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Abstract

This study reports on the optimization of a microwave-assisted distillation process to obtain Dong Van marjoram essential oil, and the determination of its composition, content of constituents, and cytotoxic and antimicrobial activities. Using the response surface method (RSM), the optimal essential oil distillation conditions were determined as material size 0.74 (cm), water to material ratio 4.14:1 (mL/g), microwave power 302.4 (W), and distillation time 2.1 hours. At optimal conditions, the mass of Dong Van marjoram essential oil obtained was 0.887 ± 0.007 g, corresponding to a content of 0.6% essential oil in the material. GC-MS and GC-FID methods showed that the main chemical constituents of Dong Van marjoram essential oils obtained were rosefuran epoxide (44.9%), caryophyllene (10.8%), germacrene D (2.6%), and α-humulene (1.3%). The essential oil exhibited moderate inhibition against both tested cancer cell lines, with IC₅₀ values of 23.9 µg/mL (for PC3) and 56.2 µg/mL (for A549). However, the oil exhibited strong effectiveness against three bacterial strains, Escherichia coli, Bacillus subtilis, and Staphylococcus aureus, and a yeast strain, Saccharomyces cerevisiae, with minimal inhibitory concentration (MIC) values ranged from 50 to 100 µg/mL.

Keywords

microwave-assisted hydrodistillation (MAHD)response surface methodology essential oils chemical composition.

Introduction

Plant essential oils have been shown to exhibit numerous useful properties, including anti-microbial, anti-fungal, antioxidant, anti-free radical, and anti-inflammatory activities.^1,2 The composition of essential oils is widely varied and might include terpene and oxide derivatives such as ketones, alcohols, and aldehydes.^3-5

Dong Van marjoram (E. winitiana var. dongvanensis Phuong., family Lamiaceae) is an herbaceous plant native to Asian countries such as China, Vietnam, and Thailand.⁶ This species is quite common in the high mountains of Ha Giang province, Vietnam.⁷ Although the plant has been used in the food industry as a seasoning in sausages, baked goods, processed vegetables, and soups, its most prevalent use is in folk medicine where the plant is used to treat colds, fever, digestive disorders, sunstroke, rheumatic arthritis, and nyctalopia.^1,7

Elsholtzia species produce essential oils with diverse chemical compositions including terpenoids, aldehydes, phenylpropanoids, and phytosterol.¹ The essential oil of E. winitiana is a rich source of citral, containing citral A (34.8%) and citral B (30.1%), suggesting its use in the cosmetic and pharmaceutical industries.⁷ Citral is mostly obtained from the upper part of the plant, but its content varies considerably depending on the harvesting time and the quality of the raw materials.⁷ The essential oils of E. winitiana also have a high concentration of rosefuran or rosefuran epoxide.^8,9

There are many traditional methods to obtain essential oils from plant materials such as hydrodistillation (HD), steam distillation (SD), and organic solvent extraction.¹⁰ However, the loss of some degradable compounds due to heating or hydrolysis can occur during HD and SD techniques, while organic solvents are toxic. Nowadays, microwave-assisted hydrodistillation (MAHD) is an alternative method for isolating essential oils because of its advantages such as cost- and time-effectiveness, environmental friendliness, and lower energy consumption.^11-13 Microwave extraction using a microwave oven can improve the performance of the process. This improvement is determined by the energy from microwaves causing internal pressure in the essential glandular cells, which helps the more efficient extraction of the essential oils.¹⁴

In this study, we used microwave-assisted hydrodistillation (MAHD) to extract the essential oil from Dong Van marjoram branches (E. winitiana var. dongvanensis Phuong.). Then, we used RSM (Response Surface Methodology) with a central composite design (CCD) to determine the maximum oil distillation performance with four parameters including material size (cm), water to material ratio (mL/g), distillation time (h), and microwave power (W). The quantitative profiles of the essential oils were analyzed using gas chromatography (GC) with flame ionization detection (FID), and GC combined with mass spectrometry (MS). Finally, we evaluated the in vitro bioactivity of the essential oil from branches of Dong Van marjoram (E. winitiana var. dongvanensis Phuong.) obtained by the MAHD method.

