Cow Urine: Plant Growth Enhancer and Antimicrobial Agent

The cow is considered a divine animal in the Hindu culture. The milk, cow dung, urine of the cow is used for various purposes. The benefits of cow urine have been depicted in ancient Hindu texts, Vedas. The present study aimed at using cow urine as a plant growth enhancer and antimicrobial agent. The plants chosen for this study were Zea mays (maize), Triticum aestivum (wheat), Cymbopogon citratum (grass), Ocimum basilicum (tukmaria) and Trigonella foenum graecum (methi). The plants were grown for 30 days using different cow urine concentrations i.e 0%(control), 5%, 15%, 25%. The various parameters such as plant height, shoot, and root length, number of leaves, the mass of the root etc were observed. The antibacterial test using different cow urine concentrations i.e 0%(control), 5%, 15%, 25% was conducted on Escherichia coli using disc diffusion method. The fungus was screened and isolated from raw coconut and was grown on YPD media to obtain the mother culture. Using Slide culture technique, the fungi were identified as Aspergillus. The media was poisoned using different cow urine concentrations i.e 0%(control), 5%, 15%, 25% and the fungi culture was inoculated. GCMS analysis was conducted to identify the compounds present in the cow urine. Among the concentrations, 5% cow urine concentration showed maximum growth when compared to other concentrations whereas 25% concentration showed more antibacterial and antifungal activity when compared to others. In GCMS Analysis, 16 compounds have been identified, in which, 6 compounds were antifungal, 3 compounds were antibacterial, and 2 compounds as plant growth enhancers. This study concludes that cow urine can be used as a plant growth enhancer and it possesses antimicrobial characteristics. Introduction The urine of Cow is commonly used to prevent and cure diseases especially by the rural people in India. Cow is considered sacred among the Hindu religion in India since ages. The use of Cow urine diminished when the western medicine has been developed, although it is still practiced as a treatment in some parts of the world. Currently, the use of Cow Urine as a therapeutic agent is being practiced to treat diseases and prevent the spread of diseases. Nonetheless, not much of the composition of the Cow Urine and its functions are known. In Vedas (Ancient Hindu texts), Cow Urine has been given a lot of significance. It was also called as Amrita, the beverage of immortality. The Cow Urine from the Indian cow breed has medicinal properties unlike the crossbred or exotic cows. Recent studies have showed that Cow Urine has an effect on the immune system by increasing the phagocytic activity of macrophages. It was also shown that it has anti-bacterial properties [1]. Some medicinal properties of Cow Urine include reduction of weight, treating kidney problems, treating indigestion and edema [2]. The practitioners of Buddhist culture in Myanmar still use Cow Urine as a traditional medicine [3]. The constituents of the Cow Urine in right proportions are the reason to cure diseases. The analysis of Cow Urine showed that it is made up of all the essential Salts, Metals, Minerals, Vitamins, Enzymes and other entities in small quantities [4]. Journal of Horticulture and Plant Research Submitted: 2018-11-19 ISSN: 2624-814X, Vol. 8, pp 30-45 Revised: 2019-10-07 doi:10.18052/www.scipress.com/JHPR.8.30 Accepted: 2019-11-01 CC BY 4.0. Published by SciPress Ltd, Switzerland, 2019 Online: 2019-11-29 This paper is an open access paper published under the terms and conditions of the Creative Commons Attribution license (CC BY) (https://creativecommons.org/licenses/by/4.0) In this study, we tried to identify if there is any effect on the Plant growth in the presence of Cow Urine. The Antimicrobial activity of the Cow Urine was also studied. The Cow Urine was also analyzed using Gas Chromatography-Mass Spectroscopy (GCMS) to determine its constituents and its effects. Materials and Methods Cow urine collection The cow urine was collected in a nearby organic farm and was filtered using filter paper. The urine was collected in urine sample kits and was stored at 4 Celsius. The soil was also collected from the organic farm. Seed collection 5 types of seeds were collected i.e 1. Zea mays – Maize 2. Triticum aestivum – Wheat 3. Trigonella foenum graecum – Methi 4. Ocimum basilicum Tukmaria (Sabja) 5. Cymbopogon citratus Lemon grass Preparation of different cow urine concentrations Various concentrations of cow urine were prepared i.e 0%, 5%, 15%, 25%. 5% cow urine was prepared by mixing 5ml in 100ml distilled water. 15% cow urine was prepared by mixing 15ml in 100 ml distilled water. 25% cow urine was prepared by mixing 25ml in 100ml distilled water. Sowing of seeds The seeds were sown in pots and were labeled using small flags which depict the name of the seed and the concentration of the cow urine used as shown in the Fig. 1. The plants were irrigated using different concentrations of cow urine (10ml) for 30 days and the growth was observed as shown in Fig. 1. Figure 1. Seeds sown in their respective blocks Parameters Various parameters of the plants were observed. The parameters include 1. Total chlorophyll [5]: Chlorophyll was extracted using acetone and spectrophotometer was used to measure the absorbance at specific wavelengths i.e 663nm and 645nm. Chlorophyll a(μg/ml) =12.7 (A663) – 2.69 (A645) Chlorophyll b(μg/ml) =22.9 (A645) – 4.68 (A663) Total Chlorophyll (μg/ml) =20.2 (A645) – 8.02 (A663) 2. Plant height: Measured using scale in cms. Journal of Horticulture and Plant Research Vol.8 31


