Optimization of a novel Renealmia ligulata (Zingiberaceae) essential oil extraction method through microwave-assisted hydrodistillation

Renealmia is a tropical plant genus within the Zingiberaceae family. In tropical South America, Renealmia plants are known for their therapeutic uses against bone and muscle pain, colds, and to counteract snake bite symptoms. Despite the biomedical importance of Renealmia metabolites, the components of their essential oils (EO) have been scarcely studied, and a cause thereof is the lack of local efficient, inexpensive, and environmentally friendly EO extraction methods. This work addressed the optimization of an EO extraction method from the aerial parts and rhizomes of Renealmia ligulata plants based on microwave-assisted hydrodistillation (MAHD) with an ultrasound-assisted extraction (UAE) pretreatment. Three MAHD extraction variables (radiation power, radiation exposure length, and solvent volume) were studied on their own and in combination using a response surface analysis to determine the value combinations leading to optimal EO yields. The results showed that the best average extraction duration time was 42.5 min, combined with a radiation power of 765 W and a solvent volume 225.9 mL for 30 g of aerial part plant material or 799 W and 145 mL of solvent for 20 g of plant rhizomes. A GC-MS analysis of the obtained R. ligulata EOs revealed that their main component was epi-Eudesmol (28% in plant aerial parts and 13% in rhizomes), which is a molecule of interest considering its reported neuro-protective properties.


Introduction
The Zingiberaceae family, distributed throughout regions of Africa and South America, comprises nearly 85 species and entails 23 genera native species of Africa and 62 of the American tropics [1].Within this family, several species of the Renealmia genus, native to the Amazon, are frequently employed as medicines by indigenous peoples.For instance, the Kichwa in Ecuador, use these plants to treat colds, as analgesics for bone and muscle pain, and to counteract snake bite symptoms [2].
The species Renealmia ligulata (Maas) occurs in the Colombian Andes at altitudes between 1,200 and 2,000 meters above sea level, bearing the vernacular names of Murrapa or Matandrea.In the Colombian department of Quindío, in the Andean middle range, this plant species has been recorded in the municipalities of Armenia, Buenavista, Calarcá, Circasia, Córdoba, Filandia, Génova, Pijao, and Salento, at altitudes between 1,450 and 2,050 m.a.s.l., and it is frequently associated with roadsides, water bodies, and inside forests at the edge of trails on slopes, forming conspicuous aggregates.
EOs are a complex mixture of secondary metabolites, entailing monoterpenes, sesquiterpenes, phenylpropanoids, and some compounds of non-terpenoid nature.Given this ample EO array in medicinal and aromatic plants, ethnic and traditional wisdom has stirred the study of their biological properties as well as their antioxidant, anti-inflammatory, antimicrobial, and anticancer activities [4].
Extraction is one of the fundamental steps in plant EO research and use.The efficiency of different conventional EO extraction techniques, like extraction with solvents, distillation, and conventional hydrodistillation (HD), is usually affected by extraction-inherent features.For instance, lengthy sample exposure to solvents or thermal treatment in HD yields, likely results in plant metabolite degradation [5,6].
Novel approaches have been applied in EO extraction from plants, such as microwave-assisted hydrodistillation (MAHD), which is a relatively inexpensive and environmentally friendly method.In addition, MAHD provides several advantages for EO extraction because it can reduce heating times, lowers energy consumption, and overcomes the inconveniences already mentioned [7,8,9,10].
Likewise, seeking to improve yields and to shorten extraction times, various researchers have applied ultrasound-assisted extraction (UAE) to EO.This is a modern extraction technology that uses the cavitation, mechanical vibration, and thermal effects produced by ultrasound to destroy plant cell walls, thus enhancing solvent diffusion and accelerating the dissolution of target compounds.This method has the advantages of simple instrumentation, easy operation, and high efficiency [11,12,13,14].
An important part in EO extraction though MAHD is the optimization of extraction variables.These include extraction time, microwave power, and the plant material-solvent ratio.The response surface methodology (RSM) is a valuable statistical and mathematical tool widely used to predict the optimal experimental parameters and construct a mathematical model to analyze the effects of interactions among variables [14].This work focused on (i) optimizing the extraction process of EO from Renealmia ligulata aerial parts and rhizomes, using RSM in the MAHD context, (ii) assessing the effect of UAE as an EO extraction pretreatment about yield percentages when applying the MAHD extraction technique, and (iii) studying the chemical composition, through gas chromatography mass spectrometry (GC-MS), of the extracted EOs.