Material and Methods

Materials

Elsholtzia winitiana var. dongvanensis Phuong. plants were collected in December 2020, in Dong Van district of Ha Giang province, Vietnam. A specimen of the plant was botanically identified by Dr Nguyen Quoc Binh, Vietnam National Museum of Nature, Vietnam Academy of Science and Technology. After harvesting, the branches were separated and dried to prepare for the extraction of essential oils. The used plant materials were of uniform quality, and free from pests and diseases.

Extract equipments

The essential oil extraction system included a microwave model R20A1 produced by SHARP; this equipment acts as the heat source of the extraction process and a Clevenger extraction apparatus. The flask containing the raw material was placed in the microwave chamber. The distillation system was located outside the oven, which condenses and separates the essential oil from the liquid phase.

The extraction process of essential oils

Firstly, 150 g of dry plant material was cut into a suitable size and added to a 2 L flask containing water for which the water to material ratio is identified. The sample was soaked for 30 minutes before the Clevenger extraction process was conducted by a microwave oven. Time was measured from when the oven was turned on. After the extraction process, the crude oil and a few condensation products were recovered. The crude essential oil was dried with Na₂SO₄, which also completely removed the remaining condensation products. Finally, the mass of essential oil was accurately determined on an analytical balance and stored at 4 °C in a refrigerator until gas chromatography (GC)-flame ionization detector (FID) and GC-mass spectrometry (MS) analyses.

The yield of essential oil obtained (%) was calculated by the following formula:

The yield (%) = \frac{W_{1}}{W_{2}} \times 100

(1)

where W₁ is the mass of essential oil obtained (g) and W₂ is the mass of dry sample originally used (g)

Experiment Design for Response Surface Methodology Optimization

Response surface methodology (RSM) was used to optimize the experimental conditions with respect to maximum oil yield. Four parameters were considered for optimization, namely: material size, water/material ratio, extraction time, and microwave power. First, a series of single-factor investigations was performed by varying one of the considered variables while fixing the others, in the following sequence: material size, water to material ratio, microwave power, and extraction time. Based on the results, experimental levels were determined to include the level center (encoded 0), level low (encoded -1), and level high (encoded +1) to build an experimental design matrix following the Box-Wilson model with Design-Expert 7.0.0 software. Twenty-five sets of experiments were designed, which were then attempted to generate the data for model estimation (Table 1 and 2). The validity of the estimated model was confirmed by performing analysis of variance (ANOVA). The optimized conditions were verified by conducting experiments with those parameters and the masses of essential oils were compared. The function that describes the mass of the essential oil with respect to experimental parameters is as follows:

Y_{k} = b_{0} {+ \sum}_{j = 1}^{k} b_{j} x_{j} + \sum_{u, j = 1}^{k} b_{u j} x_{u} x_{j} + \sum_{j = 1}^{k} b_{j j} x_{j}^{2}

(2)

Table 1.

Ranges of parameters determined by experimental design

Variable names and ranges			Research level
Actual variables	Coded variables	Δ	-α	-1	0	1	+α
Z₁: Material size (cm)	A	0.5	0.29	0.5	1	1.5	1.71
Z₂: Water to material ratio (mL/g)	B	1	2.58	3	4	5	5.42
Z₃: Microwave power (W)	C	120	70	120	240	360	410
Z₄: Distillation time (h)	D	1	0.59	1	2	3	3.41

(Coefficient α = 1.414)

With: Y and X are the predicted response and independent variables, respectively, b₀ is the intercept; b_j, b_uj, and b_jj are variable coefficients.

Table 2.

