Instant controlled pressure drop technology in cardamom essential oil extraction and antioxidant activity

Green cardamom (Elettaria cardamomum) is an outspread spice native to Asia, which is well appreciated for its sensory characteristics, delicate aroma, and unique taste. The technologies such as the instant controlled pressure drop (DIC) have been applied to reach higher yields and better quality of EO. Then, this study explores the impact of DIC as a pretreatment before hydrodistillation (HD) on the EO yield and their antioxidant activity. Obtained results showed that the coupling of DIC-HD increased the yield of essential oil and also had a positive impact on their antioxidant capacity. The EO yield of DIC-HD (140 °C and 30 s) was 4.43% vs. 2.52% for control; the AOX of DIC-HD (165 °C and 30 s) was 86% inhibition vs. 57.02% for control, and the TEAC of DIC-HD (140 °C and 30 s) was 1.44 uMTE/g EO vs. 13.66 uMTE/g EO.

Materials and Methods

Cardamom seeds were obtained from finca Argovia in Tapachula Chiapas, México. All solvents used were HPLC grade.

Moisture Content

The moisture content of the seeds was determined by a dynamic method using a laboratory air dryer (Binder FD 23). Two grams of seed were placed in the dryer at a temperature of 105 °C for 24 h.

DIC Treatment

The DIC treatment of Cardamom seeds was carried out in four steps.  Firstly, 100 g of seeds were placed into the DIC reactor, in which a vacuum of 30 mbar was established. Secondly, saturated steam was injected into the reactor until the selected study saturated steam temperature was reached (being from 0.17 up to 0.7 MPa), and this was maintained for a short time (being from 15 to 45 s). Thirdly, samples were subjected to an instant controlled pressure drop (ΔP/Δt > 0.5 MPa.s⁻¹) towards vacuum (30 mbar); in fact, this pressure drop causes the autovaporization of the water and the swelling of the matrix. Finally, the pressure was released toward the atmospheric pressure, and cardamon seeds were recovered. In this study, the used DIC equipment was a LAB DIC 0.1 model. The impact of DIC treatment on morphology, essential oil yield, and radical scavenging activity by DPPH and TEAC was evaluated using a surface response design. After DIC treatment, cardamom seeds were stored at −80 °C until further analysis.

Essential Oil Extraction via Hydrodistillation

For this study, 50 g of grounded cardamom were mixed with 600 mL of distilled water in a Clevenger-type apparatus and distilled for six hours. The essential oil was dried over anhydrous sodium sulfate and stored until analysis. The yield was calculated as grams of oil per 100 g of seed (dry basis).

Radical Scavenging Activity by DPPH

The DPPH analysis is based on the radical unpaired electron yield to an antioxidant substance; DPPH is demoted from blue-purple color to light yellow. For this assay, a stock solution of DPPH 125 μM was prepared. For the assay, 20 μL of samples and 200 μL of DPPH were added to a well in a 96-microwell plate and mixed; analysis was carried out by triplicate. The plaque was stored in the dark for 90 min at room temperature. The concentration of essential oil in the sample was 0.19 g/mL. Absorbance readings were taken at 520 nm using an xMark™ Microplate Absorbance Spectrophotometer. Scavenging was expressed as percent DPPH discoloration.

Trolox Equivalent Antioxidant Capacity (TEAC)

Trolox equivalent antioxidant capacity is based on the comparison between the antioxidant capacity to cleave the radical cation of ABTS and Trolox. ABTS stock solution by reacting 7 mMol/L and 2.45 mMol/L of potassium persulfate after incubation in the dark for 16 h. The stock solution was then diluted in ethanol to an absorbance of 0.8 ± 0.1 at 734 nm. Trolox standard solutions were prepared in methanol from 0 to 700 μmol/L and assayed under the same conditions. In each well of a 96-microplate, 200 μL of reagent and 20 μL of the sample were added and incubated for 6 min with constant agitation. Each sample was assessed in triplicate. The concentration of essential oil in the sample was 0.62 mg/mL. The readings were performed at 734 nm using xMark™ Microplate Absorbance Spectrophotometer. Trolox standard solutions were prepared in methanol from 0 to 700 μmol/L and assayed under the same conditions. The Trolox equivalent antioxidant capacity (TEAC) was calculated based on the Trolox calibration curve and reported as the μM/L needed for Trolox to decolorate at the corresponding concentration.

Results

Stereo microscopy was used to monitor the morphological changes in the seed.  These changes could be attributed to both main stages of DIC treatment: the steam condensation occurred during the saturated steam injection into the reactor (which increased the initial moisture content of the seed) and also to the autovaporization of seed water (which could trigger a cardamon seed expansion).  The control seeds performed average moisture values of 5.1% d.b, the DIC-treated seeds performed moisture contents between 10.5 to 11.3% d.b. Then, this study suggests that the DIC treatment allowed a rapid increase in moisture content, which subsequently engendered a cardamon seed expansion due to autovaporization, which positively impacts both the EO yield and the antioxidant scavenging capacity.

