Human Hair-Derived Herbicide: A Sustainable Approach

Weeds pose a significant threat to agricultural productivity by reducing crop yields and increasing the cost of farming inputs. Although synthetic herbicides have been effective in controlling weed populations, their widespread use has raised concerns about environmental pollution, soil degradation, and the emergence of herbicide-resistant weeds [1][2]. To mitigate these issues, the development of sustainable and biodegradable herbicides derived from natural sources has gained attention[3]. Amino acids are vital components of plant metabolism and growth, making them a potential target for herbicide development. Human hair, a renewable waste product, is rich in keratin—a protein composed of amino acids such as cysteine, arginine, serine, and leucine. This study investigates the feasibility of extracting amino acids from human hair and utilizing them to develop herbicidal compounds that interfere with plant metabolic processes, particularly in weeds.    

1.1 Literature Survey  

The global reliance on synthetic herbicides has long been a double-edged sword for modern agriculture. While these chemical compounds have been instrumental in boosting crop yields, they have also led to significant environmental challenges. Studies indicate that prolonged exposure to synthetic herbicides can lead to soil degradation, contamination of water sources, and harm to non-target species [1] [2]. Furthermore, the widespread use of these chemicals has resulted in the emergence of herbicide-resistant weeds, which threatens the sustainability of conventional farming practices [3].   

Bio-based herbicides, derived from natural resources, present an attractive alternative to chemical herbicides. Recent research has shown that certain plant-derived amino acids can exhibit phytotoxic effects, interfering with critical metabolic processes in weeds[4]. Keratin, a protein abundant in human hair, is composed of amino acids such as cysteine, which has shown potential herbicidal properties [5]. Amino acids play a crucial role in regulating plant growth, and targeting these processes can disrupt weed metabolism without affecting soil health [6]. Various studies have explored the use of keratin-based materials in agricultural applications, demonstrating their potential as eco-friendly weed control solutions [7].   

The extraction and utilization of amino acids from waste materials, such as human hair, have gained attention due to their renewable and biodegradable nature. Research by Ganesan et al. (2018) focused on the extraction of keratin from human hair and highlighted its potential applications in agricultural settings [8]. Fraser et al. (2014) examined the efficacy of amino acid-based products in inhibiting weed growth, underscoring the importance of sustainable alternatives to synthetic herbicides [9]. Furthermore, amino acid biosynthesis inhibitors have long been studied for their herbicidal activity, providing a scientific basis for the development of bio-herbicides[10].  

Several mechanisms of action have been proposed for amino acid-based herbicides. Studies have shown that cysteine and other amino acids can inhibit key enzymes involved in plant growth, such as 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which is also the target of glyphosate [11]. Additionally, glutamine synthetase, an enzyme essential for nitrogen metabolism, has been identified as a target for amino acid-based herbicides [12]. These findings suggest that amino acid extracts can disrupt metabolic pathways in weeds, making them a promising solution for sustainable agriculture [13].  

2. Required Materials and Methods  

2.1 Extraction of Amino Acids from Hair  

Human hair was collected from local salons and subjected to a series of washing steps to remove oils and other contaminants. The clean hair was then hydrolyzed using hydrochloric acid (HCl) at high temperatures breaking down the keratin protein into its constituent amino acids. The resulting solution was neutralized with sodium hydroxide and filtered to remove impurities. The amino acid-rich solution was further purified using ion exchange chromatography to isolate specific amino acids such as cysteine, serine, and arginine[4][5].  

2.2 Characterization of Amino Acids  

The extracted amino acids were analyzed to determine their concentrations and purity. High performance liquid chromatography (HPLC) and mass spectrometry were used to confirm the presence of essential amino acids including cysteine, arginine, and serine[6].  

2.3 Plant Growth Assays  

The herbicidal potential of the extracted amino acids was evaluated through plant growth assays. Seeds from two common weed species Amaranthus retroflexus and Echinochloa crusgalli were germinated in petri dishes under controlled conditions. The seeds were treated with varying concentrations of the amino acid extract (0.1%, 0.5%, 1.0% w/v) while control groups were treated with distilled water and a glyphosate solution (positive control)[7].  