Matrix of orthogonal plans and experimental results

Run	Actual variables				Coded variables				Y (g) (The mass of essential oils)
Run	Z₁	Z₂	Z₃	Z₄	X₁	X₂	X₃	X₄	Actual	Predicted
1	0.5	3	120	1	-1	-1	-1	-1	0.11	0.123
2	1.5	3	120	1	+1	-1	-1	-1	0.05	0.063
3	0.5	5	120	1	-1	+1	-1	-1	0.14	0.152
4	1.5	5	120	1	+1	+1	-1	-1	0.10	0.092
5	0.5	3	360	1	-1	-1	+1	-1	0.71	0.722
6	1.5	3	360	1	+1	-1	+1	-1	0.50	0.512
7	0.5	5	360	1	-1	+1	+1	-1	0.72	0.751
8	1.5	5	360	1	+1	+1	+1	-1	0.60	0.541
9	0.5	3	120	3	-1	-1	-1	+1	0.15	0.154
10	1.5	3	120	3	+1	-1	-1	+1	0.05	0.043
11	0.5	5	120	3	-1	+1	-1	+1	0.17	0.183
12	1.5	5	120	3	+1	+1	-1	+1	0.06	0.072
13	0.5	3	360	3	-1	-1	+1	+1	0.70	0.703
14	1.5	3	360	3	+1	-1	+1	+1	0.5	0.442
15	0.5	5	360	3	- 1	+1	+1	+1	0.72	0.732
16	1.5	5	360	3	+1	+1	+1	+1	0.50	0.471
17	0.29	4	240	2	-1.414	0	0	0	0.80	0.779
18	1.71	4	240	2	+1.414	0	0	0	0.51	0.541
19	1	2.58	240	2	0	-1.414	0	0	0.56	0.588
20	1	5.42	240	2	0	+1.414	0	0	0.65	0.632
21	1	4	70	2	0	0	-1.414	0	0.05	0.06
22	1	4	410	2	0	0	+1.414	0	0.65	0.679
23	1	4	240	0.59	0	0	0	-1.414	0.68	0.725
24	1	4	240	3.41	0	0	0	+1.414	0.71	0.695
25	1	4	240	3	0	0	0	0	0.81	0.832

Phytochemical Screening of Essential Oils

The essential oil components were analyzed by GC-MS and GC-FID. For GC-MS analysis, the system involved an HP7890A model GC (Agilent Technologies, Santa Clara, CA, USA) equipped with an HP5975C MS detector and an HP5 MS column (60 m × 0.25 mm, film thickness 0.25 µm) (Agilent Technologies, US). Running conditions were GC-MS: carrier gas: helium; flow rate: 1 mL/min; split ratio 100:1; injection volume of sample 1 μL; temperature of injector set at 250 °C. The temperature program began at 60 °C, then increased up to 240 °C, at 4°C/min. The electron impact ionization voltage was 70 eV, the emission current was 40 mA, and the acquisitions scan mass range was 35-450 amu. GC-FID analysis was applied using similar conditions as those used for GC-MS analysis.¹⁶

The GC-FID peak area was used to calculate the relative percentage of components in the essential oil, without any correction factor. Identification of the constituents was carried out by comparing the obtained retention indices (RI) and mass spectra with W09N08, HPCH1607 mass spectral libraries, and the data of NIST Chemistry WebBook.

Cytotoxicity Assays

The cytotoxicity of the obtained essential oils was assayed against three established cell lines. Cell lines A549 (CLS 300114) and PC-3 (ATCC CRL-1435) were purchased from the American Type Culture Collection (Manassas, VA, USA), and cell line A-549 (Item number: 300114) from Cell Lines Service GmbH (Eppelheim, Germany). The cells were maintained at the Bioassay Laboratory, Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, Vietnam. Culture media included DMEM (Dulbecco's modified Eagle's medium), EMEM (Eagle's minimum essential medium, Sigma-Aldrich, St. Louis, MO, USA), and 10% heat-inactivated fetal bovine serum (FBS). The culture was performed in a humidified atmosphere of 95% air and 5% CO₂ at 37 ^oC. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to measure cell viability.¹⁷

