Study Design and Sampling

Around 300g of the soil was collected at an aborigine village in Ulu Semenyih, Selangor, Malaysia. The soil sample was collected at the riverside, near the toilet area, and at the pond area. Contaminated soil with helminths eggs is expected within these areas knowing practice of the aborigine community practicing open defecation. Cow urine (CU) samples were obtained from a cow farm located at Lanchang, Pahang, Malaysia in the early morning from the cow care takers. Around 250mL of CU underwent an evaporation process by using a hot plate at 60°C to concentrate the compound in the cow urine.

Soil Sample Collection

A small shovel and a plastic bag were used to collect the soil samples. Roughly around 200-300 grams of soil within a depth of 4-6 inches (optimized) was collected. The soil samples were collected from the toilet area as the soil from the toilet area is loamy which is the optimum environment for the helminth eggs to grow. The preservation of soil samples took place in a refrigerator to keep the soil in its state prior experiment.

Helminth Egg Isolation from Soil Samples

Soil samples undergo floatation techniques to isolate helminth eggs from the soil. After the detection of eggs using McMaster Chamber, the eggs were cultured by using 0.1% sulfuric acid in a glass petri dish. The eggs were cultured at a temperature of 30-35⁰C for three weeks. A few drops of 0.95% sodium chloride were added to prevent the dryness of the sample within the duration.

Cow Urine Preparation

Cow urine samples were replicated and separated into 4 types of preparation. The preparations are called raw cow urine (RCU), evaporated raw cow urine diluted with distilled water (ERCUD), evaporation raw cow urine diluted with saline (ERCUS) and evaporated filtered cow urine diluted with saline (EFCUS). Cow urine repeatedly undergoes different processes in quadruple. The evaporation process was done by using a hot plate with a temperature of 60°C. As for the filtration process, Whatman filter paper was used to filter cow urine. Cow urine samples were filtered and evaporated by using a hot plate with a medium temperature (60⁰C). It took approximately 4 to 5 hours for the cow urine samples to evaporate completely. Around 5ml of distilled water and 5ml of saline were added respectively for each sample to prepare ERCUD, ERCUS, EFCUD, and EFCUS. Different preparations of cow urine were done to investigate how different preparations of cow urine can affect the anthelmintic properties of cow urine. 

It took around 3 weeks to isolate and culture both helminth eggs. From the soil samples, two types of helminths are commonly found which are Ascaris lumbricoides and Trichuris trichiura. Table 1 below shows all the summarized results post 24 hours of various treatments using the most commonly found helminths in this study.

Based on Table 1, for raw cow urine (RCU), both Ascaris and Trichuris eggs can be found after being exposed to the sample for 24 hours. There is no indication that the RCU affects both types of helminth eggs as the eggs are still attached to their structure and can be found under the microscope using the McMaster Chamber after 24 hours. In addition, for evaporated raw cow urine diluted with distilled water (ERCUD) and evaporated filtered cow urine diluted with saline (EFCUS), both results for Ascaris lumbricoides and Trichuris trichiura indicate there are no eggs that can be detected after 24 hours of being exposed to the sample. However, for evaporated raw cow urine diluted with saline (ERCUS), helminth eggs can be monitored before and after the exposure to the sample.

Based on the result shown in Table 2, the structure of healthy Ascaris lumbricoides is equivalent to a smooshed circle layered by a thick shell that consists of three layers of membrane. As regards distorted Ascaris lumbricoides structure, the outer layer of the membrane is perceptible to the eye where the membrane is impaired. Thereupon, it can be inferred that the exposure of ERCUS is proficient in disrupting the structure of helminth eggs by giving ample of time for the anthelmintic property to elapse.

Figures & Tables

Discussion

The result above shows that all tests using evaporated raw cow urine dilute with saline compound shows a prominent result than using raw cow urine. Preliminary studies indicated that cow urine in its distillation form has more potency in its larvicidal activity than cow urine [5]. This may be due to the phenol substance in cow urine compositions. Phenols have been substantiated as anthelmintic agents in previous research due to their complex form and their hydrogen binding [6]. Furthermore, distillation is an optimal water removal process as 97% of water and 50% of salts can be removed without losing a considerable amount of nutrients which can be advantageous for the final product use as fertilizer [7].

