Elucidation of biopotentiators in organic based formulation bijamrith from Gir Cow (Bos indicus) excrements and urine using GC-MS

Gir or Gyr is a milch cow and one of the prominent ‘zebu breeds’ that tolerates high temperature and highly suitable for tropical regions. It is the top most proven desi cow breed originated from mountains of Gir, Kathiawar, Gujarat, India, that produces 100% of easily digestible A2 protein of milk that plays a pioneer role in human dietetics, nutrition and is domesticated for breeding and improvement programme for its amenability in crossing with wide range of desi cow namely, Red Sindhi and Sahiwal of Indian breeds and also exotic breeds of Brazil and South America (Vitthalrao Khyade, 2021).
       
In natural farming, Gir cows play a very important and crucial role in soil health restoration and enhancement. Through the inherent feature of natural grazing habits, Gir cow very well contribute to nutrient recycling by organic matter spreading, improving soil texture, structure and fertility status. Incorporating Gir cow dung envisages a valuable resource potential of organic manures for soil essential nutrient enrichment, with increased yields in organic farming by minimizing the use of synthetic fertilizers (Adil Husnain, 2023).
       
Urine and dung are the two important biowastes excreted by cow (Bos indicus). These wastes are very cheaper and easily available to serve as bioenhancers/ biostimulant that possess the potentiality to increase yield of various crops. Organic formulations like bijamrith, jeevamrith/ Gnanajeevamrith derived from cow excrements and urine are advocated for soil and plant nutrition as a natural agriculture alternative that promotes lower production cost and can help to achieve high quality product and yield with lower or without the usage of chemical fertilizers and pesticides.  Organic liquid formulations of bijamrith showed better shoot length, root length and seed vigour index in crops such as groundnut, soybean, moth bean and green gram. Crop diseases caused by bacteria, fungi and viruses was also controlled in early seed germination stage by formulations of bijamrith which acts as bio-enhancer in increasing seed viability and germination capability (Naikwade Pratap, 2019).
       
Urine from Gir cow had been proved to contain gold (Parmar, 2016) and various trace elements such as copper, calcium, zinc, iron (Ambika et al., 2021). Urine distillates of Gir cow were found to improve hematological parameters and immuno-modulatory effects in tested rabbits (Joshi Ankita et al., 2012). Gottimukkala et al., (2019) reported that urine of cow acts both as crop growth promoting bio-enhancer and also produces antibacterial substances in crops such as maize, wheat, fenugreek, basil and lemon grass. Cereal seeds treated with urine of cow showed higher growth characteristics and provided excellent capacity to build strength for seedlings and saplings at younger ages. Joseph et al., (2022) reported the bioactive compounds like sesquiterpenoids, neophytadiene, dimethyl phthalate, methyl ester, ambrin, oleic acids, octadecanoic acid, methyl isostearate in cow dung. Number of cow waste based bio-enhancers such as Amrutpani, Bijamrut, Jivamrut, Panchagavya and Sanjivak have been developed by growers in organic farming systems and were proved to increase yield in groundnut. Seed treatment with bijamrith and basal application of farm yard manure in soil along with foliar spray of Jivamrut, Amrutpani, Sanjivak and Panchagavya assured higher net returns and benefit cost ratio in groundnut crop (Mathukia et al., 2021).
       
Hence, the excrements of cow can rightly be called as biopotentiators, since liquid based organic formulations helps for biological control for plant diseases, improve soil fertility, productivity. The elucidation of most of the volatile composition of low molecular weight organic compounds from cow wastes could be harnessed and derived from the solvent extract using GC-MS. Hence, the scientific validation of the bioenhancers present in bijamrith prepared with Gir cow excrements was documented using GC-MS.

Collection of urine and dung from Gir cow
 
Fresh urine was collected from heifers of Gir cow in a pre-sterilized vial. It was filtered through Whatman filter paper No. 1 to remove debris and other extraneous matters and used for preparation of bijamrith. The experiment was conducted in 2021-2022 at Nammazhvar Organic Farming Research Centre and Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India.
 
