Therapeutic Potential of Gir Cow Urine Distillate: Antioxidant Activity, Antibacterial Efficacy, and Molecular Docking Studies

Gir cow urine distillate also known as ‘Gomutra Ark’. It has been traditionally used in Ayurveda for its potential. Cow urine distillate has medical properties. It has antimicrobial properties in which it exhibiting antibacterial and antifungal properties. It effective against certain bacteria, viruses and fungi. Secondly CUD has anti-inflamentary properties such as CUD can help to reduce the inflammation. And CUD has major effective property is an antioxidant property.

Antioxidants are the substances which are present in CUD, that help to reduce oxidative stress in human body. When body produces more free radicals than it can neutralize with antioxidant is called oxidative stress. When an oxidative stress is increased above its certain range or limits then, it leads to cell and tissue damages and various health problems like cancer. The antioxidant activity of CUD is measured by FRAP method on the basis of principle of reducing power of antioxidant substances of cow urine. FRAP reagent contains ferric-tripyridyltriazine (Fe³⁺ TPTZ) complex.[27]

In this investigation, the antioxidant potential of cow urine distillate (CUD) was assessed. The study utilized urine collected from Indian Gir (Gujarati) breed cows. CUD is known to possess a distinctive chemical profile, with urea as its predominant constituent, creatinine as a metabolic by-product, and various minerals including potassium, sodium, and calcium. It also contains organic molecules such as phenol and indoles. Owing to this unique composition, cow urine distillate has been traditionally employed for medicinal applications.

CUD may serve as an alternative to conventional antimicrobial agents due to its status as a natural product that is extensively available and cost-effective. Empirical studies have been conducted to investigate the antimicrobial and antioxidant properties of bovine urine. Within the paradigms of traditional Indian medicine, it is posited that cow urine possesses curative capabilities for various bacterial and viral infections, as well as conditions such as pyrexia, anemia, epilepsy, abdominal pain, constipation, and wound healing. This cultural practice of utilizing folklore remedies remains prevalent in rural regions of India and Nepal. Furthermore, CUD is hypothesized to exhibit anti-inflammatory and analgesic properties, potentially contributing to the therapeutic management of certain malignancies and diabetes mellitus. In several South Asian nations, cow urine is purported to have efficacy in cancer treatment.

The molecular relationship between bovine urine and bacterial proteins may elucidate substantive evidence regarding the bactericidal properties of CUD. Nevertheless, further investigation is requisite to fully elucidate the antimicrobial potential of bovine urine and to assess the safety and efficacy of its application in this regard. The objective of this study is to evaluate the antibacterial properties of CUD utilizing both in vitro experiments and in silica methodologies, specifically molecular docking, to furnish evidence-based conclusions pertinent to its application as an antibacterial agent.

MATERIALS AND METHOD

1) Preparation of Cow Urine Distillate : Freshly collected Geer cow urine was used for the preparation of CUD. The urine was transferred into a clean round-bottom flask and subjected to simple distillation at 90 °C using a standard distillation setup. The condensate was passed through a silica column, and the purified distillate was collected in sterile glass bottles. Samples were stored at ambient temperature until further analysis.

2) Preparation of Reagents for FRAP Assay: The following chemicals were prepared: 0.2 M phosphate buffer (pH 6.6), 1% potassium ferricyanide, and 10% trichloroacetic acid, and 0.1% ferric chloride.

3) FRAP Reaction Procedure: Different concentrations of CUD in methanol (10%–100%) were prepared in glass reaction tubes. To each tube, 2.5 ml of 0.2 M phosphate buffer (pH 6.6) was added, and the mixture was vortexed thoroughly. This was followed by the addition of 2.5 ml of 1% potassium ferricyanide. The samples were centrifuged at 3000 rpm for 10 minutes to separate any precipitated proteins. The collected supernatants were mixed with 2.5 mL of a 10% trichloroacetic acid solution and then incubated at 50 °C for 20 minutes. Once cooled, 0.5 mL of 0.1% ferric chloride was introduced, and absorbance readings were taken at 700 nm using a UV–Visible spectrophotometer, with methanol and distilled water serving as reference blanks.

4) Test Microorganisms: Pathogenic bacterial strains tested included Staphylococcus aureus (Gram-positive) as well as Escherichia coli, Salmonella typhi, and Klebsiella species (Gram-negative). (Gram-negative)—were authenticated, subcultured, and maintained for experimentation. Petri dishes were cleaned, wrapped in aluminum foil, and sterilized at 121 °C for 15 minutes under 15 psi pressure.