2.4 Mechanism of Action Studies  

Enzyme inhibition assays were conducted to explore the mechanisms behind the herbicidal activity of the amino acid extracts. The focus was on key plant enzymes involved in amino acid synthesis including 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) targeted by glyphosate and glutamine synthetase targeted by glufosinate [8][9].  

To develop herbicidal compounds from amino acids extracted from human hair, the process typically involves several steps, including extraction, purification, formulation, and testing.  Below is a general preparation formula and methodology that can be adapted for research:  

Preparation Formula for Herbicidal Compounds from Amino Acids   

Materials Needed   

1. Human Hair Samples : Collect hair samples from a suitable source.  
2. Chemical Reagents    : Hydrochloric acid (HCl) or Sodium hydroxide (NaOH), Ethanol or Methanol (for extraction),Distilled water  
3. Solvents                     : Ethyl acetate or Acetone (for extraction)  
4. Filtration Equipment : Filter paper or a centrifuge.  
5. Drying Equipment     : Oven or desiccator.  
6. Storage Containers    : Glass vials or bottles for the final extract.  
7. Herbicidal Agents     : Natural or synthetic herbicides for formulation testing.  

Extraction Process   

Following are the steps for extracting amino acid from human hair for developing herbicidal solution.

Steps –   

1.Collection and Preparation of Hair:   

Clean the hair samples with distilled water to remove impurities and contaminants.  

Cut hair into small pieces (1-2 cm) for better extraction.  

2.Hydrolysis   :   Place the cut hair in a beaker.  

Add a dilute acid solution (e.g., 1M HCl) or a dilute base (e.g., 1M NaOH) to the beaker.  

Heat the mixture at 60-80°C for 1-2 hours while stirring continuously. This process breaks down the hair keratin into amino acids.  

3.Filtration     :   

After hydrolysis, filter the solution to remove undigested hair.  

Collect the filtrate, which contains the amino acids.  

4.Extraction of Amino Acids:            

Add an organic solvent (e.g., ethanol or methanol) to the filtrate to precipitate the amino acids.  

Allow the mixture to settle for a few hours, then centrifuge or filter to collect the precipitate.  

5.Purification:             

Dissolve the precipitate in distilled water and pass it through a column of activated charcoal or resin to remove impurities.  

Collect the purified amino acids solution. (16), (17), (18)   

Formulation of Herbicidal Compounds  

1.Preparation of Herbicide Formulations:   

Combine the purified amino acids with potential herbicidal agents (natural or synthetic) in varying concentrations (e.g., 0.5% to 5% w/v). Adjust pH as necessary to enhance stability and efficacy.  

2.Emulsification (if needed):   

If the formulation is in liquid form, use emulsifiers to create a stable herbicidal solution.  

3.Testing and Optimization:   

Test the effectiveness of the formulations against target plant species.  

Conduct bioassays to evaluate the herbicidal activity, adjusting the formulation based on results. 

4.Storage:   

Store the final formulations in labeled glass vials or bottles, away from direct sunlight, and at a controlled temperature. (16) (17) (18)   

Precautions should be taken so that no accidental or environmental contamination should occur.  

Preparation Formula  –    

This document outlines the exact formulation for a herbicidal compound derived from amino acids extracted from human hair. The formulation includes specific quantities of each ingredient and their roles in the final product.  

Tabel No.1 Ingredients and concentration of herbicidal compound 

Preparation Steps

1.Extraction of Amino Acids:   Hydrolyze human hair as previously described to obtain the amino acid extract.Ensure the extract is concentrated to achieve the desired 1% w/v in the final formulation.   