Plant material collection and identification
Plant material, consisting of plant aerial parts (i.e., flowers, leaves, and stems) and rhizomes, were collected in the pristine forest of La Concha (4°38' 12" N y 75°39' 06" W), within the Circasia municipality (western slope of the central mountain range of Colombia) in the Colombian department of Quindio.For taxonomic identification, a complete specimen was taken to the herbarium of the University of Quindio (HUQ) to be analyzed by a botanical expert in this plant group.The material was entered to the herbarium collection with accession number 029953, thus confirming its taxonomic identity as R. ligulata Mass material (a taxonomic description of the plant species in question is provided in supplementary information).

Material drying and grinding
R. ligulata aerial parts and rhizomes were subjected to drying in a recirculating air oven at temperatures between 40 and 45 °C for eight days; thereafter, the plant material was pulverized by using a fodder grinding mill TRF 70.

Microwave-assisted Hydrodistillation (MAHD) extraction
A household microwave oven (General Electric, JES11G, with a maximum power of 1,000 W; capable of 100-W increments and with a frequency of 2450 MHz) was modified for the procedure.The microwave oven was adapted for extraction by drilling an opening and installing a Clevengertype condenser on top.Then, a flat-bottom flask containing the pulverized plant material was placed inside the appliance and the MAHD was carried out [15].The EOs obtained were stored at 4 °C until analysis.The EO yield was calculated following Equation 1.

%𝑅 = (𝑚𝑎𝑠𝑠(𝑔)𝑜𝑓 𝐸𝑂) (𝑚𝑎𝑠𝑠(𝑔)𝑝𝑙𝑎𝑛𝑡𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙)
* 100 The adapted MAHD system was specific for the chosen microwave oven brand.The modifications made to the appliance may not apply when adapting another of a different brand.Thus appliance technical specification must be taken into account when constructing a similar experimental setup.This is due to the lack of empirical evidence supporting a linear correlation between their power and efficiency concerning energy conversion rate.

MAHD extraction optimization via RSM with a Box-Behnken experimental design
A Box-Behnken design (BBD) was implemented with three factors: length of microwave radiation, microwave power, and amount of solvent, on three levels each [low (-1), medium (0), and high (+1)] to investigate simultaneously the effects of the independent factors on EO yields in the context of MAHD extraction.These factors were coded, as shown in Table 1.The variables were taken from different works conducted with some modifications [14,16,17,18].This design evaluated the EO percentage of yield as the response variable.The study predicted 12 runs, together with three random replicates at the central point (CP) applied to test the experimental error.

MAHD with ultrasound pretreatment (MAHD+US)
Ultrasound pretreatment was applied on R. ligulata aerial parts (30 g) and rhizomes (20 g).Each plant material type was subjected to an ultrasound bath for 30 minutes in a Bransonic equipment series M1800-H, set to a frequency of 40-KHz and 145-W power [19].

GC-MS Analysis
The obtained EO were analyzed via GC-EM in a SHIDMAZU-QP2010 apparatus employing helium as carrier gas with a flow of 1 mL/min.All samples were prepared by diluting in 1:20 v/v ratio with dichloromethane and 1 l of each sample was injected in Split mode.A Zebron ZB-5MS 5 % Polysiralene -95 % Polydimethylsiloxane column was used (30 m × 0.25 mm ID × 0.25 m).
The temperatures of the column, injector, and transfer line were adjusted to 40, 300, and 300 °C, respectively.The heating ramp was programmed in the following manner: initially, the column temperature was set at 40 °C constant for 2 min, then increased to 260°C with 5°C/min steps; and finally rose up to 310 °C with increasing steps of 20 °C/min; remaining unaltered for 3 min.Mass spectrometry was conducted with an electron impact ionization system at 70 eV, in Scan mode, with an analysis range from 40 m/z to 450 m/z.The EO components were identified comparing the obtained retention times and mass spectra to the equipment's 2013 NIST library and through comparison of Kovats indices, which were determined based on the retention times of n-alkanes C 8 -C 25 , according to Adams [20].

