Exploitation of Cannabis sativa L. Roots Grown under Aeroponics Cultivation -

Exploitation of Cannabis sativa L. Roots Grown under Aeroponics Cultivation

Cannabis sativa L. has been used for a long time to obtain food, fiber, and as a medicinal and psychoactive plant. Today, the nutraceutical potential of C. sativa is being increasingly reappraised; In this study quantified the presence of valuable bioactives (namely, β-sitosterol, stigmasterol, campesterol, friedelin, and epi-friedelanol) in the root extracts of C. sativa, a finding which might pave the way to the exploitation of the therapeutic potential of all parts of the C. sativa plant. To facilitate root harvesting and processing, aeroponic (AP) and aeroponic-elicited cultures (AEP) were established and compared to soil-cultivated plants (SP). Interestingly, considerably increased plant growth—particularly of the roots—and a significant increase (up to 20-fold in the case of β-sitosterol) in the total content of the aforementioned roots’ bioactive molecules were observed in AP and AEP.

Material and Method

C. sativa seedlings of uniform size, the first two true leaves being equal to about 3.0 cm, were selected. The base of the stem of each seedling was fixed with a sponge, placed in a reticulated pot for aeroponics (with a diameter of 6.0 cm and a height of 6.0 cm), and this was placed on the hole of the lid of a tub for aeroponic cultivation in black PVC (50.0 cm × 50.0 cm × 34.0 cm in height), capable of hosting five plants each time, at a distance of 15 cm from each other. Complete Hoagland’s nutrient solution was sprayed at the roots of the plants for duration of 15 min every hour, and recovered via a closed circuit. The electrical conductivity (EC) and pH of the nutrient solution were controlled at 0.6–0.7 ms/cm and 6.0, respectively. The aeroponics culture system was maintained in a climatic cell with a photoperiod as stated above with high pressure sodium lamps (Sonlight AGRO 250W grown + bloom). The photosynthetic photon flux density (PPFD) at the plant canopy was about 150 µmol/m2/s. Temperatures during the periods of light and dark were maintained at 27 ± 1 ◦C and 22 ± 1 ◦C, respectively. The relative humidity was 65 ± 5% and the mean CO₂ concentration was 670 ± 30 µmol/mol. For the traditional cultivation in pots, the seedlings with two real leaves (about 3 cm long) were placed in a plastic pot (with a diameter of 30.0 cm and a height of 30.0 cm), containing a mixture as stated above (50% vermiculite and 50% peat), three plants per pot, spaced 15 cm from each other, and watered periodically with whole Hoagland’s nutrient solution. The pots were placed in a climatic cell with the conditions described for aeroponics and at the same time as this. Three culture systems of C. sativa Kompolti were set up: SP (soil plant), carried out only for the vegetative phase; AP (aeroponic plant), carried out only for the vegetative phase; AEP (aeroponic-elicited plant), carried out only for the vegetative phase supplemented with the elicitor technique with salicylic acid. AEPs were obtained by adding salicylic (25 µM final concentration) acid to the nutrient solution of one-week old plants.

Biomass Production

Five plants from each culture system were harvested three times after 8 weeks of culture. It should be noted that the plants were kept in the vegetative phase by maintaining the photoperiod constant (18 h). Under these conditions, plants could not flower because flowering requires a gradual reduction of the photoperiod (from 18 to 12 h). The root systems were carefully washed with tap water, dried with absorbent paper to remove excess water, and their fresh weights were immediately recorded. The other parameters taken into consideration were as follows: dry weight of the roots (g); fresh and dry weight of the aerial parts (g); height of the aerial parts (cm); diameter of the stems (mm); and surface of the collected leaves at half height (cm).

Extraction of Dry C. Sativa Roots

Powdered C. sativa roots (300.0 mg) and 100 µL of the IS solution (cholesterol, 1.128 mg/mL in ethyl acetate) was extracted in ethyl acetate (40 mL) under magnetic stirring for 1.5 h at room temperature, followed by centrifugation at 5000 rpm for 8 min. The supernatant was collected in a flask and the residue was extracted once again in the same manner. The collected organic phases were washed with water (2 × 10 mL) and brine (10 mL) then dried (Na2SO4 anhydrous), filtered, and evaporated to dryness in vacuo at 30 ◦C. The residue was dissolved in 10 mL of ethyl acetate and kept at 4 ◦C until GC-MS and GC-FID analyses.