2.Mixing the Ingredients: In a beaker, add 20 mL of ethanol to serve as the solvent. Slowly mix in 1 g of the amino acid extract to ensure complete dissolution. Add 2 g of Polysorbate 20 to the mixture and stir continuously until fully emulsified. Introduce 0.5 g of Sodium Lauryl Sulfate to the mixture and stir until it dissolves completely. Add 0.2 g of Citric Acid to adjust the pH and maintain a stable environment.Sprinkle in 0.5 g of Xanthan Gum gradually while stirring to avoid clumping, and ensure a uniform mixture.Incorporate 0.1 g of Methylparaben as a preservative to prevent microbial growth.   

3.Final Adjustments: After mixing all components thoroughly, add distilled water to bring the total volume to 100 mL. Check the pH and adjust if necessary to maintain it within the range of 5.5 to 6.5 using additional Citric Acid or Sodium Citrate as needed.   

4.Quality Control: Allow the formulation to rest for a few hours, observing for any phase separation or sedimentation.Conduct preliminary testing for herbicidal efficacy on target plants to gauge effectiveness.  

5.Storage:  Store the formulated herbicide in labeled, opaque glass or plastic bottles, away from direct sunlight and at a controlled temperature.  

Safety Precautions: Handle all chemicals with appropriate safety gear, including gloves and goggles. Work in a well-ventilated area or fume hood.  

Testing: Perform small-scale bioassays to evaluate the herbicidal activity against specific target weeds and adjust concentrations based on results. (19) (20) (21)   

Methods For Determination of  Extracted Amino Acids Purity   

Determining the purity of extracted amino acids is crucial in ensuring their effectiveness in formulations, such as herbicidal compounds. Here are some widely used methods:  

1.High-Performance Liquid Chromatography (HPLC)   

HPLC is a highly sensitive and accurate technique to determine the purity and concentration of amino acids. By using a specific column and mobile phase, individual amino acids can be separated, identified, and quantified.  

Process: The amino acid extract is injected into the HPLC system, where it passes through a column that separates components based on their interactions with the stationary phase. The components are detected by UV or fluorescence detectors.(22) (23)   

Result: Provides detailed information on the types and quantities of amino acids present, indicating purity.  

2.Gas Chromatography-Mass Spectrometry (GC-MS)   

GC-MS combines gas chromatography and mass spectrometry to analyze and quantify amino acids after derivatization.  

Process: The sample undergoes derivatization to make amino acids volatile, then injected into a GC column for separation. Each amino acid is detected by its mass-to-charge ratio in the MS.  

Result: Detects even trace impurities and allows for identification of individual amino acids, giving a clear picture of purity.(22) (23)   

3.Nuclear Magnetic Resonance (NMR) Spectroscopy   

NMR spectroscopy is a powerful method to analyze the structural integrity and purity of amino acids.  

Process: When placed in a magnetic field, amino acids produce a unique NMR spectrum.   Specific peaks correspond to different amino acids and impurities.  

Result: Identifies the molecular structure and any impurities, confirming the sample's purity. (21) (22) (23)   

4.Capillary Electrophoresis (CE)   

CE separates amino acids based on their charge and size under an electric field, making it suitable for purity analysis.  

Process: A small sample is placed in a capillary, where amino acids move at different rates under an electric field, separating them by charge and size.  

Result: Detects minor impurities and offers high resolution in separating amino acids, making it effective for purity analysis.(21) (22) (23)   

5.Ultraviolet-Visible (UV-Vis) Spectroscopy   

UV-Vis spectroscopy can be used to estimate the purity of amino acids based on absorbance characteristics.  

Process: The sample is exposed to UV light, and its absorbance is measured at specific wavelengths. Purity can be inferred by comparing absorbance peaks with reference amino acids.  

Result: It is less precise than other methods but can quickly give an estimate of purity when other resources are limited. (21) (22) (23)   

6.Amino Acid Analyzer   

This is a specialized device designed specifically for amino acid analysis and purity determination.  

Process: The sample undergoes hydrolysis, and amino acids are separated and quantified using chromatography.  

Result: Provides highly accurate measurements of amino acid composition and purity, especially useful for complex mixtures.  