Preparation of organic based formulation bijamrith
 
Bijamrith was prepared from fresh dung and urine of Gir cow. About 5 kg of dung was taken in a muslin cloth and tied with a jute thread as a small bundle and hanged overnight (12 hours) in 20 liters of water. In another container 50 g of lime was dissolved in 1 litre of water and kept separately overnight. On the next day morning, cow dung was squeezed in water and added with a handful of field soil and to this 5 liters of cow urine and lime water (supernatant alone) was added and stirred well to prepare the formulation (Palekar, 2005).
 
Analysis of nutrient parameters and bioactive compounds in bijamrith
 
Bijamrith was analyzed for pH, Electrical Conductivity (EC), total Nitrogen, Phosphorous, Potassium and organic carbon content. The pH and EC of bijamrith was measured using a glass electrode and conductivity bridge instruments respectively (Falcon et al., 1987). Organic carbon content of bijamrith was estimated by the wet digestion method (Walkley and Black, 1934). About 0.5 ml of  bijamrith was taken in a 500 ml conical flask and added with 10 ml of 1 N K₂Cr₂O₇ and 20 ml of conc. H₂SO₄. The contents were allowed to stand for 30 min. Then, distilled water (200 ml) orthophosphoric acid (10 ml) and diphenylamine (1ml) indicator were added and was titrated against 0.5 N Fe.(NH₄)₂(SO₄)₂ towards the end point of a bright green colour. The total nitrogen content was analysed by distillation method. Bijamrith was digested with diacid (H₂SO₄ and HClO₄ at 5:2) and volume was made up with distilled water. The extract was analyzed by Kjeldahl distillation method as per the procedures of Humphries (1956) and Biswas et al., (1977). Total phosphorus content was analyzed by vanadomolybdate colorimetric (ELICO-SL159) method and the triacid extract were analyzed for total potassium by flame photometer (Jackson, 1973).
 
Elucidation of compounds in bijamrith by GC-MS profiling
 
A known quantity of bijamrith formulation was extracted using diethyl ether solvent for separation of compounds (liquid- solvent extraction method) and allowed for evaporation overnight.
       
Perkin Elmer Clarus SQ8C GC-MS model fitted with DB5 MS capillary standard non-polar column available at Department of Agricultural Microbiology, Agricultural College and Research Institute, Tamilnadu Agricultural University, Coimbatore, Tamil Nadu, India was used for elucidation of bioactive compounds from the solvent extractant of bijamrith (Sridharan et al., 2021).
       
The instrument was set with injector port temperature to 220°C, interface temperature  at 250°C, source kept at 220°C. The oven temperature was set to, 75°C for 2 mins, 150°C @ 10°C/min, upto 250°C @ 10°C/min. Split ratio set as 1:12 and the injector used was splitless mode. The DB-5 MS capillary standard non-polar column with dimension 0.25 mm OD ´ 0.25 μm ID ´ 30 meters length procured from Agilent Co., USA was used for elution. Helium gas was used as the carrier gas at 1 ml/min. The MS was set to scan from 50 to 550 Da. The source was maintained at 220°C and 4.5e^-6 motor vacuum pressure. The ionization energy was -70eV. The MS was also having inbuilt pre-filter which reduced the neutral particles. The data system has inbuilt libraries for searching and matching the spectrum. NIST MS Search 2.2v contains more than five lakh references. Interpretation of the mass spectrum of GC-MS was done using the database of National Institute Standard and Technology (NIST14). The spectrum of the compounds was compared with the spectrum of the known components stored in the inbuilt library. 

Chemical characterization of bijamrith from Gir cow
 
The pH, EC, N, P, K and organic carbon content of bijamrith formulation were 7.08, 4.26 dSm^-1, 2.38%, 0.13%, 0.49% and 0.93% respectively.
 