EO yields from MAHD-assisted extraction
The Box-Behnken design to assess three variables affecting EO extraction efficiency (i.e., percentage of EO yield) with the MAHD method, resulted in the data shown in Table 2 and Table 3, which reveal R. ligulata aerial parts and rhizomes, respectively, as EO sources.
The EOs from R. ligulata revealed a better yield from plant aerial parts compared to rhizomes.The results obtained in the experimental design were subjected to analysis of variance (ANOVA) in the Stat graphics program, as shown in Table 4 and Table 5.    4 and 5 show the effect of the sources of variance on EO yield, when extracted from R. lingulata aerial parts and rhizomes; testing the statistical significance of each extraction variable effect by comparing its mean square against an estimate of the experimental error.In addition, the R 2 statistic indicates that the model adjusted explains 95.6 % and 99.4 % of the variability in EO yield from plant aerial parts and rhizomes, respectively.Likewise, the p value for the lack of fit in the ANOVA table is > 0.05, suggesting that the model is adequate for the data observed within the 95% confidence interval (CI).
As shown in Fig. 1, four effects were significant (p < 0.05) on EO exaction yields from plant aerial parts, whereas eight effects were significant on EO exaction yields from rhizomes.These effects were significantly different from zero, within the 95 % CI, as shown in the Pareto diagram in Figs 1A and 1B.

MAHD extraction variable effects as revealed by surface response graphics
To assess the interactions between the extraction parameters and the response variable (i.e., EO extraction yield), multiple response surface (RS) graphics were constructed and analyzed Fig. 2.
Figures 2A and 2B show the effect of microwave radiation, in terms of radiation power and the length of radiation exposure, on EO yield, from plant aerial parts and rhizomes, respectively.A microwave exposure time above 35 with a radiation power between 720 and 800 W resulted in the best best yields.These outcomes may be due to the microwave effect, promoting rotation of the molecular dipoles and, thus, internal heating within a short time, resulting in a high-pressure gradient within the plant material, releasing EO [19].
Regarding the combination of the length of radiation exposure and solvent amount, the RS graph ics (Figs.2C and 2D) revealed that an exposure time above 35 min with solvent volumes between 180 and 240 mL for the aerial parts and between 120 and 140 mL for rhizomes resulted in best EO yields.The highest solvent volumes slowed down the process and led to suboptimal EO yields.Hence, at a longer extraction time with the least and upper solvent amounts can shorter extraction time but may lead to incomplete release and possibly degradation of metabolites [21].Lastly, addressing the interaction between microwave radiation power and solvent volume for plant aerial parts and rhizomes, figures 2E and 2F respectively revealed that high radiation power affects positively EO yield, and that increasing solvent amount counters EO extraction yields, likely because the upper microwave power tests was not sufficient to promote and accelerate the effect of the mass transfer [19].

Optimal EO extraction parameters
EO extraction parameter data were analyzed via statistical prediction thus narrowing the optimal conditions that maximize its yield, depending on the plant parts from which the EO were obtained, as shown in Table 6.Bearing in mind the optimal parameters obtained, triplicates of optimized experiments were performed resulting in a yield improvement of 0.2186 % ± 0.0160 % for extractions from R. ligulata aerial parts and 0.071 % ± 0.023 % for extractions from plant rhizomes.These values were close to those predicted by the model.Besides, we demonstrated that R. ligulata aerial parts had an EO content three times higher than that of its rhizomes.

