Chemical composition and antibacterial activity of Dracocephalum kotschyi essential oil obtained by microwave extraction and hydrodistillation

ABSTRACT

Dracocephalum kotschyi essential oils obtained by hydrodistillation (HD), microwave-assisted hydrodistillation (MAHD), and solvent-free microwave extraction (SFME) were investigated by GC-FID (Gas Chromatography-Flame Ionization Detector) and GC-MS (Gas Chromatography-Mass Spectrometry). The percentage of oxygenated compounds was significantly increased from 62.52% in HD to 76.47% in MAHD, and 84.52% in SFME. Conversely, the monoterpene hydrocarbons were decreased from 30.84% in HD to 13.71% in MAHD, and 5.85% in SFME. The main compound in the essential oil obtained by HD is limonene, which accounted for more than 30% of the oil, while the percentage of this compound was reduced to 9.52% in MAHD, and 5.60% in SFME. The percents of other oxygenated compounds such as neral, geranial, geraniol, geranyl acetate, α-terpineol, trans-verbenol, carvon, and trans-carveol were noticeably higher in microwave methods than those present in HD method. In aqueous medium, linear oxygenated monoterpenes may be converted to a monocyclic terpinyl cation, which it could lose a proton to give limonene. The main biosynthetic pathway of these compounds, as well as a possible route of their conversion into limonene, due to prolonged heating in the HD method, was proposed. The antimicrobial activity of D. kotschyi essential oils against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria was evaluated by broth micro-dilution susceptibility manner. The most sensitive bacteria to these oils was found to be S. aureus with the lowest minimum inhibitory concentration value of 2 mg mL⁻¹ for MAHD and SFME specimens. The results indicated that oils obtained by microwave methods were more active against S. aureus.

KEYWORDS:

• Antibacterial activity
• Biosynthetic pathway
• Dracocephalum kotschyi
• Essential oil
• Limonene
• Microwave extraction
• Terpinyl cation
• Thermal degradation

Introduction

The genus Dracocephalum, belonging to the Lamiaceae family, is represented in Iran by eight species.^[1] Dracocephalum kotschyi is one of the important endemic species and is distributed in many parts of Iran.^[2] It is locally known as Badrandjboie-Dennaie or Zarrin-giah, and the aerial parts of the plant have been used as an additive to improve the taste and scent of tea and yogurt. In Iranian folk medicine, the plant is used as a remedy for stomach disorders. Its usefulness for headache, congestion, and liver disorders has also been described in traditional medicine documents.^[3] It is one of the constituents of Spinal-Z, a traditional Iranian anticancer remedy, which was used by traditional healers as a plant concoction for the treatment of many forms of cancer in humans.^[4] Pharmacological studies have confirmed some medicinal properties of the plant, including antihyperlipidemic,^[5] immunomodulatory,^[6] antinociceptive,^[7] and cytotoxic^[8] effects. In recent years, it has been reported that neuroprotective and immunoinhibitory effects of the plant are due to its methxylated flavonoid, calycopterin.^[9–12] Furthermore, methoxylated flavonoids of the plant enhance selective tumour inhibition and confirm the anticancer activity of this plant species.^[13] The plant is also a source of essential oil and trypanocidal terpenoids.^[14] Constituents of the oil of the plant collected from different parts of Iran were reported previously.^[15–18] These results show a completely different chemical composition of oil from the plant collected in different regions.

Essential oils are complex mixtures of volatile substances generally present at low concentrations.^[19,20] Distillation is the most conventional method for the extraction of essential oils.^[21] During distillation, plant materials, exposed to boiling water or steam, release their essential oils through evaporation. Recovery of the essential oil is facilitated by distillation of two immiscible liquids, viz. water and essential oil, based on the principle that at the boiling temperature, the combined vapour pressures equal the ambient pressure. Thus, the essential oil ingredients, whose boiling points are normally in the range 200–300°C, are evaporated at a temperature close to that of water.^[22] But, in this process, the elevated temperatures and prolonged extraction time can cause chemical modifications of the oil components and often a loss of the most volatile molecules.^[23] These shortcomings have led to consideration of the use of new techniques in essential oil and aroma extraction, which typically enhance the quantity of essential oil, preserve its quality, and use less energy. There has recently been widespread interest in the application of microwave heating to extract of essential oils.^[24–27] Microwave heating is defined as the heating of a substance by electromagnetic energy operating in the frequency range. The frequency range, which also can be used for heating, is very large. But microwave energy, with a frequency of 2.45 GHz, is well known to have a significant effect on the rate of various processes in the chemical and food industry.^[28] It is important to recognize that the energy delivered by microwaves is insufficient for breaking covalent chemical bonds. During microwave at high-frequency heating, many material properties affect the heating performance. There is a fundamental difference in the nature of microwave heating when compared with conventional methods of heating materials. Conventional heating relies on one or more of the heat transfer mechanisms of convection, conduction, or radiation to transfer thermal energy into the material.^[29]