Mueller–Hinton Agar (MHA) was formulated by dissolving 19 g of the medium in 500 mL of distilled water, while nutrient broth was prepared by dissolving 1.3 g in 100 mL of distilled water. Both media were boiled to dissolve, plugged with cotton, wrapped with foil, and sterilized by autoclaving at 121 °C for 15 minutes. The nutrient broth was dispensed into sterile test tubes, inoculated with pure bacterial cultures using sterile loops, and incubated under aseptic conditions. For agar plates, MHA was cooled to 45–50 °C, poured into sterile Petri dishes, allowed to solidify, and stored at 4 °C until use.

5) Preparation of Filter Paper Discs: Whatman No. 1 filter paper was punched into 6 mm discs, placed in sterile Petri dishes, and sterilized in a hot-air oven at 161 °C for 2 hours.

6) Antibacterial Assay (Disc Diffusion Method): Antibacterial activity was assessed using the agar disc diffusion method in three steps:

• Inoculation: Bacterial suspensions were uniformly spread onto MHA plates under aseptic conditions in a laminar flow hood. Each plate was divided into five marked zones: positive control (ciprofloxacin 5 µg), negative control (DMSO), and three test zones (CUD at 5%, 10%, and 15%).
• Disc Placement: Sterile filter paper discs were impregnated with test samples or controls and placed in their respective zones.
• Incubation: Plates were incubated at 35 °C for 24 hours, and antibacterial activity was determined by measuring the diameter of inhibition zones. Experiments were conducted in triplicate.

7) Determination of Minimum Inhibitory Concentration (MIC): MIC values were determined using the broth dilution method. Mueller–Hinton broth (10 mL) was dispensed into sterile test tubes and autoclaved. Bacterial suspensions were prepared according to the 0.5 McFarland standard (~1.5 × 10? CFU/mL). Test compounds were serially diluted to obtain concentrations of 200, 100, 50, 25, and 12.5 μg/mL. After incubation at 37 °C for 24 hours, the MIC was recorded as the lowest concentration with no visible bacterial growth.

8) Molecular Docking: Structures of bioactive metabolites from fresh cow urine were sourced from peer-reviewed literature and PubChem in SDF format. These were optimized using the MMFF94 force field in MGL Tools and converted to PDBQT format using Open Babel.

The DNA gyrase B ATP-binding domain from E. coli (PDB ID: 4KFG; resolution 1.60 Å) was chosen as the target. The protein was prepared by removing water molecules and native ligands, followed by the addition of polar hydrogens in Discovery Studio Visualizer 2021. Docking simulations were performed with AutoDock Vina v1.2.0, using ciprofloxacin as the reference ligand. Grid dimensions were set to 50 × 50 × 50 (spacing 1 Å), centered at X = 14.299, Y = 18.687, Z = −12.407. Redocking of the native ligand validated the docking protocol, with RMSD values below 2 Å considered acceptable.

9) Identification of CUD Components: Relevant research articles were retrieved from Scopus-indexed journals using “GC–MS of cow urine” as the search term. Ten relevant studies were reviewed, and seven common chemical constituents were selected for molecular docking analysis.

Figure 1: 3D structure of DNA gyrase protein (PDBID: 4KFG).

RESULTS

1. Cow Urine Distillate (CUD)

The CUD sample was obtained through a simple distillation process. For characterization, total dissolved solids (TDS), pH, and toxicity were assessed, along with physical attributes such as color and odor. The measured pH of 8.0 falls well within the safe range for CUD applications, indicating a non-corrosive nature and compatibility with disinfectant use. The TDS value of 409 mg/L lies comfortably within the “excellent” category for potable water quality and is far below any levels of concern. The sample was clear, free from odour, and non-toxic—meeting both safety and aesthetic requirements for drinking or utility-grade standards. No abnormal taste, colour, or contaminants were observed, reflecting the characteristics expected from a high-quality CUD preparation.

Table 1 CUD Sample

2. Antioxidant Activity (FRAP)

Dilutions of CUD derived from Geer cow urine were prepared at concentrations ranging from 10% to 100%. Absorbance measurements were taken at 700 nm using a UV–Visible spectrophotometer. A clear trend of rising absorbance was recorded as CUD concentration increased, with the maximum value observed in the undiluted (100%) sample. This pattern suggests a higher abundance of chromophoric and antioxidant-active compounds in more concentrated CUD preparations.