Introduction
The urine of Cow is commonly used to prevent and cure diseases especially by the rural people in India. Cow is considered sacred among the Hindu religion in India since ages. The use of Cow urine diminished when the western medicine has been developed, although it is still practiced as a treatment in some parts of the world. Currently, the use of Cow Urine as a therapeutic agent is being practiced to treat diseases and prevent the spread of diseases. Nonetheless, not much of the composition of the Cow Urine and its functions are known.
In Vedas (Ancient Hindu texts), Cow Urine has been given a lot of significance. It was also called as Amrita, the beverage of immortality. The Cow Urine from the Indian cow breed has medicinal properties unlike the crossbred or exotic cows. Recent studies have showed that Cow Urine has an effect on the immune system by increasing the phagocytic activity of macrophages. It was also shown that it has anti-bacterial properties [1]. Some medicinal properties of Cow Urine include reduction of weight, treating kidney problems, treating indigestion and edema [2]. The practitioners of Buddhist culture in Myanmar still use Cow Urine as a traditional medicine [3].
The constituents of the Cow Urine in right proportions are the reason to cure diseases. The analysis of Cow Urine showed that it is made up of all the essential Salts, Metals, Minerals, Vitamins, Enzymes and other entities in small quantities [4].
In this study, we tried to identify if there is any effect on the Plant growth in the presence of Cow Urine. The Antimicrobial activity of the Cow Urine was also studied. The Cow Urine was also analyzed using Gas Chromatography-Mass Spectroscopy (GCMS) to determine its constituents and its effects.

Cow urine collection
The cow urine was collected in a nearby organic farm and was filtered using filter paper. The urine was collected in urine sample kits and was stored at 4 Celsius. The soil was also collected from the organic farm.

Preparation of different cow urine concentrations
Various concentrations of cow urine were prepared i.e 0%, 5%, 15%, 25%. 5% cow urine was prepared by mixing 5ml in 100ml distilled water. 15% cow urine was prepared by mixing 15ml in 100 ml distilled water. 25% cow urine was prepared by mixing 25ml in 100ml distilled water.

Sowing of seeds
The seeds were sown in pots and were labeled using small flags which depict the name of the seed and the concentration of the cow urine used as shown in the Fig. 1. The plants were irrigated using different concentrations of cow urine (10ml) for 30 days and the growth was observed as shown in Fig. 1.
3. Root Length: Measured from the shoot ending to the root tip using scale. 4. Shoot Length: Measured shoot using scale in cms. 5. Leaf Length: The leaf with highest length was considered. 6. Leaf breadth: The breadth of the highest leaf length was considered. 7. No of branches 8. Root weight: dried weight(gm) of the root is considered.

Soil analysis
The soil samples is given for analysis in ECOICONS, ECIL, to determine what changes have been made by the cow urine in the soil. Various parameters and methods used for soil analysis are tabulated in Table 1.

Antimicrobial analysis
Bacteria • E. coli bacteria was obtained from the SNIST Biotechnology Department.
• E. coli was grown on the nutrient medium to obtain the mother culture.
• Antbacterial activity was conducted using various cow urine concentrations (5%, 15%, 25%) using well diffusion method. Fungi • A fungal species was isolated from a raw coconut. Based on its morphological character, it was tentatively identified as Aspergillus. • Fungi was grown on the YPD medium to obtain the mother culture.

Gas chromatography mass spectroscopy analysis
Gas chromatography-mass spectroscopy analysis was conducted to determine the compounds present in the cow urine which may have made the changes in the plant growth, soil and antimicrobial properties.
1. The urine sample was extracted in dichloromethane using the principle of liquid-liquid extraction. 2. The cow urine was mixed with dichloromethane in 1:1 ratio and was left in shaker for 3hrs. 3. The difference in their densities resulted in a two-layer liquid i.e top layer-cow urine, bottom layer-dichloromethane. 4. The dichloromethane was extracted using a pipette and was transferred into 2ml vials.
The protocol for GCMS [6]: The GC-MS system started with the initial oven temperature of 60 °C for 5 min, increasing to 240 °C at a rate of 15 °C for 5 min, and then to 300 °C at a rate of 15 °C for 5 min. The injector temperature was maintained at 200 °C. The interface temperature was 250 °C. Helium was used as a mobile phase at a flow rate of 1.0 mL/min. Mass spectral detection was carried out in electron ionization mode by scanning at 40-600 (m/z).
The GCMS analysis was performed at IICT, Tarnaka.

