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Selective Extraction of Cannabinoid Compounds from Cannabis Seed Using Pressurized Hot Water Extraction

Phytochemicals of Cannabis sativa mainly for the use in the different industries are that of delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Pressurized hot water extraction (PHWE) is seen as an efficient, fast, green extraction technique for the removal of polar and semi-polar compounds from plant materials. The PHWE technique was applied to extract cannabinoid compounds from Cannabis sativa seed. Response surface methodology was used to investigate the influence of extraction time (5–60 min), extraction temperature (50–200 °C) and collector vessel temperature (25–200 °C) on the recovery of delta-9-tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD), cannabichromene (CBG) and cannabigerol (CBC) from Cannabis sativa seed by PHWE. The identification and semi quantification of cannabinoid compounds were determined using GCXGC-TOFMS. The results obtained from different extractions show that the amount of THC and CBN was drastically decreasing in the liquid extract when the temperature rose from 140 to 160 °C in the extraction cell and the collector′s vessel. The optimal conditions to extract more CBD, CBC, and CBG than THC and CBN were set at 150 °C, 160 °C and 45 min as extraction temperature, the temperature at collector vessel, and the extraction time, respectively. At this condition, the predicted and experimental ratio of THCt (THC + CBN)/CBDt (CBD + CBC+ CBG) was found to be 0.17 and 0.18, respectively. Therefore, PHWE can be seen as an alternative to the classic extraction approach as the efficiency is higher and it is environmentally friendly.

1. Introduction

Cannabis L Sativa is a plant belonging to the family of Cannabaccae and grows to around 2–5 m in height [1]. The plant is considered dioecious since it contains both male and female parts [2]. It exhibits several psychoactive and medicinal activities. The use of Cannabis has been recorded throughout history in many industries, such as food, cosmetics, paper, clothing, and pharmaceutical [3,4]. The phytochemicals within the plant have gained interest overtime for its medicinal purposes, mainly for its use against cancer-inducing effects [4,5].

Up today, there are above 500 compounds within the Cannabis L Sativa plant which are known, the most interesting is a set of compounds known as cannabinoids, there are about 70 different cannabinoids which are unique to the Cannabis L Sativa plant [5,6]. Among these, the most psychoactive compound being delta-9-tetrahydrocannabinol (THC), the metabolic breakdown of the compound, which is cannabinol (CBN), is still psychoactive, but less than that of THC. The second most abundant compounds are that of cannabidiol (CBD), cannabichromene (CBG) and cannabigerol (CBC). These compounds are synthesized from cannabinoid acids [1,7]. CBD, as explained, is one of the main anticancer components found in the Cannabis L Sativa plant. It is mainly found in the form of cannabidiolic acid (CBDA) and changes to CBD through decarboxylation [7,8].

The problem with the use of cannabis in pharmaceuticals and other endeavors is the presence of the THC and CBN in the plant, which is considered the more psychoactive and toxic for a human [8]. It can also affect the brain of young adults under the age of 21 years, and it has shown to affect school performance by reducing IQ [9]. Hence, various extraction techniques were applied to isolate or suppress the psychoactive compounds from the extract [1,10].

The extraction of cannabinoid compounds from cannabis has attracted the attention of many researchers [2,5,11,12]. Several extraction techniques of cannabinoid compounds from Cannabis L Sativa rely on the use of of conventional extraction approaches such as distillation, solvent extraction, Soxhlet, maceration, and sonication, however, these techniques are time-consuming and use an important volume of organic solvent with toxicity for environmental and human well-being [13,14]. Hence, the development of modern extraction techniques like ultrasound-assisted extraction (UAE), supercritical extraction (SFE) and pressurized hot water extraction are emerging as an alternative [15,16]. These techniques present a significant advantage over conventional methods. These advantages are less degradation, elimination of additional sample clean up, reduction in organic solvent consumption, concentration steps before chromatographic analysis, selectivity, improvement in kinetics, and extraction efficiency and ease of automation [17,18]. In other hand, UAE is much faster and low cost than the traditional extraction techniques. Nevertheless, the efficiency of the technique depends on the nature of the target analytes. Besides, it should be kept in mind that the distribution of the ultrasonic wave in the vessel is not uniform. It is limited to the vicinity of the ultrasound probe, making its application on a big scale a difficult task. SFE was more selective than the UAE method. It provides the advantages of high diffusivity and low viscosity. The main disadvantage is a high running cost [5,12,19,20].