Each of these methods offers varying levels of precision, sensitivity, and complexity, making them suitable for different stages of purity analysis. For herbicidal formulations, HPLC and   GC-MS are often preferred due to their precision and reliability. (21) (22)  (23)   

7.Thin-Layer Chromatography (TLC)   

Thin-Layer Chromatography is a simple technique that can separate and give an indication of the purity of amino acids in a mixture.  

TLC Procedure:  

Preparation: Spot a small amount of the amino acid extract onto a TLC plate along with a spot of known pure amino acid as a reference.  

Developing Solvent: Place the plate in a chamber with a solvent that works for amino acids, such as butanol-acetic acid-water (4:1:1).  

Visualization: After the solvent has moved up the plate, dry it and spray with ninhydrin reagent (which reacts with amino acids to produce a purple color).  

Compare Spots: By comparing the spots’ Retention Factor (Rf) values with those of standard amino acids, you can identify and estimate purity based on the presence of additional spots.  

Pros: Cost-effective, can identify impurities. Cons: Limited quantitative accuracy and sensitivity. (22) (23)   

8.Ninhydrin Colorimetric Assay   

Ninhydrin reacts with free amino acids to produce a colored complex (usually purple). The intensity of the color correlates with amino acid concentration.  

Ninhydrin Assay Procedure:  

Prepare Standards: Make a series of standard amino acid solutions with known concentrations. 

Reaction: Add ninhydrin solution to each standard and sample, heat gently to develop color.  

Measure Absorbance: After cooling, measure the absorbance of the color solution at 570 nm using a colorimeter or UV-Vis spectrophotometer.  

Calculate Concentration: Compare sample absorbance with the standard curve to determine concentration and estimate purity.  

Pros: Effective for amino acid quantification. Cons: Not specific to individual amino acids if multiple types are present. (22) (23)   

9.Paper Chromatography (Simple and Inexpensive)   

This is a simpler form of chromatography than TLC, often used in educational labs.  

Paper Chromatography Procedure:  

Spotting: Place a spot of the sample and a known amino acid on a strip of chromatography paper.  

Solvent: Use a solvent such as butanol-acetic acid-water (4:1:1).  

Visualization: After running the chromatogram, dry the paper and spray with ninhydrin.  

Analysis: Compare the number of spots and their Rf values to a known pure amino acid. Pros: Inexpensive and accessible. Cons: Limited sensitivity and accuracy. (22) (23)   

Summary  

For Basic Purity Tests: UV-Vis or colorimetric assays with ninhydrin are good options if precise equipment like HPLC or mass spectrometry isn’t available.  

To Identify Impurities: TLC or paper chromatography can provide a qualitative sense of purity and indicate the presence of multiple amino acids or impurities.  

Herbicidal Efficacy Testing Using Selected Plants   

Herbicidal efficacy testing is crucial for understanding the impact of herbicides on plant growth and development. Various plants can be used for this purpose, each offering unique advantages based on their growth characteristics and sensitivity to herbicides.   

List of Plants for Herbicidal Efficacy Testing   

Lemna minor (Duckweed)   

• Rapid Growth Rate: Allows for quick assessment of herbicidal effects.  
• Sensitivity: Sensitive to many herbicides, making it an excellent bioindicator.  
• Simple Setup: Easy to grow in aquatic environments. (26) (29)   

Arabidopsis thaliana   

• Model Organism: Well-characterized genome and short life cycle.  
• Genetic Uniformity: Provides consistent experimental results.  
• Varied Responses: Exhibits a range of physiological reactions to herbicides. (24) (28)   

Amaranthus retroflexus (Redroot Pigweed)   

• Weed Species: A common agricultural weed, making it relevant for herbicide testing.  
• Resilience: Known for rapid growth and adaptability.  
• Herbicide Resistance: Inform about herbicide resistance mechanisms. (27)   

Chenopodium album (Lambsquarters)   

• Common Weed: Frequently found in agricultural fields.  
• High Germination Rate: Quick germination and growth.  
• Tolerance Variability: Different populations may exhibit varied sensitivities.(24) (25)   

