reported are means of four independent experiments, whose statistical significance was assessed using t-test performed through Microsoft Excel (2013). P values ≤0.05 were considered to be statistically significant.

Results
Worms fed on Panchgavya for 24 or 48 h registered no different (p>0.05) survival rates in the face of bacterial challenge as compared to control worms (Appendix A and Appendix B). However, worms with 72 or 96 h Panchgavya exposure registered a 15-27% (p<0.05) better survival upon challenge with different pathogenic bacteria, except for S. pyogenes as compared with control worms (Figure 1; Appendix C and Appendix D). These results demonstrate the prophylactic potential of Panchgavya against four different gram-positive and gram-negative bacterial infections, wherein previous exposure of C. elegans to this formulation was found to confer statistically significant protection on this worm against subsequent bacterial attack.
However, when administered to C. elegans already infected by these pathogens, Panchgavya was not found to offer any survival benefit to the nematode host (Appendix E). Additionally, the Panchgavya-exposed worm population was able to generate progenies in absence as well as presence of pathogenic bacteria, which did not happen in control wells containing Panchgavya-unexposed worms, suggesting overall higher fitness of Panchgavya-exposed worms.  (D) 23±1.50% (p=0.002) higher survival on fifth day, against S. marcescens; (E) Panchgavya-exposure was not found to confer any protection on C. elegans against S. pyogenes challenge. Results pertaining to 72 h and 96 h exposure of worms to Panchgavya, prior to bacterial challenge, were not statistically different. Values reported are means of four independent experiments, whose statistical significance was assessed using t-test performed through Microsoft Excel. P values ≤0.05 were considered to be statistically significant.

Conclusions
Though there are few reports mentioning in vitro antimicrobial activity of either Panchgavya mixture (Gajbhiye et al., 2018) or its individual components (Deepika et al., 2016), to the best of our knowledge, the present study is the first report demonstrating in vivo anti-infective efficacy of Panchgavya mixture. The observed protective effect of Panchgavya against bacterial infection may in part stem from its immunomodulatory potential (Gajbhiye et al., 2015). This short study validates the therapeutic potential of Panchgavya mentioned in Ayurved (Susruta Samhita, 1885). Further studies for characterization (e.g. generating its metagenomic, which may reveal presence of beneficial microbes, and chemical profile) of this ancient formulation can provide insights into the mechanisms underlying its anti-infective efficacy.

Grant information
The author(s) declared that no grants were involved in supporting this work.
No competing interests were disclosed. Competing Interests:

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
26 October 2018 Referee Report https://doi.org/10.5256/f1000research.18019.r39214

Prasun Kumar
Department of Chemical Engineering, Chungbuk National University, Cheongju, South Korea Antibiotic-resistant infections are a global threat, and novel antibiotic, as well as non-antibiotic approaches to deal with the problem of antimicrobial resistance (AMR), are urgently needed. In this pursuit, taking insights from traditional wisdom seems to be a logical effort. This particular research note describes an investigation of the prophylactic efficacy of a traditional Indian formulation -, Panchgavya and using the nematode worm model has demonstrated such a prophylactic effect to be there against some of the pathogenic bacteria. I appreciate that they have selected important pathogens like P.

aeruginosa S. marcescens
Despite being an ancient formulation, it seems to be under-investigated by modern-day Panchgavya scientists. Studies like this one attempting to validate the traditional medicine claims are welcome. As mentioned in the conclusion part by authors, they should take up in near future further characterization of this formulation. If such a standardized formulation can be made available for public use, it may help in reducing the overall infection burden of human populations. -Differential behavior on Gram +ve bacteria is quite interesting and must be studied further.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Authors have used beta-lactamase producing antibiotic-resistant strains in their study. That is good, as resistant strains of and have also been listed by WHO as of P. aeruginosa S. marcescens high priority. Use of as a model host by authors is also logical, as there is some overlap among the C. elegans virulence factors of pathogenic bacteria damaging , and those damaging human cells.

C. elegans
While the current study may be approved for publication as a research note, in future authors should try to investigate the mechanisms through which ' imparts protection to 'Panchgavya C. against infectious bacteria. elegans

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.