The alternative to these techniques is pressurized hot water extraction (PHWE). It is considered a green extraction method. Instead of CO2, water is used under its supercritical form as a solvent that exhibits the same solvability properties as methanol and ethanol [21]. Pressurized hot water extraction has proved to be an excellent approach for the recovery of the polar and semi-polar bioactive compounds from plant materials [21,22]. PHWE technique is based on the use of high temperature and pressure to keep the water in the supercritical fluid form during the entire extraction process [23]. The technique has attracted attention due to its benefits as compared to other conventional and non-conventional extraction approaches [21]. PHWE has been widely used for the extraction of phenolic compounds [24]. In this technique, temperature is very important; it directly affects extraction efficiency and the mass transfer during the extraction process [25,26].

Since THC and CBN are the main psychoactive compounds from cannabis plant, several researcher have developed techniques for a high selectivity extraction of non-psychoactive compounds over the psychoactive one. For example, Romano and Hazekamp [20] have used an infusion extraction approach to recover more CBD than THC selectively. The final extract obtained by them was heated up to evaporate THC and keep CBD in the solution. Perrotin-Brunel et al. [27], and Grijo et al. [28] have explored the solubility properties of CO2 and pressure for an efficient extraction of CBD. The results showed that the solubility of cannabinoids compounds increase with the increase of the pressure. To our knowledge, there are no studies that have applied PHWE for selective extraction of cannabinoid compounds from Cannabis L Sativa seeds. Therefore, the present study includes a pressurized hot water extraction process that yields a formulated cannabinoid nutraceutical. The PHWE technique was applied to cannabis seed in order to extract more CBD and lessen the THC and CBN, reducing the psycho-activity of cannabis products.

2. Materials and Methods

2.1. Chemicals and Reagents

Chemicals used in this study were of analytical grade. Methanol was used as a solvent and purchased from Sigma-Aldrich (Johannesburg, South Africa).

2.2. Plant Material

One species of cannabis was subjected to extraction and analysis. Seed samples of Cannabis L Sativa were collected from farmers in Gauteng province in August 2019. Prior to processing, seeds were dried at 50 °C for 4 h and the water content was found to be 5.5–7.9% weight. The dried samples were crushed to powder and kept in a sealed container until extraction.

2.3. Response Surface Methodology

The influence of extraction temperature (80–200 °C), the temperature at collector vessel (25–200 °C) and extraction time (15–60 min) on the selective recovery of cannabinoid compounds were investigated using response surface methodology (RSM). Full factorial design, comprising of 30 experiment run with three center point replicates, was created in MODDE Pro (Sartorius Stedim Biotech, Malmö, Sweden), to assess the recovery of THC, CBN, CBD, CBG, and CBC from the Cannabis sativa seeds. During the optimization process, the pressure and flow rate were kept constant at 105 bar and 0.5 mL min −1 , respectively, according to our previous work [24]. The partial least square regression was applied to evaluate the fitting of the model and response surface. The adequacy of the models was evaluated by the R 2 and Q 2 values (where R 2 shows the model fit and Q 2 shows an estimate of the future prediction precision). The F-test was used to assess the significance of the coefficients of regression. The modeling was done with a quadratic model like linear, squared, and interaction terms.

2.4. Pressurized Hot Water Extraction

Pressurized hot water extraction instrument was constructed in the laboratory and equipped with an extraction cell, gas chromatography (GC) oven for controlling the extraction temperature and time, and a collector vessel ( Figure 1 ). This was in accordance with the method described by [24] with some modifications. For each run, a mass of 5 g of Cannabis sativa seed powder was filled into the PHWE extraction cell. Before each extraction, the cell loaded with the powder was pre-heated at a specific extraction temperature for 10 min. The dynamic extractions were done under various extraction conditions as specified by the CCD matrix ( Table 1 ). The approach used in the pressurized hot water extraction for cannabinoid compounds is to extraction.

Pressurized hot water extraction setup with showing trap solution and oil heat bath for collector.

Table 1

Experimental design with the response of independent factors (relative peak area).