In cow urine, there are more than 10 types of enzymes which are alkaline phosphatase (110.110 KA unit), lactate-dehydrogenase (21.780 unit/L), amylase (90.236 unit), acid phosphatase (456.620 XA unit), vitamin B1 (444.125 mg/L), vitamin B2 (0.6339mg/L), vitamin C (216.408mg/L), uric acid (135.028 mg/L), protein (0.1037 g/L), lactate (3.7830 mmol/L), creatinine (0.9970 g/L), aromatic hydroxyl acid (2.7030mg/100ml), calcium (5.735 mmol/L), phosphorus (0.4805 mmol/L), phenol (4.7580mg/100ml), free volatile phenol (0.7130 mg/100ml) and compound volatile phenol (1.3420mg/100ml) [8]. Cow urine properties such as volatile and non-volatile components which are urea, aurum hydroxide, phenols, carboxylic acid, creatinine, calcium, and manganese are well known for their ability as antimicrobial, antifungal, and germicidal [9]. Moreover, both biogenic volatile inorganic and organic compounds such as methanol, propanol, acetone, CO2, NH3, CH4, and some metabolic secondary nitrogenous products are formed during the distillation process of cow urine [10].

Previous research shows that cow urine can cause paralysis and death in worm’s dependent on a dose which indirectly shows the anthelmintic activity of cow urine were affected by its concentration [10]. In this study, cow urine was marked anthelmintic by affecting the structure of the eggs within a period. Cow urine influences a wide range of fields as it has been proven that it is also effective as a pest controller and larvicide [11]. Cow urine is a substantial source of macronutrients and micronutrients and thus purifies the environment and increases soil fertility with disinfectant and prophylactic properties [12]. With so many benefits of cow urine, the ability to enrich the soil, fertility of the soil, and productivity of the soil is important. It modifies the texture of the soil and has a high content of oxygen and nitrogen that is used to produce superior-quality compost to improve agricultural yields and save humans from the residual effects of harmful pesticides and fertilizers [13].

The beneficial effect of cow urine on health is caused by its chemical components, therefore ingestion of cow urine distillate or any other form is beneficial not only for human physiology but also as an antiseptic, fertilizer, and other therapeutic values [14]. Moreover, cow urine has been granted U.S patents (No. 6,896,907 and 6,4,10,059) for its therapeutic properties, especially for its ability as an antibiotic agent to control bacterial infections and to fight cancer [15]. It has struck a pose as its therapeutic value has been proven across every medical field including as an anthelmintic agent. Cow urine is well known for its many therapeutic value, and great pharmacological significance, and its medicinal usefulness has been extensively described in Ayurveda in detail [16]. Thus, cow urine is highly valuable for the use thereof its anthelmintic property in all other spheres.

It is hypothesized that evaporated raw cow urine possessed biochemical agents that can be used in the future as anthelmintic agents. It has been proven that cow urine can affect helminth egg structure in its evaporated form. Further studies can explore more possibilities to investigate cow urine as a potential medicine. Different methods such as photo-activation of cow urine, high-performance liquid chromatography (HPLC), distillation, and evaporation can be done in the future to compare the result and provide a better recommendation.         

Conflict of Interest

The authors declare that there is no conflict of interest.

Author Contributions

Dr. Mehru Nisha and Ruziana conceived of the presented idea. Ruziana developed the theory and performed the computations. Dr. Mehru Nisha and Dr. Pang Jyh Chyang verified the analytical methods and encouraged Ruziana to investigate anthelmintic property of cow urine and supervised the findings of this work. All authors discussed the results and contributed to the final manuscript. Ruziana carried out the experiment and wrote the manuscript with support from Dr. Mehru Nisha and Dr. Pang Jyh Chyang.

References