Chemical composition and biological activity of Mentha citrata Ehrh., essential oils growing in southern Algeria

Abstract

Three techniques, hydro distillation, steam distillation and microwave-assisted distillation, was used in this work to extract volatile secondary metabolites essential oils (EOs), from the aerial part of Mentha citrata Ehrh., plant (steams, leaves and flowers). The plant material was collected from a location called El-kobna in El-Oued province, southern Algeria during June 2018. The extracted Mentha citrata EOs, were analyzed using both gas chromatography coupled with flame ionization detector and gas chromatography attached with mass spectrometry detector techniques. The antioxidant activity of these EOs were evaluated in vitro using DPPH (2,2-diphenyl-1-picrylhydrazyl) free radicals scavenging activity and cytotoxic test. The results of the EOs analysis showed large variability of the chemical compositions for all techniques. Moreover, a promising cytotoxic activity on colon cancer cells was found.

Introduction

The aromatic plants of Mentha are belonging to family Lamiaceae, an industrial and economically important genus, that comprises about twenty-five species were widely distributed worldwide with high prevalence in the Mediterranean region (Lawrence 2007). The Algeria medicinal herbs and plants were widely used in traditional medicine, especially in rural communities to treat several illnesses (Messaoudi and begaa 2019). Particularly the Mentha species have been used in the form of tea to relieve many diseases (Benabdallah et al. 2018; Begaa and messaoudi 2019). It has many documented biological activities such as antiemetic, antispasmodic, in treatment of many respiratory and gastrointestinal tract disorders, also it has substantial antimicrobial, and antifungal activities (Butnariu and Sarac 2018; Hassan et al. 2013; Begaa et al. 2018; Leal et al. 2013; Mimica-Dukic et al. 2003; Begaa and messaoudi 2018; Mimica-Dukic and Bozin 2008; Brahmi et al. 2017). Previous phytochemical studies on the genus afforded a diverse group of secondary metabolites, mainly terpenoids as essential oils, in addition to phenolic acids, flavonoids, and lignans (She et al. 2010; Saeidnia et al. 2004; Zheng et al. 2007; Butnariu and Sarac 2018). There are about fifteen species of Mentha in Algeria (Quézel and Santa 1962). Mentha citrata Ehrh. grows in southern Algeria (El-Oued) which are distributed from Morocco to Egypt (Boulos 1999). Thorough exploring of the current literature, recently, one report could be found concerning the chemical composition of the essential oil (Verma et al. 2016). However, nothing could be traced regarding a variation in the essential oil (EOs) chemical composition of the specie Mentha citrata Ehrh., growing in Algeria by changing the extraction method which might affect the biological value of the oil. In addition, no report about their biological activity could be found. Therefore, the goal of this study was to investigate qualitatively and also quantitatively the chemical composition of the EOs by these two techniques of GC–FID and GC–MS with an effort to determine the best extraction technique. In parallel, the antioxidant and cytotoxic effect of the extracted essential oil were examined to verify the validity of traditional uses.

Materials and methods

Plant material

The aerial parts of Mentha citrata Ehrh (Fig. 1), was collected from the city of El-kobna, El-Oued Southeast Algeria (33°32′00N and 6°43′13E) considered as a desert climate zone, in the month of June 2018. Mentha citrata samples were washed with distilled water to remove the dust, then dried in shade at room temperature over 2 weeks.

Fig. 1.

Aerial parts and leaves of Mentha citrata Ehrh. (family Lamiaceae)

Extraction of the essential oil

Hydrodistillation and steam distillation

The samples of Mentha citrata were subjected to distillation either with water to prepare hydro-distillation essential oil (HD-EOs), or steam to prepare steam distillation essential oil (SD-EOs) for 3 h by using the apparatus of Clevenger-type. The EOs, was collected and dried over anhydrous sodium sulfate (Na₂SO₄) to eliminate the weter. After that, they were stored at 4 °C in sealed-structure of glass vials until analysis.

Microwave-assisted distillation

The plant material (100 g) was cut into small pieces, then placed in a flask with 250 mL of distilled water. Then we put them in home appliance microwave (Model: Midea AG823ABI) with few modifications to accommodate the Clevenger-type device. The time was set at 30 min and operated at 800 W as adapted before (Ferhat et al. 2006).