Antimicrobial Activity

Eight microbial strains obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) were used to evaluate the antimicrobial activity, including Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 25923, Bacillus subtilis ATCC 27212, Staphylococcus aureus ATCC 12222, Aspergillus niger ATCC 9763, Fusarium oxysporum ATCC 48112, Saccharomyces cerevisiae ATCC 16404, and Candida albicans ATCC 10231. The antimicrobial activity of the samples was determined by a minimum inhibitory concentration (MIC) assay against the above fungal and bacterial strains. The Gram-positive and -negative bacteria were cultured in tryptic soy broth (TSB; Merck KGaA, Darmstadt, Germany), while fungi were grown in SDB (Merck, Germany) to a final inoculum size of about 150 × 106 colony-forming units (CFU) per mL (or 0.5 McFarland standard at λ = 550 nm). A sample of essential oil at various concentrations ranging from 12.5 to 200 μg/mL was loaded into 96-well microplates containing fresh cultures, and the plates were incubated at 37 °C for 24 hours. Blank controls were prepared identically with essential oils being replaced by 5% DMSO.¹⁸ Positive controls included doxycycline for Gram (-) and gentamicin for Gram (+) bacteria, and nystatin for fungi.

Results and discussion

Optimization of experimental conditions using response surface method (RSM)

Table 3 displays analysis of the ANOVA results for the second order regression model describing essential oil extraction with respect to four factors. Among all the variables, only the terms A, C, AC, A², B², C², D² had coefficients that were statistically significant. The obtained R² value was 0.9923, which is reasonable and in agreement with an Adjusted R² value of 0.9815. In addition, the adequate precision value, which is used to evaluate the interfering signal by 26.829, shows that this model can be used to describe the response and that no further model specification is required. Therefore, it can be concluded that the regression model built by Design Expert 7.0.0 software is appropriate to predict the performance of Dong Van marjoram essential oils extraction. Besides, based on Figure 1, there were many data points with predicted values (Predicted) and actual values (Actual) closely distributed to the original straight lines with a slope of 1. In addition, the experimental model was considered a reasonably fit as calculated residuals follow a random pattern, as shown in Figure 2, suggesting that actual responses were approximately close to the value predicted by the regression equation. The second order model is described by the following equation:

\begin{aligned} \overset{⌢}{Y} = & 0.8 – 0.074 A + 0.25 C – 0.027 A C \\ – 0.07 A^{2} – 0.095 B^{2} – 0.22 C^{2} – 0.05 D^{2} \end{aligned}

Figure 1.

Plot comparing actual values and predicted values

Figure 2.

Normal plot of residuals with run number

Table 3.

Analysis of variance (ANOVA) for the quadratic model

Source	Sum of Squares	Df	Mean Square	F-value	p-value	Remarks
Model	1.89	14	0.14	92.14	<0.0001	significant
A	0.11	1	0.11	73.60	<0.0001	significant
B	0.0067	1	0.0067	4.59	0.0577	not significant
C	1.23	1	1.23	840.63	<0.0001	significant
D	0.00007	1	0.00007	0.048	0.8309	not significant
AB	0.0004	1	0.0004	0.27	0.6131	not significant
AC	0.012	1	0.012	8.24	0.0166	significant
AD	0.0025	1	0.0025	1.70	0.2212	not significant
BC	0.00002	1	0.00002	0.017	0.8988	not significant
BD	0.00122	1	0.00122	0.83	0.3825	not significant
CD	0.00122	1	0.00122	0.83	0.3825	not significant
A²	0.039	1	0.039	26.70	0.0004	significant
B²	0.072	1	0.072	49.17	<0.0001	significant
C²	0.4	1	0.4	269.73	<0.0001	significant
D²	0.02	1	0.02	13.62	0.0042	significant
Residual	0.015	10	0.00146
Cor total	1.91	24
Std. Dev.	0.038		R²	0.9923
Mean	0.45		Adjusted R²	0.9815
C.V. %	8.55		Adeq. Precision	26.829