Essential Oil Yield

HD and DIC-HD extracted oils presented a clear appearance and a pleasant aroma. The highest extraction percentages were 4.43%, 4.40%, and 4.39% from DIC 12 (140 °C, 30 s, 0.36 MPa), DIC 11 (140 °C, 15 s, 0.36 MPa), and DIC 8 (122 °C, 41 s, 0.21 MPA). In contrast, the lowest extraction yields were provided by the control, DIC 1 (165 °C, 30 s, 0.7 MPa) and DIC 2 (140 °C, 45 s, 0.36 MPa), with yields of 2.52%, 2.54%, 2.56%, respectively. The oil yield extraction could be enhanced under a temperature range between 110 and 140 °C and a treatment time between 10 to 20 s.

Cardamom essential oil has been used as an important part of folk medicine, rituals, cosmetics, and perfumes. Due to its great importance and the economic significance of cardamom, any increase in EO yield is welcomed. The highest yield was obtained in the sample DIC 12 (140 °C, 30 s, 0.36 MPa) with a 4.44%. Studies provide a wide range of values among the same matrices. By comparing EO yields from only HD to DIC-HD, the percentage of EO extracted from treated samples was overall higher than those of the untreated. It is worth noting that the EO yield is a multifactorial response and can be influenced by multiple factors such as plant variety, growing conditions, harvesting conditions, post-harvest processing (i.e., drying method), geographic area, etc. For cardamon seeds, the increase in the essential oil yield can also be attributed to the increase in the porosity in the epidermis.

DPPH Free Radical Scavenging Capacity

The strongest antioxidant activity was obtained under DIC 3 (140 °C, 30 s, 0.36 MPa) with a 68%. The lower AOX was obtained under DIC 1 (165 °C, 30 s, 0.7 MPa), DIC 8 (122°C, 41 s, 0.21 MPa), and DIC 12 (140 °C, 30 s, 0.36 MPa) treatments with 62.28%, 65.38% and 65.38% respectively. By comparing the AOX of essential oil extracted from treated seeds to control, it can be shown an increase in AOX; while DIC 3 (140 °C, 30 s, 0.36 MPa) performed an AOX of 68%, the AOX of control performed 57%. DIC preserves the quality of secondary metabolites thanks to the water’s autovaporization that guarantees rapid cooling, which prevents the thermal degradation of sensitive compounds.

ABTS Trolox Equivalent Antioxidant Capacity Determination (TEAC)

Contrary to yield and AOX, in which a maximum value is desirable, the least TEAC means a better antioxidant capacity in this study. The better performers for TEAC were found by DIC 6 (130 °C, 30 s, 0.36 MPa) with 1.99 uMTE/g EO and by DIC 3 (140 °C, 30 s, 0.36 MPa) with 2.04 uMTE/g EO. On the other hand, the untreated seed (control) gave 13.66 uMTE/g EO, indicating low antioxidant capacity. In this study, the best TEAC was obtained at lower time and temperature values; in contrast with poblano pepper, they reached the highest TEAC values of both steam pressure and holding time. An interesting approach would be to test the effect of more variables such as the initial moisture content, the treatment time, the temperature, and the number of DIC cycles, to have a complete picture of their interactions and how they affect the response variables.

Conclusions

Traditionally essential oil extraction of cardamon seeds has been achieved via hydrodistillation (HD); this study has evaluated the effect of coupling DIC technology to HD on EO’s extraction efficiency and biological activities. In this respect, results showed a clear improvement in cardamon yield and antioxidant activity by coupling DIC technology to hydrodistillation. DIC-HD (140 °C, 30 s, 0.36 MPa) does show an increase in the EO yield of 1.7 times (4.44% from DIC vs. 2.52% for only HD). In addition to this, DIC-HD essential oils improved antioxidant capacities with HD. Regarding AOX by DPPH, under the selected studied parameters of saturated steam temperature and thermal processing time, all applied DIC treatments allowed for increased the AOX of extracted essential oils. The highest AOX was found under DIC 3 (140 °C, 30 s, 0.36 MPa), being 68%, vs. the control with 57%. Concerning TEAC, the results showed that DIC pretreatment improved the antioxidant activity, being found the best value of TEAC under DIC 6 (130 °C, 30 s, 0.36 MPa) with 1.988 uMTE/g EO; control performed a TEAC value of 13.66 uMTE/g EO, indicating a lower antioxidant capacity. Regarding the morphological changes in cardamon seeds, it could be suggested that the increase in the essential oil yield and antioxidant activity can be attributed to the increase in the porosity of the matrix; however, more scanning electron microscopy studies are needed to explain the microstructural changes of the seed attributed to the DIC treatment.

Reference:

Teresa-Martínez, G.D., Cardador-Martínez, A., Téllez-Pérez, C., Allaf, K., Jiménez-Martínez, C. and Alonzo-Macías, M., 2022. Effect of the Instant Controlled Pressure Drop Technology in Cardamom (Elettaria cardamomum) Essential Oil Extraction and Antioxidant Activity. Molecules, 27(11), p.3433.