Study investigates the antioxidant activity of hemp leaves

By Vijay Kumar MalesuReviewed by Danielle Ellis, B.Sc.Jul 11 2023

In a recent study published in Antioxidants, researchers explored the antioxidant potential and extraction efficiency of active compounds from hemp leaves using different solvents and mixtures. Identifying cannabis varieties with superior antioxidant properties could produce antioxidant-rich leaves, promoting sustainability and minimizing waste.

Background

Cannabis is a versatile plant with industrial, nutritional, ornamental, medicinal, and recreational applications. The classification of cannabis plants is based on the level of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), a prominent phytocannabinoid. Cannabis contains a diverse group of active compounds, including cannabinoids, terpenes, and flavonoids, which exhibit multidirectional biological activity and can influence each other’s pharmacological profile.

Cannabinoids like cannabidiol (CBD) and tetrahydrocannabinol (THC) have demonstrated potential therapeutic properties, while terpenes and flavonoids offer medicinal benefits and antioxidant activity. Hemp leaves, often considered waste in the industry, have been found to contain high levels of active compounds, making them a valuable resource for extraction. Utilizing hemp leaves can contribute to a zero-waste approach and sustainable development of the industry.

About the study

In the present study, three hemp varieties Bialobrzeskie, Henola, and Tygra were obtained from an experimental station. The plants were cultivated following recommended agriculture practices, including soil preparation, sowing, herbicide application, and fertilization. The extraction of cannabinoids from the hemp leaves was performed using two methods: maceration and ultrasound-mediated extraction. Various solvents and solvent mixtures were used, and the extracts were filtered and concentrated.

Chromatographic analysis was conducted using an ultra-high-performance liquid chromatography technique to determine the cannabinoid profile of the extracts. The antioxidant activity of the extracts was evaluated using four different assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), cupric reducing antioxidant capacity (CUPARC), and ferric reducing antioxidant power (FRAP).

Each assay measured different aspects of the extract’s antioxidant potential. Trolox, a known oxidant compound, was used as a reference for comparison. The obtained data were analyzed using statistical methods such as skewness, kurtosis, ANOVA, and post hoc test. Principal component analysis (PCA) was employed to explore the relationship between the cannabinoid profile and antioxidant activity of the extracts. Multidimensional comparative analysis (MCA) was also performed to assess and rank the extracts based on their overall antioxidant activity.

Study results

The researchers investigated the content of four cannabinoids (CBD, Δ⁹-THC, CBG, and CBC) and the antioxidant activity of cannabis leaf extracts. The results showed that the content of cannabinoids varied among different hemp varieties and extraction methods.

The highest CBD content was found in Henola leaves extract attained by maceration with methanol, whereas the maximum CBG content was observed in Tygra leaves extract, which was obtained by maceration with ethanol and methanol. Bialobrzeskie leaves extract prepared by ultrasound-mediated extraction with methanol exhibited the highest Δ⁹-THC content and Tygra leaves extract prepared by ultrasound-mediated extraction with isopropanol recorded the highest CBC content.

Further, the antioxidant activity was evaluated using four different assays: DPPH, ABTS, CUPRAC, and FRAP. The results indicated that the extracts obtained through different extraction methods and solvents showed varying antioxidant potential. In the set of extracts obtained by ultrasound-mediated extraction with single-component extractants, Bialobrzeskie leaves extract obtained with methanol exhibited the highest antioxidant activity in DPPH, FRAP, and CUPRAC assays.

Tygra leaves extract obtained with methanol showed the highest antioxidant potential in the ABTS assays. Among macerated extracts with different solvents, methanol Bialobrzeskie leaves extract demonstrated the highest antioxidant activity in the DPPH, ABTS, and FRAP assays. In the CUPRAC assay, high potential antioxidant was observed in Tygra leaves extract macerated with isopropanol.

In the set of extracts prepared by ultrasound-mediated extraction with two-component extractants, Bialobrzeskie leaves extract prepared with ethanol and methanol (50:50, V/V) showed the highest antioxidant potential in the DPPH, CUPRAC, and FRAP assays, while Tygra leaves extract prepared with ethanol and methanol (50:50, V/V) exhibited the highest antioxidant activity in the ABTS assay.

Researchers also performed MCA to compare all the extracts and determine the strongest antioxidant extract. The results of MCA identified the Bialobrzeskie leaves extract obtained by ultrasound extraction with methanol as the most potent antioxidant. PCA was conducted to explore the relationship between cannabinoid content and antioxidant activity.

The analysis revealed that the variation in the samples was primarily explained by the first principal component (PC1), which negatively correlated with antioxidant results. PC2 negatively correlated with antioxidant activity and positively correlated with cannabinoid content.

The study highlights the potential interplay between cannabinoid content and antioxidant activity in cannabis leaf extracts. While Δ⁹-THC and CBD showed a higher correlation with antioxidant activity, CBC and CBG exhibited a relatively weaker correlation. It should be noted that cannabinoids interact in the human body, and their cooperative relationships enhance the therapeutic potential and efficacy of cannabis-based treatments.

Conclusions

To summarize, the study highlights the antioxidant potential of cannabis leaves, specifically Bialobrzeskie, Tygra, and Henola varieties, using different extraction methods. The extracts demonstrated significant antioxidant properties, offering protection against oxidative damage induced by free radicals. Among the extracts, Bialobrzeskie leaves extract obtained through ultrasound-assisted extraction with methanol exhibited the strongest antioxidant activity.

Further research is necessary to explore additional biological activities of cannabis leaves and investigate in vivo applications. The study suggests the need to explore alternative extraction techniques, diverse applications, and delivery methods to harness the therapeutic potential of cannabis leaves fully.