Response Surface Analysis, Optimization, and Model Verification

Based on a quadratic polynomial model determined from experimental data by reaction surface method, the three-dimensional response surfaces showing the influence of the double interaction effect of the pairs of technological factors on the objective function (the mass of essential oils obtained) are shown in Figure 3. The X- and Y-axes of the three-dimensional response surface represent two factors, for material size and water to material ratio (microwave power 240 W and distillation time 2 h), material size and microwave power (water to material ratio 4:1 (mL/g) and distillation time 2 h), material size and distillation time (water to material ratio 4:1 (mL/g) and microwave power 240 W), water to material ratio and microwave power (material size 1 cm and distillation time 2 h), water to material ratio and distillation time (material size 1 cm and microwave power 240 W), microwave power and distillation time (material size 1 cm and water to material ratio 4:1 (mL/g)). The Z-axes represent the mass of essential oils obtained.

Figure 3.

Response surface of essential oil mass

Using the RMS method to optimize the distillation process, the optimal values of the independent variables were obtained by solving second-order regression equations to maximize the mass of essential oils obtained. The results of optimal technological parameters are as follows: Material size 0.74 (cm), water to material ratio 4.14 : 1 (mL/g), microwave power 302.4 (W), and distillation time 2.1 (h). With optimal conditions, the maximum mass of essential oil obtained was 0.893 g (Table 4 and Figure 4).

Figure 4.

Optimum conditions by solution of ramps.

Table 4.

Values of the independent variable and the real variable under optimal condition

Independent variables				Real variables
A	B	C	D	Material size (cm)	Water to material ratio (mL/g)	Microwave power (W)	Distillation time (h)
-0.53	0.14	0.52	0.1	0.74	4.14 / 1	302.4	2.1

From the optimal technological conditions found by RSM, as in Table 4, we repeated the experiment 3 times under these conditions. The average mass of essential oil obtained was 0.887 ± 0.007 (g), whereby the yield of the essential oils obtained was 0.6%. The experimental and theoretical calculation results are almost equivalent, showing that the built model is compatible with the experiment.

Chemical Composition of the Essential Oils

The chemical composition of the essential oil obtained by the MAHD method is represented in Table 5. Twenty-five compounds were detected, accounting for 98.6%. The main constituents were rosefuran epoxide (44.9%), followed by caryophyllene (10.8%), germacrene D (2.6%), α-humulene (1.3%), and rosefuran (1.2%). In addition, there was an unknown chemical component, accounting for 26.6% of the oil (m/z 95, 194; RI 1336).

Table 5.

Chemical composition of the essential oil extracted from Elsholtzia winitiana var. dongvanensis Phuong. by the MAHD method.

Chemical name	RI*	RI	Percentage (%)
Rosefuran	1095	1099	1.2
Linalool	1104	1103	0.3
Unknown 1 (95, 166, RI 1175)		1175	1.7
Rosefuran epoxide	1177	1180	44.9
α-Terpineol	1200	1200	0.1
Unknown 2 (96, 168, RI 1213)		1213	4.3
Unknown 3 (95, 166, RI 1222)		1222	Nd
Piperitone	1268	1265	0.2
Dehydroelsholtzia ketone	1306	1310	0.2
Unknown 4 (95, 184, RI 1334)		1334	Nd
Unknown 5 (95, 194, RI 1336)		1336	26.6
α-Copaene	1388	1389	0.4
β-Bourbonene	1398	1400	0.2
α-Gurjunene	1419	1425	0.4
Caryophyllene	1437	1437	10.8
α-Humulene	1474	1471	1.3
γ-Muurolene	1485	1490	0.3
Germacrene D	1497	1498	2.6
β-Selinene	1509	1504	0.2
Bicyclogermacrene	1532	1514	0.7
γ-Cadinene	1534	1530	0.2
δ-Cadinene	1540	1536	0.5
Scapanol	1586	1594	0.2
Caryophyllene oxide	1607	1605	1.2
α-Cadinol	1673	1674	0.1
Total			98.6