Ultrasound pretreatment efficiency comparison
To evaluate ultrasound treatment effects on plant material before conducting EO extractions with the MAHD method, experiments were conducted and compared with those from previously optimized EO extractions.Results are shown in Fig. 4.
Ultrasound pretreatment improved yield by 12.8 % in R. ligulata aerial parts and by 33.3 % in R. ligulata rhizomes.Such contrast is likely due to the mechanical effect of ultrasound, accelerating the release of the EO through a physical process called cavitation.The effects of caviation can induce different alterations in plant tissues, such as matrix fragmentation, cell erosion, pore formation, increased absorption, shear force, and changes in swelling index.Cavitation produces shock waves, micro-jets, shear force, and turbulence, modifying plant matrix and accelerating EO extraction [22,23,24,25].
The GC-MS analysis of the obtained R. ligulata EOs led to the identification of their constituent metabolites revealing component variation, which is likely dependent employed plant part, as comprehensively shown in Table 7 and the chromatograms in Fig. 5.The compound epi-Eudesmol was the dominant component of EO in R. Ligulata aerial parts and rhizomes (Table 7 and in Figure 5).This result is of interest, because this molecule is considered a neuroprotector with potential pharmacological, some studies on EO rich in -Muurolol and epi-Eudesmol have shown a powerful bactericidal effect on Escherichia coli and Salmonella typhimirium, opening a window for studies of its biological activity and future applications [13].Furthermore, to the best of our knowledge, ours is the first report on the optimization, extraction, and metabolic characterization of EO of R. Ligulata.
Regarding the chemical composition of the obtained R. Ligulata EO, monoterpenes constituted 11 % of the extract with and without US pretreatment on plant rhizomes; whereas, aerial parts did not contained monoterpenes.Furthermore, the main structural family in EO from aerial parts and rhizomes were sesquiterpenes with areas from 80 % to 90 %, depending on the plant organ and the extraction method.Likewise, it was possible to verify that the main components of EO from from aerial parts and rhizomes were Caryophyllene oxide, -Muurolol, and epi-Eudesmol.

Conclusions
The MAHD essential oil extraction parameters with the best response surface optimization results were a microwave power of 765 W and a solvent volume of 225.9 mL for 30 g of R. Ligulata aerial parts and a microwave power of 799 W and 145 mL of solvent for for 20 g of R. Ligulata rhizomes.The best average extraction time was 42.5 min.

Figure 1 .
Figure 1.Standardized effects of MAHD EO extraction variables for A) R. lingulata aerial parts and B) rhizomes.Bar colors denote positive and negative direction effects.The Pareto diagram shows that the linear terms time (A) and power (B) and the quadratic factor time (B2), have greater effect on the MAHD.

Figure 2 .
Figure 2. Response surface estimates of the MAHD process variables tested for EO extraction from R. lingulata aerial parts (A, C, and E) and rhizomes (B, D, and F).

Fig. 3
Fig.3displays the value space in which the combination of two EO extraction parameters (microwave power and radiation time) maximizes the desirability function in the for each EO source, revealing the combination of factor values at which the optimum is achieved.

Figure 4 .
Figure 4. Essential oil yield percentages with and without ultrasound (US) treatment before MAHD extraction from R. ligulata rhizomes (Ri) and aerial parts (AP).

Figure 5 .
Figure 5. GC/MS chromatogram of R. Ligulata essential oil and with the chemical structure of the dominant compound given.

Table 1 .
Evaluated EO extraction variables and their levels depending on plant part employed

Table 2 .
Result of Box -Behnken experimental design for EO extraction yield from R. ligulata aerial parts.

Table 3 .
Result of Box -Behnken experimental design for EO extraction yield from R. ligulata rhizomes.

Table 4 .
ANOVA for EO extraction yields from R. lingulata aerial parts

Table 5 .
ANOVA for EO extraction yields from R. lingulata rhizomes

Table 6 .
Optimal conditions for MAHD-assisted essential oil extraction form R. ligulata parts.

Table 7 .
Chemical composition of EOs.