Run Order Extraction Time (min) Extraction Temp. (°C) Collector Temp.(°C) THC (%) CBD (%) CBC (%) CBG (%) CBN (%)
19 15 80 25 0.5700 0.2989 0.2033 0.1345 0.1308
25 37.5 80 25 0.7900 0.4589 0.3121 0.2065 0.1812
7 60 80 25 0.9584 0.8978 0.6105 0.4040 0.2204
1 15 80 113 0.3989 0.2890 0.1965 0.1301 0.0917
4 37.5 80 113 0.9088 0.4556 0.3098 0.2050 0.2091
9 60 80 113 0.4278 0.8926 0.6070 0.4017 0.0984
21 15 80 200 0.2078 0.2699 0.1835 0.1215 0.0478
5 37.5 80 200 0.1889 0.3174 0.2158 0.1428 0.0435
18 60 80 200 0.1089 0.3878 0.2637 0.1745 0.0251
12 15 140 25 3.1504 1.4589 0.9921 0.6565 0.7246
10 37.5 140 25 2.8062 2.9819 2.0277 1.3419 0.6454
29 60 140 25 1.8639 3.7532 2.5522 1.6889 0.4287
15 15 140 113 1.4193 6.3856 4.3422 2.8735 0.3264
16 37.5 140 113 1.3032 7.0702 4.8077 3.1816 0.2997
20 60 140 113 1.0187 9.9046 6.7351 4.4571 0.2343
6 15 140 200 0.9537 1.0347 0.7036 0.4656 0.2194
2 37.5 140 200 0.6547 1.8502 1.2581 0.8326 0.1506
30 60 140 200 0.5809 2.9506 2.0064 1.3278 0.1336
11 37.5 140 113 0.7637 6.9309 4.9930 4.3189 0.1757
27 37.5 140 113 0.8428 5.6506 5.0824 3.1928 0.1938
14 37.5 140 113 0.6904 7.8078 5.5893 4.7135 0.1588
17 15 200 25 0.1444 0.1229 0.0836 0.0553 0.0332
23 37.5 200 25 0.1015 0.3353 0.2280 0.1509 0.0233
22 60 200 25 0.0927 0.4536 0.3085 0.2041 0.0213
8 15 200 113 0.0702 0.1099 0.0747 0.0495 0.0162
26 37.5 200 113 0.0497 0.1653 0.1124 0.0744 0.0114
3 60 200 113 0.0312 0.2221 0.1510 0.0999 0.0072
13 15 200 200 0.0201 0.0384 0.0261 0.0173 0.0046
24 37.5 200 200 0.0148 0.0407 0.0277 0.0183 0.0034
28 60 200 200 0.0105 0.0751 0.0511 0.0338 0.0024

The responses were expressed in percentage relative peak area, which is the peak area of each compound over the total peak area in the chromatogram; these percentages are generated by the instrument. During the extraction process, the collector′s vessel was heated at different temperatures in order to investigate the effect of these temperature variations on the removal of cannabinoid compounds in the obtained liquid extract.

2.5. GCXGC-TOFMS Method

A LECO 2D GCXGC chromatography with a time-of-flight mass spectrometer (LECO, Johannesburg, South Africa) was used for the identification and analysis of cannabinoid compounds from Cannabis sativa seed. This experimental GCXGC-QTOF-MS/MS method developed by Marrotti et al. [29] was used with some modification. The separation was carried out in a 30 m × 0.25 mm × 0.25 μm BPX5 column (SGE Analytical, Johannesburg, South Africa). The injection was carried out in spit mode (ratio 20:1). The carrier gas was helium (99.999% purity) and was used as at a flow rate of 1 mL min −1 . The oven temperature was set at 100 °C (for 2 min) and increased to 280 °C with rate of 15 °C min −1 . Temperatures applied were 300 °C for injector, 250 °C for transfer line, 250 °C for ion source, and 150 °C for quadrupole. Data were acquired in the full scan mode with mass ranging from 45–600 amu. The total ion chromatograms (TIC) were integrated. Raw data (m/z) generated by UHPSFC were processed using the ChromaTOF software version 4.5.1. (LECO Corp, St. Joseph, MI, USA).