Utilizing microwaves with hydrodistillation (HD) is one way of using microwaves for the extraction of essential oils.^[30–34] This method, called microwave-assisted hydrodistillation (MAHD), is performed at atmospheric pressure using water as solvent. Another method, solvent-free microwave extraction (SFME) is based on the combination of microwave heating and dry distillation, and is performed at atmospheric pressure.^[35–39] This method involves placing plant material in a microwave reactor without any added solvent or water. The internal heating of the in situ water within the plant material distends the plant cells and leads to rupture of the glands and oleiferous receptacles. This process thus frees essential oil which is evaporated by the in situ water of the plant material. A cooling system outside the microwave oven condenses the distillate continuously. The excess of water is refluxed to the extraction vessel in order to restore the in situ water to the plant material.^[40,41] The SFME is neither a modified microwave-assisted extraction (MAE) which uses organic solvents nor a modified HD which uses a large quantity of water.

In this study, we investigated the chemical composition of D. kotschyi oil obtained by MAHD and SFME. The chemical composition of D. kotschyi oil obtained by HD was also studied and compared. Therefore, the comparison of the three techniques in terms of isolation times, yields, and composition were reported. The antibacterial activity of the oils was tested against two bacteria (Staphylococcus aureus and Escherichia coli). To our knowledge, the antibacterial activity of D. kotschyi oil is reported for the first time.

Materials and methods

Plant materials

The aerial parts of D. kotschyi were collected at full flowering stage from Dizin region in the north of Tehran, Iran, in June 2011, at an altitude of 2400 m. A voucher specimen (MPH-1414) has been deposited at the herbarium of the Medicinal Plants and Drugs Research Institute, Shahid Beheshtin University, Tehran, Iran.

Hydrodistillation (HD) apparatus and procedure

HD was performed according to the method described in the European Pharmacopoeia. Hundred grams of air-dried aerial parts of D. kotschyi were submitted to a Clevenger-type apparatus and extracted with 700 mL of water for 3 h (until no more essential oil was obtained). The essential oil was collected, dried under anhydrous sodium sulphate, and stored in sealed vials in the dark, at 4°C, until used.

Microwave-assisted hydrodistillation

MAHD was performed in a commercial microwave laboratory oven (Ethos MR, Milestone, Italy). This is a multimode microwave reactor with a built-in magnetic stirring, which uses 2.45 GHz frequency. Microwaves are generated by a dual magnetron system with a maximum delivered power of 1000 W variable in 10 W increments. The dimensions of the PTFE-coated cavity are 35 cm × 35 cm × 35 cm. During experiments, time, temperature, pressure, and power can be controlled with the “easy-WAVE” software package of the system. Temperature was monitored by a shielded thermocouple inserted directly into the flask and by an external infrared (IR) sensor. Temperature was controlled by a feedback to the microwave power regulator. The MAHD experiment was performed at atmospheric pressure. Then, 50 g of dried aerial parts of the plant were placed in a 2 L two-neck flask and mixed with 800 mL of distilled water. The flask was placed within the microwave oven cavity and a Clevenger refrigerator provided with a glass stopcock and a circulating water condenser was connected to the sample holder to collect the extracted essential oil. The mixture was heated at a fixed power of 800 W and 100°C for 30 min. This period was sufficient to extract all the essential oils from the sample. The essential oil was collected, dried under anhydrous sodium sulphate, and stored at 4°C until analysed.

Solvent-free microwave extraction

For SFME, 50 g of dried D. kotschyi was soaked in 700 mL distilled water at room temperature for 1 h in order to hydrate the external layers of the plant material, and the excess water was drained off. The moistened plant material was placed in 500 mL two-neck flask connected to a Clevenger apparatus. The mixture was heated at the same condition of MAHD process. During the process, the vapour passed through the condenser outside the microwave cavity and condensed water was refluxed to the extraction vessel in order to provide uniform conditions of temperature and humidity for extraction. The extraction was continued for 30 min until no more essential oil was obtained. The essential oil was collected, dried under anhydrous sodium sulphate, and stored at 4°C until being analysed.