RI*: Retention index compared between software predictions

Nd: Not detected

Similar to the oil of Dong Van Marjoram used in this study, that obtained from E. densa Benth. in India also had rosefuran as its major component, in this case forming 86% of the total.¹⁹ Thirty-nine chemical components were identified in the essential oil obtained from E. stauntonii Benth. in North America, of which the main ones were rosefuran epoxide (40.4%) and rosefuran (41.7%).²⁰ In contrast, rosefuran epoxide was not the main component of the essential oils of some other Elsholtzia species in India. In that of E. strobilifera Benth., 65 chemical components were identified, the major of which was pinocarvone, accounting for 51.9%.²¹ In the essential oil of E. eriostachya var. pusilla, 21 chemical components were detected, the major ones being geranial (52.5%) and neral (39.1%).²² The essential oil of E.cristata Willd. contained 23 components with the major one being 3-methyl-2-(3-methylbut-2-enoyl)-furan, accounting for up to 88.7%.²³

The results of this study have contributed to clarifying the chemical composition of the essential oil of marjoram dong van from Ha Giang province, Vietnam.

In vitro Bioactivity of Essential Oils

The essential oil obtained by the MAHD method was evaluated in terms of its anti-proliferative activity on two cancer cell lines (PC3, and A549), as well as its anti-microbial activity on eight strains of fungi, yeast, and bacteria.

Moderate inhibition was observed against both tested cell lines, with IC₅₀ values of 23.9 µg/mL (for PC3) and 56.2 µg/mL (for A549) (values for paclitaxel 3.5 and 3.7 ng/mL, respectively). However, the essential oil showed strong activity against three bacterial strains (E. coli, B. subtilis, S. aureus), and a yeast strain (S. cerevisiae) with minimal inhibitory concentration (MIC) values ranging from 50 to 100 µg/mL (Table 6). In general, these data show that essential oils from Dong Van marjoram extracted by the MAHD method have potential in a biological application.

Table 6.

Antimicrobial activity of Dong Van marjoram essential oil

Sample	MIC (μg/mL)
Sample	E. coli	P. aeruginosa	B. subtilis	S. aureus	A. niger	F. oxysporum	S. cerevisiae	C. albicans
Essential oils	50	>200	50	100	>200	>200	100	>200
Positive control^*	6.0	9.8	9.4	17.8	6.2	3.0	8.1	2.3

Positive control: doxycycline for Gr (-) and gentamicin for Gr (+) bacteria, nystatin for fungi.

Conclusion

We have studied and optimized the technological parameters of the distillation process using microwaves to collect Dong Van marjoram essential oil. The optimal technological parameters found were material size 0.74 (cm), water to material ratio 4.14:1 (mL/g), microwave power 302.4 (W), and distillation time 2.1 (hour). With optimal conditions, the mass of Dong Van marjoram essential oils obtained was 0.89 ± 0.007 (g), corresponding to the content of the essential oil of 0.6% in the material. Dong Van marjoram essential oil was shown to contain a high content of rosefuran epoxide (44.9%). The essential oil exhibited only moderate inhibition against the two tested cancer cell lines (PC3 and A549), but strong effectiveness against three bacterial strains, E. coli, B. subtilis, and S. aureus, and a yeast strain, S. cerevisiae. These data show that essential oils from Dong Van marjoram extracted by the MAHD method have the potential for biological application.

Footnotes

Acknowledgements

This study was supported by Vietnamese Ministry of Education and Training under the project code: B2019- TNA-06.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

Ethical Approval is not applicable for this article. Statement of Human and Animal Rights: This article does not contain any studies with human or animal subjects

Statement of Informed Consent: There are no human subjects in this article and informed consent is not applicable.

ORCID iD

Tan Phat Dao

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