2.6. Quality Assurance

Deionized water was used throughout the study. Glassware was properly cleaned, and all the reagents were of analytical grade. All extractions were done in triplicates. All measurements were inter-day repeatability measurements taken over a week. In addition, the optimum extraction condition was partially validated for repeatability and reproducibility using relative peak areas.

2.7. Model Fitting and Predictive Efficiency

The model fitting and predictive efficiency between the experimental and predicted data at optimum extraction conditions were investigated by using the absolute average deviation (AAD), root mean square error (RMSE), mean absolute error (MAE), standard error of prediction (SEP), model predictive error (MPE), and chi-square statistic (χ 2 ), and correlation coefficients (R 2 ). The equations used to calculate these factors are described in Table 2 .

Table 2

Equations of error functions.

Error Equation
Absolute average deviation AAD = [ ∑ i = 1 n ( ( | Y i e x p − Y i c a l | ) Y i e x p ) n ]
Root mean square error RMSE = ∑ i = 1 n ( Y i , e − Y i , p ) 2 n
Mean absolute error MAE = i n ∑ i = 1 n | Y i , e − Y i , p |
Standard error of prediction (%) SEP ( % ) = RMSE Y i , e × 100
Model predictive error (%) MPE ( % ) = 100 n ∑ i = 1 n | Y i , e − Y i , p Y i , p |
Chi-square (χ 2 ) χ 2 = ∑ i = 1 n Y i , p − Y i , e Y i , p
Correlation R 2 R 2 = ∑ i = 1 n ( Y i , p − Y i , e ) ∑ i = 1 n ( Y i , p − Y i , e ) 2

AAD: Absolute average deviation, RMSE: Root mean square error, MAE: Mean absolute error, SEP: Standard error of prediction, MPE: Model predictive error, χ 2 : Chi-square statistic, R 2 : Correlation coefficients.

3. Results and Discussion

3.1. Identification of Cannabinoid Compounds

Cannabinoid compounds were identified using GC–TOF/MS. The chromatogram and fragmentation patterns are presented in Figure 2 , Figure 3 and Figure 4 . Five cannabinoid compounds such as THC, CBN, CBD, CBC, and CBG were identified from the plant extracts. Five cannabinoid compounds have been identified in the cannabis extracts. Figure 2 shows a chromatogram obtained for a cannabis extract.

Methods of CBD Extraction, Storage and Tincture Blending

Just like hemp farmers are perfecting methods of crop cultivation, the CBD processing industry has developed various methods of extraction. Throughout the R&D process, safety and efficacy are the key concerns for pros and consumers alike. Below are 4 key factors that can determine the best choice for CBD extraction:

  • Solvent – What will be used to separate the cannabinoids from the biomass
  • Design – The equipment needs to be designed to handle the solvent being used
  • Purpose – The equipment needs to be constructed for its intended use
  • Facility – They must be appropriate for the extracted solvent
  • Consumption – The solvent should be safe for consumption by humans


This may be the oldest form of extraction. CBD can be stripped from the plant by soaking it in ethanol or a high profile alcohol. Not only is this an efficient way of extracting CBD from the hemp plant, but it’s also classified as GRAS (Generally Regarded as Safe) by the FDA. This method is great for creating a full spectrum of hemp extracts and tinctures as it aims to extract all cannabinoids, not just the CBD.


Soaking certain materials in alcohol can lead to a change in their biochemistry. When alcohol is applied and left for a certain amount of time, it draws out the cannabinoids like CBD and other compounds of the plant that are also beneficial. It is important to use a high-proof as it allows minimal damage of the cannabinoids, and suspending them in a liquid that prevents them from damage like oxidation.

The processing for ethanol extraction does require more work and involves the use of several different methods of refinement and filtration. Although if it is done properly, then there is no need for winterization, the process to remove undesirable elements which were also extracted from the plant. For those just starting in the industry, this process is easily scalable and obtainable at a small scale.


Technology continues to advance and the cannabis and hemp industries, in many ways, are driving it. The most advanced and widely used method of extraction is CO2 Extraction. This is a process that uses pressurized carbon dioxide to pull the desired photochemical from a plant. This comes in two forms: subcritical and supercritical. Both yield results based on temperature and pressure.