Oil analysis procedure

GC analysis of the oils was conducted using a Thermoquest gas chromatograph with a flame ionization detector (FID). The analysis was carried out on fused silica capillary DB-5 column (30 m × 0.25 mm i.d., film thickness 0.25 μm). The injector and detector temperatures were kept at 250°C and 280°C, respectively. Nitrogen was used as carrier gas at a flow rate of 1.1 mL min^–1; oven temperature program was 60–250°C at the rate of 4°C min^–1 and finally held isothermally for 10 min.

GC-MS analysis was carried out by use of Thermoquest-Finnigan gas chromatograph equipped with fused silica capillary DB-5 column (30 m × 0.25 mm i.d., film thickness 0.25 μm) coupled with a TRACE mass (Manchester, UK). Helium was used as carrier gas with ionization voltage of 70 eV. Ion source and interface temperatures were 200°C and 250°C, respectively. Mass range was from 35 to 456 amu. Oven temperature program was the same given above for the GC.

Identification of compounds

The constituents of the essential oils were identified by calculation of their retention indices under temperature-programmed conditions for n-alkanes (C₆–C₂₄) and the oil on a DB-5 column under the same chromatographic conditions. Identification of individual compounds was made by comparison of their mass spectra with those of the internal reference mass spectra library (Wiley, 7.0) or with authentic compounds and confirmed by comparison of their retention indices with authentic compounds or with those reported in the literature.^[42] For quantification purpose, relative area percentages obtained by FID were used without the use of correction factors. Compounds that were identified according to retention indices reported in the previous publications are presented in Table 1, together with their references.

Table 1. Chemical Composition of Dracocephalum kotschyi Boiss. Essential Oils Obtained by Hydrodistillation, Solvent Free Microwave Extraction and Microwave Assisted Extraction.

No.	Compound	RI	LRI	% HD oil	% SFME oil	% MAHD oil
1	α -Pinene^a	938	932	-	0.25	0.41
2	o-Cymene	1026	1022	0.25	-	-
3	Limonene^a	1032	1024	30.59	5.60	9.52
4	Linalool^a	1096	1095	-	0.25	0.33
5	cis-Thujone	1109	1101	-	-	0.15
6	trans-p- Mentha-2,8-dien-1-ol	1122	1119	0.52	0.94	0.88
7	α-Campholenal	1128	1122	1.04	1.02	1.38
8	cis-p-Mentha-2,8-dien-1-ol	1136	1133	0.52	0.97	1.04
9	trans-p-Limonene oxide	1139	1137	0.41	0.17	0.54
10	trans-Pinocarveol	1146	1135	-	0.26	-
11	trans-Verbenol	1148	1140	0.64	1.66	1.92
12	Pinocarvone	1167	1160	-	-	0.15
13	α-Terpineol	1193	1186	0.92	1.47	1.50
14	trans-p-Mentha-1(7),8-dien-2-ol	1190	1187	0.33	-	-
15	cis-Dihydrocarvone	1200	1198	-	-	1.22
16.	Verbenone	1216	1206	0.36	0.40	-
17.	trans-Carveol	1220	1215	3.36	7.20	4.41
18.	cis-Carveol	1232	1226	0.39	1.20	0.30
19.	Nerol	1231	1227	-	1.45	-
20.	cis-p-Mentha-1(7),8-dien-2-ol	1231	1227	-	-	0.22
21.	Neral^a	1242	1235	4.89	6.77	7.14
22.	Carvone	1250	1239	4.55	5.91	6.59
23.	Geraniol^a	1253	1249	-	4.52	2.04
24.	Geranial	1271	1264	6.42	7.20	8.27
25.	Perilla aldehyde^a	1279	1269	26.00	24.80	25.50
26.	Borneyl acetate	1290	1285	0.26	-	0.15
27.	Limonene-10-ol	1297	1288	-	2.59	-
28.	Menthyl acetate	1294	1294	-	-	0.16
29.	Perilla alcohol	1299	1294	-	-	0.34
30.	Methyl geranate	1323	1322	10.80	11.30	11.30
31.	Eugenol	1359	1356	-	0.45	-
32.	α-Copaene	1387	1374	0.21	0.29	-
33.	Geranyl acetate	1378	1379	1.16	3.18	2.90
34.	Germacrene D	1494	1484	0.15	0.68	0.36
35.	β-Bourbonene	1398	1387	-	-	0.21
36.	3Z-3-Hexenyl benzoate	1573	1565	-	0..39	0.16
37.	Globulol	1610	1590	-	-	0.82
38.	Veridiflorol	1610	1592	-	0.62	0.61
	Isolation (min)			240	30	30
	Oxygenated compounds			62.52	84.52	76.47
	Hydrocarbons compounds			31.20	6.82	14.33
	Monoterpene hydrocarbons			30.84	5.85	13.71
	Oxygenated monoterpenes			62.52	83.51	75.04
	Sesquiterpene hydrocarbons			0.36	0.97	0.62
	Oxygenated sesquiterpenes			0	1.01	1.43
	Others compounds			0	0	0
	Total identification (%)			93.72	91.34	90.80

RI: retention index. LRI: retention indices reported from literatures.

a. Identification was confirmed by comparison of the date with those of authentic compound.

