Optimization of Extraction Process of Cannabidiol from Hemp Leaves by Response Surface Methodology and Content Distribution in Different Parts
Cannabis sativa L., also known as Chinese hemp, thread hemp, etc., is an annual herbaceous plant of the Cannabis genus in the Moraceae family. The main active ingredient in hemp is cannabinoid. Currently, more than 110 natural cannabinoids have been isolated from plants of the cannabis genus, among which tetrahydrocannabinol (THC) and cannabidiol (CBD) (see Figure 1 for structure) are the two main cannabinoid active ingredients in hemp. Research shows that CBD has antioxidant activity, sedation, antidepressant, anti-cancer, obesity treatment, anti-inflammatory properties, vasodilation and other potential effects, and has no hallucinogenic and addictive effects. It has become a new drug for clinical treatment of breast cancer and epilepsy. Currently, in many countries and regions around the world, cannabidiol has been approved for medicinal and food use.
Natural CBD has a left-handed structure, while chemically synthesized CBD is usually a 1:1 mixture of two isomers, left-handed and right-handed. Due to their very similar properties, separation is very difficult. The chemical synthesis steps are cumbersome and cannot effectively produce bioactive compounds with specific structures, and the cost is high. Therefore, extracting cannabidiol from natural plants is still the most economical source.
At present, the extraction of cannabidiol from hemp is mainly based on leaching and hot reflux, and there are still problems such as long time consumption and low efficiency in the detection method of CBD; Moreover, the challenges of low extraction efficiency, poor stability, and low content urgently need to be overcome. The ultrasound assisted extraction method used in this article has better repeatability and is simpler and easier to operate than the Soxhlet extraction method. With the continuous expansion of the legalization scale of hemp cultivation, research on CBD has also deepened, and people’s understanding of the effectiveness and safety of CBD has greatly increased. CBD will also better benefit humanity.
This article takes hemp leaves as the research object and establishes a simple, rapid, and accurate HPLC detection method. This method has a fast peak time for cannabidiol, good effect, and simple flow matching ratio; Using ultrasound assisted extraction method to obtain natural cannabidiol, exploring the influence of different factors on the extraction content, and using response surface methodology to optimize the conditions for ultrasound assisted extraction of CBD, the yield of cannabidiol was improved, which has high application value. And the content of CBD in various parts of Daqing hemp was studied, providing theoretical basis for the extraction of CBD from hemp and the comprehensive utilization of hemp, and promoting the development of hemp industry.

This study used hemp leaves as experimental materials and established a simple and rapid HPLC detection method for cannabidiol in hemp leaves using Agilent HC-C18 column. The method has high sensitivity, is simple and easy to implement, and the results are accurate and reliable. Compared with the liquid-phase method of cannabidiol established by Chen et al., which peaks after 9 minutes, this liquid-phase method has a faster peak time of 7-8 minutes, excellent separation effect, and ideal peak shape; Compared with Xie et al., the average recovery rate of the liquid-phase method established in this study was 97.49% (RSD of 0.45%), with a recovery rate of 91.2% to 97.6% (RSD of 3.0%). This method has good stability and repeatability, and can efficiently detect the content of cannabidiol in hemp. It is suitable for quantitative analysis of cannabidiol and provides a reliable guarantee for evaluating the quality of hemp.
On the basis of single factor analysis, the optimal process conditions for ultrasound assisted extraction of cannabidiol from hemp leaves were obtained using Box Behnken Design response surface methodology: methanol was used as the extraction solvent, the solid-liquid ratio was 1:22 (g/mL), the extraction power was 320W, the extraction temperature was 81 ℃, and the extraction time was 26min. Under these conditions, the cannabidiol content was 79.53 ± 0.26mg/g. Compared with Yan et al.’s research, further specific studies were conducted on the conditions of ultrasound extraction, including extraction power, extraction material to liquid ratio, and extraction temperature, to clarify and supplement the conditions for ultrasound assisted extraction of cannabidiol from hemp. Compared with Hao et al.’s study of extracting cannabidiol using hot reflux technology for 3 times every 2 hours, the method used in this study requires less time and consumes less solvent to obtain the same amount of cannabidiol; Compared with Gao et al.’s research, the extraction time is shorter, and under the optimal process conditions, the amount of cannabidiol extracted is also higher. The optimal extraction process conditions obtained in this experiment have to some extent shortened the extraction time, avoided the impact of long-term extraction on the active ingredients, and improved the extraction amount and efficiency of cannabidiol.
The high-performance liquid chromatography method established by our research institute was used to determine the content of cannabidiol in various parts of cultivated hemp varieties in Daqing, Heilongjiang. The content of cannabidiol was highest in tender leaves, followed by whole leaves, old leaves, seeds, and stems, and lowest in roots. The research results can provide data for the extraction of cannabidiol from hemp and the further development and utilization of high value-added hemp products.