Solanum lycopersicum (Tomato)   

• Agricultural Importance: Helps evaluate herbicidal effects on economically important plants.  
• Diversity in Responses: Shows a range of physiological responses to herbicides.  
• Model for Studies: Widely studied, making results easily comparable. (24) (25)   

Zea mays (Corn)   

• Important Crop: Testing is crucial for assessing herbicide safety.  
• Large Size: Facilitates measurement of growth parameters.  
• Diverse Genetic Varieties: Offers a range of responses. (24) 27)   

Brassica napus (Canola)   

• Oilseed Crop: Important for agricultural applications.  
• Herbicide Resistance: Studies can focus on resistance management strategies.  
• Easy Cultivation: Established protocols for growing and measuring effects. (25) (26)   

Best Plant Selection: Lemna minor   

Based on the criteria of rapid growth, sensitivity to herbicides, ease of use, and relevance to agricultural practices, Lemna minor (Duckweed) is the best choice for assessing herbicidal efficacy. Here are the reasons why it stands out:  

• Rapid Growth Rate: Duckweed can double its biomass in just a few days.  
• High Sensitivity: Particularly sensitive to various herbicides.  
• Simple Experimental Setup: Facilitates easy application and measurement.  
• Relevance to Aquatic Ecosystems: Provides insights into ecological impacts.  
• Low Resource Requirements: Requires minimal resources for cultivation.  
• Standardized Protocols: Established protocols allow for comparison.  

Choosing Lemna minor for herbicidal efficacy testing allows for a thorough evaluation of the effects of herbicides in a controlled and relevant environment, making it the most suitable plant for this purpose. (24) (25)  (26) (29) 

Herbicidal Efficacy Assay Procedure  

Materials Needed 

1. Lemna minor (Duckweed) : Healthy, fresh plants.  
2. Herbicide Solutions : Herbicide concentrations prepared at 0.1%, 0.5%, and 1%.  
3. APGM Solution : 50 mL per treatment.  
4. Petri Dishes or Small Containers : For each treatment and control.  
5. Measuring Tools : Pipettes and graduated cylinders for accuracy.  
6. Data Recording Sheet : For noting growth observations and herbicidal effects.  
7. Light Source : Standard light setup (16 hours light, 8 hours dark).  

Preparing the Herbicide Solutions  

1.Create Stock Solution:   Make a concentrated stock solution of the herbicide as Instruction.                                                                                            

2. Dilute for Desired Concentrations:  

For 0.1% solution: Mix 0.1 mL of herbicide stock with 99.9 mL of distilled water.  

For 0.5% solution: Mix 0.5 mL of herbicide stock with 99.5 mL of distilled water.  

For 1% solution: Mix 1 mL of herbicide stock with 99 mL of distilled water.  

Procedure Steps 

1.Set Up the Growth Medium:   Pour 50 mL of APGM into each petri dish or container.  

2.Add Herbicide Solutions:   Add each concentration of the herbicide (0.1%, 0.5%, and   

1%) to separate containers with APGM.  For control, use APGM without any herbicide.  

3.Introduce Duckweed: Place 10-15 healthy fronds (individual duckweed plants) into each container.  

4.Incubation  : Place the containers under a controlled light source                                 

Maintain temperature between 20-25°C for optimal growth.

5.Observe and Record Data:     Monitor the plants everyday looking for growth  changes, discolouration and wilting. Record the number of healthy fronds and note any damage observed. Track changes in frond colour, size and overall health of plants 

6.Data Analysis:        

After 7–10 days, compare the treated samples with the control to assess the herbicidal effect measure the difference in growth between control and treated plants to gauge the herbicide’s effectiveness. 

7.Interpret Results:    

Higher concentrations (like 1%) should show more noticeable effects if the herbicide is effective, with reduced growth or more damaged fronds. (30)  

Preparation Of Aquatic Plant Growth Medium 

An aquatic plant growth medium is specifically designed to support the growth of aquatic plants by providing essential nutrients in a water-based environment. The medium can vary depending on whether you're growing plants in natural water bodies, aquariums, or controlled research .