Antibacterial activity testing

In vitro antibacterial activity of the essential oil was determined against S. aureus ATCC 25923 and E. coli ATCC 25922 as models of Gram-positive and Gram-negative bacteria, respectively. Broth micro-dilution susceptibility manner using 96-well trays was performed to specify the minimum inhibitory concentration (MIC) of essential oil needed for inhibition of visible growth of the test strains. For this intent, standard protocol of CLSI (Clinical Laboratory and Standards Institute) was used with some reformation.^[43] The inoculants of the bacterial strains were accumulated from freshly cultured species, by using sterile normal saline which were adjusted to 0.5 McFarland standard turbidity and then were more diluted (1:1000 for bacteria) by sterile Mueller–Hinton broth (MHB) just before adding to the wells containing a desired range of lenis essential oil. Essential oil was evaluated in a concentration confined from 64 to 0.06 mg mL^–1 in wells containing MHB medium supplemented by 0.5% Tween-80.^[44] Inoculated trays were incubated for 20 h at 37°C and then the MICs were registered as the minimum concentrations that could inhibit the visible growth of microorganisms.

Results and discussion

Chemical composition of essential oil

D. kotschyi essential oils obtained by HD, MAHD, and SFME were investigated by capillary GC and GC-MS. The analysis allowed the identification of 38 different components (21 HD, 30 MAHD, and 27 SFME), which accounted for 93.72%, 90.80%, and 91.34% of HD, MAHD, and SFME oils, respectively. The retention indices of the oil constituents and their relative percentages are listed in Table 1. The composition of the oils was almost similar and was consisted mostly of monoterpenenoids (93.36% HD, 88.75% MAHD, and 89.36% SFME). However, the percentage of oxygenated compounds has significantly increased from 62.52% in HD to 76.47% in MAHD, and 84.52% in SFME. Conversely, the monoterpene hydrocarbons have been decreased from 30.84% in HD to 13.71% in MAHD, and 5.85% in SFME. The main compound in the essential oil obtained by HD is limonene, which accounted for more than 30% of the oil. However, the percentage of this compound has reduced to 9.52% in MAHD, and 5.60% in SFME. Another two main constituents in the essential oils were perilla aldehyde and methyl geranate, while their relative ratio has remained almost constant in the oils obtained by three methods. Perilla aldehyde has changed between 24.80% and 26.00%, and methyl geranate variations were between 10.80% and 11.30%. But the percents of other oxygenated compounds such as neral, geranial, geraniol, geranyl acetate, α-terpineol, trans-verbenol, carvon, and trans-carveol were noticeably higher in microwave methods than those in HD method. As we know, neral, geranial, geraniol, and geranyl acetate are linear monoterpens and α-terpineol is a mono-cyclic monoterpene. All of these compounds are produced in biosynthesis pathway, earlier than limonene. Carvon and carveol are also structurally related to limonene. Often, most of these compounds are converted to limonene, due to prolonged contact with water in HD method. The main biosynthesis pathway of these compounds,^[45] as well as a possible route of their conversion into limonene, due to prolonged heating in the HD method, is presented (Fig. 1). On heating and protonation in aqueous medium, all linear oxygenated monoterpenes are likely to be converted into a monocyclic terpinyl cation, which it could lose a proton to give limonene.

Figure 1. Biosynthetic Pathway of Oxygenated Compounds [45], as Well as a Possible Route of Their Conversion into Limonene, due to Prolonged Heating in the Hydrodistillation Method.

The higher abundance of oxygenated compounds in SFME oil than in HD oil is related to the rapid heating of polar substances by microwaves and to the smaller amount of water used, which prevented the decomposition of principal oxygenated constituents by thermal and hydrolytic reactions.^[38] The lower amount of oxygenated compounds in MAHD oil than in SFME oil also confirmed the rule of the amount of water in degradation of oxygenated compounds. Water is a polar solvent which accelerates many reactions, especially reactions via carbocation as intermediates. We know that, the chemistry of terpenoids is related to carbocations and their conversions are carried out by carbocation intermediates.