GC-MS elucidation of bioactive compounds in bijamrith from Gir cow
 
The present findings of GC-MS elucidation of bioactive compounds in Gir cow urine and dung based bio-formulation bijamrith (Table 1, Fig 1) showed 40 different types of organic compounds, out of which 13 compounds were classified as antimicrobials and other compounds were found to be plant growth promoters, antioxidants/anti-inflammatory. The data on application/activity of the compound eluted were retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/ and https://www.trc-canada.com/.
 

 

       
The present research findings of bijamrith prepared from urine and dung of Gir cow elucidated the occurrence of entirely different types of compounds such as cyclohexanol and tetradecenol. Similar type of studies on the ethyl acetate extract fractions of urine from Sahiwal cow at different physiological stages such as heifer, lactating, pregnancy period showed the presence of 511 sequences of peptides and aqueous extract showed about 46 antimicrobial compounds (Kumar et al., 2023). Bioactive fractions of Badri cow urine, exhibited antibacterial activity against human pathogenic bacteria, GC-MS elucidation showed the presence of 12 important compounds and the prime molecules were heneicosanol, heptadecanol, nonadecanol, hexanol, pentadecanal, tetra siloxane, pyrazole, cycl octasiloxane and FTIR analysis further confirmed the presence of alcohol, amide, alkene, alkyl halide functional groups (Nautiyal and Dubey, 2021).
       
Bijamrith formulation prepared from Gir cow urine and dung showed the presence of cyclohexanol, 3,3,5-trimethyl-, compounds exhibiting properties of abcissic acid. Similarly, Radha and Rao (2014) demonstrated that bio-dynamic formulations from cow dung and urine such as cow pat pit and panchagavya attributed to plant growth promoting properties with the production of auxins, IAA, GA, phosphorus solubilizing ability and control of various soil and root borne pathogens in maize. Combined application of cow dung and urine formulations reduced blight disease incidence in a plant called Ribwort (Plantago lanceolata).
       
In the present elucidation of compounds in bijamrith, sex pheromone compounds namely Z,Z-3,15-octadecadien-1-ol acetate has been identified. Lestari and Andi Andrian (2017) illustrated that plant growth parameters such as plant height, leaf length and leaf width of sorghum were increased by application of 100cc/l of cow urine. The plant growth promoting compounds in the urine might have contributed to improved plant growth parameters. Urine of Punganur cow at various physiological stages showed that the hormones such as growth hormones, estrogen in heifers, cortisol in milch cows, thyroxine in dry cow were released depending upon the feed intake (Chitteni et al., 2022).
       
The GC-MS results of the current study showed the occurrence of fungicidal compounds such as heptadecanoic acid, 16-methyl-, ester compounds. It was found that 15% concentration of cow urine showed inhibition of plant pathogenic fungi such as Fusarium, Rhizoctonia, Sclerotium and served as a potential biopesticide (Jandaik et al., 2015). In the present GC-MS findings of Gir cow based bijamrith, the same type of antimicrobial compound has been elucidated. The secondary metabolites of the Colletotrichum siamense  analyzed from the GC-MS analysis revealed the presence of compounds namely methyl 9,12-epithio-9,11-octadecanoate that were reported to have antibacterial, antifungal and antiviral activities (Vaijayanthi et al., 2023).
 

The Gir cow based organic formulation namely bijamrith prepared using dung and urine elucidated 40 different types of plant growth promoting, antibacterial and antifungal compounds. Amongst all, 9-octadecenoic acid (Z)-, ester compound namely 2,3-dihydroxypropyl were the major biologically active molecules identified in bijamrith formulation. The present study on scientific validation of bijamrith from Gir cow opens a broader avenue for the application of bijamrith in seed coating, pelleting or seed priming for the development of designer seeds in near future.

The authors gratefully acknowledge the Director of Research, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India for granting core research grant for scientific evaluation of various organic based formulations in a successful manner.

The authors declare no conflict of interest and all the authors contributed equally.