Subcritical: This method has a lower solvency power which leads to pulling mostly lighter oils and leave behind most resins, paraffin, and waxes. This process is not as common as supercritical CO2 extractions as extraction takes longer and yields are generally lower. Relative to the ‘sub’ name this extraction is cold, this makes it very effective in extracting temperature-sensitive volatile oils and terpenes.

Supercritical: When talking about the most commonly used extraction this one tends to be the one that comes to mind. Through this method it allows you to receive a completely different end-product with more cannabinoids to add further health-promoting properties. With a stronger solvency power, it also makes the extraction time faster, through high temperatures and pressures.

Supercritical CO2 extraction has allowed the extract makers to isolate, capture and scientifically research cannabinoids. With every benefit, however, there is a drawback. Although it lets you quickly pull out compounds, you could potentially extract chlorophyll as well.

CO2 extraction is very selectable and tunable for different molecular weights and leaves no residual solvent. With minimal facility safety requirements and costs, automation is easy and available.


Water, the universal solvent. Some manufacturers use water vapor or even ice to extract cannabinoids from hemp. This is an inexpensive method with a high yield if done correctly. However, this is not simple to practice and can be time-consuming. Most have perfected this so what is left of the plant is a very fine extract.


This process can be simply described in 3 steps:

  1. Finely chopped plant trimmings are mixed with ice or dry ice and agitated to help separate extracts
  2. Water is added and the entire moisture is strained through a mesh bag often strained multiple times
  3. The extract settles as the bottom of the mixture and the excess water is drained from the top

Manufacturers continue to move away from using specific substances like alcohol to extract CBD as the industry continues to agree, the cleaner the process the better the product.


As a hemp testing facility, first and foremost we’re scientists and we know first-hand the various methods to extract cannabinoids all take time and lots of care. All of this would be for nothing if the extracts are not taken care of after processing. Properly stored CBD oil has a shelf life of approximately one year. Something to consider before deciding how to store your oils is researching the carrier liquid your CBD oil is in.

All commercially available CBD oil products have more than just CBD. They contain a carrier oil which can come from a variety of plant-based fats. This does not mean that your CBD oil is less “pure” as some may think, instead it makes the CBD more efficient. For example, olive oil is a more efficient carrier oil for orally ingested CBD because it’s high fatty acid content allows it to travel through the lymphatic system thus, distributing CBD more thoroughly.

So all you need to store your CBD oil is an upright position spot with a stable temperature away from extreme light, heat or moisture. Basically, a dry pantry where you would ironically store your bottle of olive oil. Use glass, not plastic and dark-tinted glass is preferred to reduce light penetration.

Another option that many go to when thinking of storing CBD oils is refrigeration. Many believe this will increase the shelf life of their product. There is only one drawback to this and that is that if the CBD oil gets too cold, it may thicken to the point that you would need to heat it a little before use.

Storing your CBD products is the easy part of the process, which is great news compared to the tedious process that comes with extraction.


Tinctures are cannabis extracts generally made from high-CBD strains of hemp blended with a carrier oil (coconut is common). Tinctures were the main form of cannabis medicine until prohibition was enacted in the 1920s.

CBD tinctures can even be made at home through methods like steeping CBD-rich hemp flowers in high-proof grain alcohol, then cooking the mixture over low heat for several hours.

Typically administered under the tongue, tinctures generally takes effect much faster than edibles because there are blood vessels under the tongue where cannabidiol can enter the bloodstream immediately. The quality is the greatest factor in bioavailability, which is how well your body absorbs and then uses what you consume. The second factor is how you use it.


Primarily used to help relieve anxiety or ease pain, doses can be adjusted by the number of drops taken. These are specifically designed to allow for maximum absorption and use in the body and have a very long shelf life.


ACS Laboratory is a clinical-grade third party CBD and Hemp testing laboratory that is ISO 17025 Certified and CLIA Accredited. Our scientists ensure all products and oils entering the market are clean and safe of any potential contaminants. We take pride in our work at ACS and offer a wide array of tests to ensure the best and safest CBD. After all the chemical components are extracted and treated, the CBD oils are ready for use. For a hemp analysis laboratory such as ourselves, the brand new scientific advances and cutting edge technology are game-changing in the process of creating premier oils. Overall, this industry is changing at a rapid speed and we are witnessing the first of many changes in our own CBD testing facility.