Table 2

Figure 2

3. Antibacterial Activity: The antimicrobial potential of CUD was assessed against four pathogenic bacteria: Staphylococcus aureus (Gram-positive) and Escherichia coli, Salmonella typhi, and Klebsiella spp. (Gram-negative). Dimethyl sulfoxide (DMSO) served as the negative control, while ciprofloxacin was used as the standard reference antibiotic.

4. Minimum Inhibitory Concentration (MIC): MIC values for CUD against the tested bacterial strains are summarized in Table 3. CUD demonstrated significant inhibitory effects, with S. aureus and E. coli showing the highest susceptibility. The MIC values ranged between 12.5 and 50 μg/mL, confirming broad-spectrum antibacterial action. However, when compared to ciprofloxacin, CUD exhibited higher MIC values, indicating relatively lower potency than the reference drug.

5. Molecular Docking Analysis: DNA gyrase, a key bacterial enzyme essential for transcription and the regulation of DNA supercoiling, was chosen as the molecular target. The ATP-binding domain of the DNA gyrase B subunit from E. coli (PDB ID: 4KFG) was selected for docking studies. The native ligand of 4KFG, DOO, contains aromatic and heterocyclic groups similar to bioactive constituents identified in CUD, making it an appropriate structural model for this investigation.

Docking outcomes, including binding energies and hydrogen-bond interactions, are presented in Table 4. Most tested ligands exhibited favorable interactions with the target protein. Among the seven compounds screened, 2-hydroxycinnamic acid showed the strongest binding affinity (ΔG = −6.9 kcal/mol; Figure 4), forming two hydrogen bonds with Val71 and Asp73 at distances of 1.93 Å and 3.90 Å. While its binding energy was slightly weaker than ciprofloxacin (ΔG = −7.4 kcal/mol; Figure 5), it established more hydrogen bonds. Ferulic acid (Figure 6) had a binding energy of −6.8 kcal/mol, interacting with Val71 and Asp73 at 2.06 Å and 3.08 Å, respectively. Gallic acid demonstrated the highest number of hydrogen bond interactions—binding to Asp45, Glu42, and Ser108—with a ΔG of −6.4 kcal/mol.

Phenol displayed the lowest affinity (ΔG = −5.2 kcal/mol), forming a single hydrogen bond. The root mean square deviation (RMSD) values for all ligand-protein complexes ranged from 1.05 to 1.87 Å, indicating stable docked conformations.

6. Major Constituents of CUD: Spectroscopic characterization confirmed the presence of multiple aromatic and heterocyclic molecules in CUD, potentially contributing to its antimicrobial activity. Key identified compounds included gallic acid, ferulic acid, cinnamic acid, allantoin, and 1-heneicosanol.

Table 3: Zone of inhibition in diameter and concentration of CUD in %.

Table 4: Minimum inhibitory concentration (MIC) of CUD.

Table 5: Binding energy and hydrogen bond interaction amino acids

Sr. No.	Name	Structure	H-bond interactive amino acid	Binding energy (Kcal/mol)	RMSD value
1	Gallic acid		Asp45 Glu42 Ser108	−6.4	1.22
2	Ferulic acid		Val71 Asp73	−6.8	1.60
3	Hydroxycinnamic acid		Val71 Asp73	−6.9	1.05
4	Cinnamic acid		Val71 Asp73	−6.7	1.15
5	Salicylic acid		Asp73 Asn46	−6.1	1.37
6	Allantoin		Asp73 Thr165	−6.0	1.43
7	Phenol		Val118	−5.2	1.87
8	Ciprofloxacin (reference)		Lys189	−7.4	1.51

The Ferric Reducing Antioxidant Power (FRAP) assay was conducted to quantitatively evaluate the antioxidant potential of cow urine distillate (CUD). After performing the FRAP procedure, ultraviolet–visible (UV–Vis) spectrophotometric measurements were taken. Results showed a direct relationship between CUD concentration and absorbance values; higher concentrations yielded greater absorbance readings. This trend indicates that antioxidant activity increases proportionally with the concentration of CUD in the diluted series.