Gas Chromatography (GC-MS, GC-FID)

GC-MS analyses were carried out using a Trace GC Ultra gas chromatograph coupled to an ion-trap mass spectrometer (ITMS) detector Polaris Q (Thermo Fisher Scientific, Italy) and equipped with a split–splitless injector. Quantification of the analytes in the dry C. sativa roots was performed using the internal standard method based on the relative peak area of analyte to IS (cholesterol) from the average of three replicate measurements. When standards were unavailable, the quantification of the target analyte was carried out using the relative response factor of the available standards of similar chemical structure. The retention indices (RIs) were calculated by comparing the retention time of each compound with those of a homologous series of n–alkanes standard (C7–C40) under the same chromatographic conditions.

Results

Biomass Production:

The growth of C. sativa var. Kompolti plants used in this study was significantly influenced by the two systems tested, i.e., conventional vs. aeroponic, with aeroponics promoting a significantly more rapid and intense growth of both the aerial parts and root systems. On average, after 8 weeks of parallel cultivation, the roots of APs showed a 64-fold and 13-fold higher fresh (FW) and dry weight (DW) as compared to SP, respectively; the aerial parts showed a 39-fold and 44-fold higher FW and DW; the stems’ average diameter and the mean leaves area increased by 3.89-fold and 8.9-fold, respectively. AP and AEP reached almost double the height (ca. 70 cm) as compared to SP (ca. 30 cm). Finally, the addition of the elicitor salicylic acid to the nutrient spray did not result in any significant variation in these parameters in AEP as compared to AP

Extract Characterization:

The content of the main roots’ bioactive constituents was comparatively determined in SP, AP, and AEP plants by GC-MS. The main compounds identified were the phytosterols campesterol, stigmasterol, and β-sitosterol, and the triterpenes epi-friedelanol and friedelin. On a per DW basis the amount of bioactives was higher in SP as compared to both AP and AEP. Regarding the single constituents, the amount of epi-friedelanol and friedelin was far higher in SP, that of campesterol and stigmasterol was similar in the three types of cultures, while β-sitosterol was higher in AP and AEP. On a percent basis friedelin and epi-friedelanol were the most expressed compounds in SP, while β-sitosterol was the most expressed in AP and AEP; finally, the amount of β-sitosterol decreased and the amount of epi-friedelanol increased in AEP as compared to AP.

The biomasses of the plants grown in aeroponics were heavier (13-fold to 64-fold for DW and FW roots, respectively), both AP and AEP contained significantly higher amounts of root bioactives. The amounts of β-sitosterol from AP (10.86 ± 0.72 mg) and AEP (9.89 ± 2.17 mg) roots were 23 and 20 times higher than in SP (0.49 ± 0.05mg), respectively; friedelin, whose concentration on a per weight basis was significantly higher in SP, exhibited higher per plant values in AEP and AP (4.55 ± 0.47 mg; 5.67 ± 0.4 mg, respectively) than SP (2.37 ± 0.3 mg). Similar proportions were observed for campesterol, stigmasterol, and epi-friedelanol.

Conclusions:

In summary, the main constituents of C. sativa roots, namely, β-sitosterol, friedelin, and epi-friedelanol, possess converging or complementary biological activities in such a way that their co-presence in C. sativa root extracts may result in additive or even synergistic effects, which could be used as adjunctive treatment in several pathological and physiopathological conditions. Hence, the aforementioned considerations make C. sativa roots obtained through aeroponic cultivation a valuable material. Aeroponics, an easy, standardized, contaminant-free cultivation technique, facilitates the harvesting/processing of roots along with a greater production of their secondary bioactive metabolites, which could be utilized in the formulation of health-promoting and health-care products.

Reference:

Ferrini, F., Fraternale, D., Donati Zeppa, S., Verardo, G., Gorassini, A., Carrabs, V., Albertini, M.C. and Sestili, P., 2021. Yield, characterization, and possible exploitation of Cannabis sativa L. roots grown under aeroponics cultivation. Molecules, 26(16), p.4889.