Growth observation
The plants were placed on white sheet of paper and the parameters were measured as per protocol mentioned in the methods. The growth observation of various plants is shown in Fig. 2

Physical parameters comparison
The plants have shown different physical changes with different cow urine concentrations. This shows that cow urine has effect on the growth of the plants.
The data from the physical parameters was tabulated in Table 2, Table 3, Table 4, Table 5. The graphs were plotted for each of the plant under various concentrations. The graphs are depicted in Fig. 6-Maize, Fig. 7-Grass, Fig. 8-Wheat, Fig. 9-Methi.

Data from soil analysis
The data from the soil analysis was tabulated in Table 6. The following parameters have shown increase in content with increase in cow urine concentration: • Moisture content • Electrical conductivity • Bulk density • pH

Antibacterial tests
The antibacterial tests were conducted using disc diffusion method and the results are depicted in Fig.10. The data from the antibacterial tests are shown in Table 7.

Concentration
Diameter of the inhibition zone 0% cow urine concentration 0.0cm 5% cow urine concentration 0.6cm 15% cow urine concentration 1.4cm 25% cow urine concentration 2.1cm From the data, it can be inferred that the diameter of the inhibition zone increases with increase in cow urine concentration. This depicts that cow urine acts as an antibacterial agent with increase in concentration.

Antifungal tests
The fungal sample was collected from a raw coconut (Fig. 11) and was inoculated in YPD medium which supports the growth of fungi. Figure 11. Growth of fungi on raw coconut The mother culture was obtained by growing the fungi on YPD media- Fig. 12. Figure 12. Fungi grown on YPD medium for mother culture Using slide culture technique (Fig. 13), the fungus was identified as Aspergillus based on its morphological characteristics. The antifungal tests were conducted by poisoning the media with cow urine. The cow urine concentrations i.e 5%- Fig. 14, 15%-Fig 15, 25%-Fig 16, was added into the YPD media and was sterilized using autoclave. The fungi were inoculated on the media and was kept in an incubator for 6 days at a temperature of 30 Celsius.

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JHPR Volume 8 Figure 14. Antifungal test using 5% concentration The results are shown in Table 8. The growth of fungi was inhibited with the increase in cow urine concentration.

Gas chromatography and mass spectroscopy analysis
The GCMS analysis was conducted as per the protocol mentioned in the methods and the graphs obtained.
The compounds identified in the analysis were, 1.
1H The following compounds (16) were identified and their properties are mentioned in the Table 9.

Discussion
The plants were grown using different concentrations (control (0%), 5%, 15%, 25%) of cow urine for 30 days and the growth was observed. The plants showed varied growth at different concentrations and the physical parameters were measured and tabulated. According to the measurements, the 5% cow urine concentration showed more growth when compared to other concentrations. The soil was sent for soil analysis to check for the changes made to the soil by cow urine. The various parameters of the soil were analysed. The parameters i.e pH, Bulk density, Electrical conductivity, Moisture content were increased with increase in cow urine concentrations. The antimicrobial properties of the cow urine were examined. The antibacterial test was conducted on E. coli using disc diffusion method. The inhibition zone increased with increase in cow urine concentration indicating the presence of antibacterial compounds in cow urine. The fungus was isolated from raw coconut and slide culture technique was used to determine its morphological features. Based on its morphological features, it was identified as Aspergillus. The antifungal tests were conducted by poisoning the media with various cow urine concentrations and inoculating the fungi. The diameter of the fungi culture decreased with increase in cow urine concentration indicating the presence of antifungal agents. Gas chromatography mass spectroscopy analysis was performed to identify the compounds in the cow urine. The cow urine was extracted by liquid-liquid phase extraction using dichloromethane and was sent for analysis. 16 compounds were identified in the sample and their respective pubchem IDs were searched. 2 compounds didn't have any pubchem ID indicating the presence of new compounds. The compounds were searched for their properties using literature review. Out of 16 compounds, 6 compounds were identified as antifungal, 3 compounds were identified as antibacterial, and 2 compounds as plant growth enhancers.
From the above study, we can conclude that cow urine has the properties of plant growth enhancer and antimicrobial properties. Most of the compounds identified were produced synthetically. This can be avoided by extracting the selected compounds from the cow urine which can save money as well as protect our environment by avoiding the synthetic means of production.