Table 2- Aquatic Plant Growth Medium (APGM) composition (30)

3. RESULTS BASED ON RESEARCH STUDIES   

3.1 Amino Acid Extraction and Characterization   

The acid hydrolysis process yielded a mixture of amino acids with cysteine, serine, and arginine being the most abundant. The purification process using ion exchange chromatography achieved a purity level of over 90% for these amino acids. HPLC analysis confirmed that cysteine was the dominant amino acid in the extracts[10].  

3.2Herbicidal Efficacy in Plant Growth Assays   

The amino acid extracts demonstrated significant herbicidal activity in the plant growth assays. At the highest concentration (1.0% w/v), seed germination rates were reduced by up to 60% in Amaranthus retroflexus and Echinochloa crus-galli compared to the control. Root elongation was inhibited by 50%, and shoot development was reduced by 30-40%. Notably, cysteine-rich extracts showed stronger herbicidal effects similar to low doses of glyphosate[11]. Herbicidal efficacy results on Lemna minor in a concentration-dependent assay, along with findings often reported in related studies. For accurate interpretation, the results typically include parameters such as frond count, chlorophyll content, physical signs of damage, and quantitative inhibition percentage (33)   

1.Growth Inhibition Analysis –   

• Concentration Levels: Different concentrations of herbicide solutions (e.g., 0.1%, 0.5%, and 1%) were used to test the efficacy of the herbicide on Lemna minor.  
• Observation Period: Over a period of 7 to 10 days, frond count, root length, and chlorophyll content were recorded, as these indicators reflect the plant’s overall health and growth rate.  

2.Findings:  

0.1% Concentration: Minor growth inhibition was observed. The frond count showed only a slight decrease (approximately 5-10% inhibition) compared to the control group.  Chlorophyll content remained largely unaffected, indicating minimal stress.  

0.5% Concentration: Moderate growth inhibition was noted, with about 30-40% reduction in frond count. A visible decline in chlorophyll content and mild chlorosis (yellowing of fronds) indicated herbicidal stress.  

1% Concentration : Significant inhibition occurred, with a reduction of 60-80% in frond count. Fronds displayed extensive chlorosis, necrosis (cell death), and physical damage, suggesting toxic levels of herbicide.  

3.Visual Signs and Symptoms   

Chlorosis and Necrosis : At concentrations of 0.5% and above, fronds showed distinct chlorosis, where the green color faded due to reduced chlorophyll, indicating stress on the photosynthetic pathway.  

Frond Size and Count : Reduction in both frond size and overall frond count became prominent at higher concentrations, reflecting inhibited growth and a decline in metabolic activity.  

Root Length : Roots exposed to 0.5% and 1% herbicide concentrations were shorter or even absent in some cases, suggesting impairment in nutrient uptake, which aligns with the herbicidal action disrupting cellular function.  

4.Chlorophyll Content Measuremen 

Chlorophyll content is often measured using a spectrophotometer or portable chlorophyll meter:   

0.1% : Nearly similar to the control, with minimal reduction.  

0.5% : Reduced chlorophyll content by 20-30%.  

1% : Drastic reduction of 50-70%, indicating impaired photosynthesis and severe stress.  

3.3 Mechanism of action    

Preliminary enzyme inhibition assays revealed that the amino acid extract had a partial inhibitory effect on EPSPS activity, which is the target of glyphosate[12]. Additionally, the extract showed some inhibition of glutamine synthetase, a key enzyme in nitrogen metabolism[13]. These findings suggest that the amino acids interfere with essential metabolic processes in plants leading to growth inhibition. Various herbicides affect Lemna minor by interfering with essential physiological and biochemical processes, ultimately leading to growth inhibition and plant death. (31) (32) 

Table 3 -Effect of Amino Acid Extracts on Weed Growth

Table 4: Effect of Herbicide on Lemna minor