The antibacterial properties of CUD were assessed through both experimental (in vitro) and computational (in silico) approaches. In vitro testing was carried out against Staphylococcus aureus (Gram-positive) and Escherichia coli, Salmonella typhi, and Klebsiella spp. (Gram-negative) using CUD concentrations of 5%, 10%, and 15%. The strongest antibacterial effect was recorded at 15% concentration. The largest inhibition zones were noted for S. typhi (20.8 ± 0.6 mm) and S. aureus (18.6 ± 0.42 mm), in comparison with ciprofloxacin. For E. coli, the inhibition zone at 15% CUD measured 13 ± 0.8 mm—approximately 50% smaller than that observed with ciprofloxacin (24 ± 1.0 mm).

When compared with previous studies, our results partially align with existing literature. For example, Sathasivam et al. reported smaller inhibition zones for S. typhi (10.4 ± 1.2 mm), whereas Majhi and Bardvalli documented comparable results. Poornima et al. found greater efficacy of CUD against Gram-positive species, consistent with our observations. Conversely, Jarald et al. demonstrated that crude cow urine possessed stronger antibacterial effects than its distillate, potentially due to loss of active components during distillation. Ahuja et al. reported a similar inhibition zone for E. coli (14 mm), which supports the present findings.

MIC testing revealed that CUD inhibited S. aureus and E. coli at 12.5 μg/mL, while Klebsiella pneumoniae and S. typhi required higher concentrations—25 μg/mL and 50 μg/mL, respectively. For comparison, ciprofloxacin showed an MIC of 6.25 μg/mL against all tested strains. These results suggest that although CUD demonstrates antibacterial activity, its potency is lower than that of ciprofloxacin. Since MIC values represent only one measure of antimicrobial potential, further evaluation—such as time-kill kinetics, cytotoxicity studies, and in vivo testing—would be necessary to determine the clinical applicability and safety of CUD as an antibacterial agent.

Figure 3: Interaction of compound 2-hydroxycinnamic acid (3D and 2D interactions).

Molecular docking is a widely used computational approach that predicts how a ligand interacts with a protein, particularly the binding mode of a small molecule within the active site of a target receptor [44]. To better understand the antibacterial mode of action of cow urine distillate (CUD), we performed in silico docking analyses of bioactive constituents from CUD against bacterial proteins involved in protein synthesis. Although CUD has shown measurable antibacterial activity, its underlying mechanism remains incompletely defined. This activity is likely linked to chemical constituents such as urea, ammonia, osmolytes, and organic acids, which can denature proteins, compromise cell membranes, cause cellular dehydration, and exert direct antimicrobial effects.

Docking results identified 2-hydroxycinnamic acid (ΔG = −6.9 kcal/mol, Table 4) and ferulic acid (ΔG = −6.8 kcal/mol, Table 4) as the highest-affinity ligands for the bacterial protein target, suggesting that these compounds may play a key role in CUD’s antibacterial action. Both molecules formed hydrogen bonds with critical amino acid residues—Val71 and Asp73—supporting their potential to disrupt bacterial protein function.

Previous research has linked the antimicrobial properties of cow urine to compounds such as 2-hydroxycinnamic acid, ferulic acid, gallic acid, cinnamic acid, phenol, carbolic acid, and allantoin. These metabolites, together with certain peptides and derivatives, can increase bacterial cell surface hydrophobicity, thereby enhancing bactericidal efficacy. Cow urine has also been reported to stimulate macrophage phagocytic activity [47], contributing to the innate immune response.

Additionally, evidence suggests that cow urine may help prevent the emergence of antibiotic resistance by inhibiting the R-factor—a plasmid-borne genetic element involved in horizontal gene transfer among bacteria [48]. Nautiyal and Dubey [35] reported that CUD exhibits antimicrobial activity against a wide range of bacterial and fungal species and has long been used in sanitation practices.

The urea present in cow urine could further support its antibacterial properties by denaturing bacterial proteins, altering their secondary and tertiary structures, impairing function, and ultimately hindering bacterial growth [49, 50]. Moreover, CUD contains cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial signalling molecule associated with biofilm formation [51]. Literature indicates that c-di-GMP from cow urine may inhibit the expression of SdiA, a quorum-sensing protein, thereby interfering with biofilm development [52].

Figure 4: Interaction of reference ciprofloxacin with protein 4KFG (3D and 2D interactions).

Figure 5: Interaction of compound ferulic acid with protein 4KFG (3D and 2D interactions).