Monoterpene hydrocarbons are less valuable than oxygenated compounds in terms of their contribution to the fragrance of the essential oil. Conversely, the oxygenated compounds are highly odoriferous and, hence, the most valuable. Also, they have been proved to possess strong antibacterial and antifungal activities.^[46–48] Concerning the comparison of the three techniques in terms of isolation times and yields, both microwave extraction and distillation were clearly fast (each 30 min), while 3 h were required for HD. Furthermore, it is noticeable that an extraction time of 30 min with microwave methods provided an appreciably high yield comparable with that obtained after 3 h by means of HD (0.1% HD, 0.09% MAHD, 0.08% SFME).

Recently, Sodeifian et al. presented the chemical components of the essential oil of D. kotschyi Boiss using supercritical carbon dioxide and the HD methods. The main components in the supercritical carbon dioxide method were (E)-citral (21.23), (Z)-citral (15.91), α-pinene (15.42), and limonene (8.02), whereas the principal ingredients in the HD method were (E)-citral (17.83), limonene (15.23), (Z)-citral (10.64), α-pinene (9.53), methyl guarantees (8.25), and geranyl acetate (8.14).^[49] In another research, the main ingredients of the essential oils of D. kotschyi growing wild in Kamu Mountain, Isfahan province, Iran, were reported. The main components were limonene (23.56%), carvacrol (14.65%), γ-terpinene (12.99%), α-pinene (12.62%), 2-methyl 1-octen-3-yne (9.73%), camphene (4.66%), myrcene (3.65%), and α-terpinene (3.12%).^[50] Also in 2016, variation in essential oil of D. kotschyi was studied at three phenological stages. GC-MS analysis of the essential oils identified 15 components, with (E)-β-ocimene as principal ingredient, whose percentage varied pursuant to the phonological stage (53.28 ± 0.7, 47.2 ± 0.7, and 33.0 ± 0.3 for vegetative, flowering, and fruiting, respectively). The other principal compounds identified were nerol at the vegetative (36.38 ± 0.7), nerol/methyl geranate (15.5 ± 0.2 and 8.3 ± 0.1) at flowering, and α-pinene/geranial/geraniol (16.7 ± 0.2, 14.8 ± 0.2, and 11.5 ± 0.2) at fruiting stage.^[51]

With a glimpse can be realized that quantity and quality of components were different than our research. These reports highlight the extensive diversity of the composition of these essential oils. The essential oils component is severely affected by intrinsic agents like species, ecotype, and ecological agents as geographical conditions, soil, biotic, climatic situations and technological factors, types of accumulation processes, storage status of crude materials, and special methods of extraction. Therefore, wild herbs from one species but from various backgrounds can display different characters and chemical constituents.^[52]

Antibacterial activity

The results of antibacterial assay of essential oils are shown in Table 2. Broth micro-dilution method was used for determination of MIC of the samples against Gram-positive and Gram-negative bacteria, S. aureus and E. coli, respectively. The most sensitive microorganism to the oil was found to be S. aureus with the lowest MIC value of 2 mg mL^–1 for MAHD and SFME samples. This may be somewhat due to the reality that the microwave extracted oils included more oxygenated compounds and these categories of compounds have been demonstrated to possess strong antibacterial and antifungal activities.

Table 2. Comparison of Minimum Inhibitory Concentrations (MIC_S; mg ml⁻¹) of Oils by Microdilution Method.

Test Bacteria	HD	MAHD	SFME
	MIC	MIC	MIC
Staphylococcus aureus (ATCC 25923)	4	2	2
Escherichia coli (ATCC 25922)	4	16	4

Conclusion

The potential of the microwave techniques has been compared with the conventional HD method, for the extraction of essential oil from D. kotschyi. Analysis of the extract highlighted differences in the composition of the essential oils. For SFME and MAHD, it was found that the limonene fraction decreased significantly, while it was the main compound in the HD extract. A possible route for conversion of the oxygenated monoterpenes into limonene, due to prolonged heating in the HD method, was proposed. For the first time, the antibacterial activity of D. kotschyi essential oil was also reported.

Nomenclature

EO	=	essential oil
HD	=	hydrodistillation
MAE	=	microwave-assisted extraction
MAHD	=	microwave-assisted hydrodistillation
SFME	=	solvent-free microwave extraction

Funding

Financial support by the Shahid Beheshti University Research Council is gratefully acknowledged.

Supplemental data

Supplemental data for this article can be accessed on the publisher’s website.

Additional information

Funding

Financial support by the Shahid Beheshti University Research Council is gratefully acknowledged.