Gas chromatography

GC–FID analysis

GC–FID (gas chromatography–flame ionization) analysis of the essential oil (EOs) was conducted using an Agilent Technologies equipped (7890A) with a FID (flame ionization detector). The separation was carried out on Rtx-5MS fused bonded column with properties (5% phenylmethylpolysiloxane, 30 m × 0.25 mm i.d., 0.25 µm film thickness). Nitrogen was used as the carrier gas (flow rate of 1 mL/min). The temperature programming was kept as: 5 min at 60 °C, then rising at 3 °C/min to 250 °C, held for 5 min isothermal. The temperature of the detector was adjusted at 300 °C. The injector temperature was kept at 250 °C, the injection volume was 0.1 µL and the split ratio was 1:50. A mixture of aliphatic hydrocarbons (C₅–C₂₈) was used to estimate the retention index (RI) of each components. The percentage composition of the individual components was obtained from electronic integration amounts using flame ionization detection.

GC/MS (gas chromatography–mass spectrometry) analysis

Mass spectral analysis was noted by using Shimadzu GCMS-QP2010 (Tokyo, Japan) equipped with Rtx-5MS fused bonded column (30 m × 0.25 mm i.d. × 0.25 µm film thickness) (Restek, USA) directly coupled to a quadrupole mass spectrometer. Briefly, the starting temperature was kept at 45 °C for 2 min, then the temperature was elevated to 300 °C (at a 5 °C/min rate), and then kept at 300 °C for 5 min, where the temperature of injector was about 250 °C. Helium carrier gas flow rate was 1.5 mL/min. The totally of mass spectra were noted applying the following condition: the filament emission current, 60 mA; ionization voltage, 70 eV; ion source, 200 °C. Diluted samples (1% v/v) were injected with split mode (split ratio 1:15). The determination of oil components was based on comparing their spectra with spectral mass spectroscopy for research in the MS Library (NIST and WILEY). The retention index (RI) was calculated using the n-alkanes series, C₈–C₂₈, and then compare with published data and to confirm the identity of the identified components (Adams 2007).

Biological evaluation

Cytotoxicity assay

Hepatic cancer Hep G2 [HEPG2] (ATCC^® HB-8065™) and colon cancer HCT116 (ATCC^® CCL-247™) cell lines were grown in RPMI 1640 media (l-glutamine augmented with 100 U/mL penicillin, 100 U/mL streptomycin, and 25 µg/mL amphotericin B) cells, were grown and developed at the temperature 37 °C in a 5% CO₂ humidified atmosphere. All the investigates were carried out in the logarithmic growth phase of the cells. Cytotoxicity was determined in triplicate using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay; 2 × 10⁴ cells/well of exponentially growing cells of both HepG2 and HCT116 cell lines were seeded in a 96-well plate (van de Loosdrecht et al. 1991). The cells were cultivated for 48 h and then incubated with various concentrations of the tested samples (0.78–100 μg/mL) at 37 °C for 48 h. Then, it was incubated with 0.5 mg/mL MTT for 4 h. The formed formazan crystals were dissolved in 200 μL DMSO (DMSO final concentration was less than 0.2%). The absorbance was detected at 595 nm and a reference wavelength of 620 nm with a (Bio-Rad Laboratories Inc., Model 3350, Hercules, California, USA). The cell viability (%) of three tests independent was considered by the following Eq. (1):

$$\text{Cell}\text{viability}(\%)=\left(\text{OD}\text{of}\text{treated}\text{cells}\right)/\left(\text{OD}\text{of}\text{control}\text{cells}\right)\times 100\%$$ 1

Doxorubicin was used as a positive control. Statistical significance was tested between the samples and the negative control (cells with vehicle) using an independent t test by the SPSS11 program.

Note: In this study of cytotoxic activity test (in vitro bioassay on human tumor cell lines) was conducted and determined by the Bioassay-Cell Culture Laboratory, National Research Centre, Cairo Egypt.

Antioxidant activity

The antioxidant activity of essential oil obtained by HD were evaluated using DPPH (2.2-diphenyl-1-picrylhydrazyl) test as per the method of (reference showed mentioned). (final concentration 1–300 mg/mL). Then we take 0.5 mL from each one and mix it with 1 mL of diphenyl-picrylhydrazine (DPPH 0.2 mM). Leave the mixture at room temperature for 30 min in the dark, then we thoroughly mix the latter (each sample). First, 0.5 mL of different EOs concentration were mixed with 1 mL of methanolic or ethanolic-DPPH˙ solution (0.2 mM), thereafter, the mixter was vortexed then allowed to stand in the dark at room temperature for half an hour. the absorbance was measured at 517 nm against a negative blank using UV–vis spectrophotometer (UV-1800, Shimadzu, Japan). Finally, inhibition percentage was calculated as follows:

$$\text{I}\%=\left(\left({\text{A}}_0-{\text{A}}_1\right)/{\text{A}}_0\right)\times 100$$ 2

where A₀ and A₁ were the absorbance of DPPH solution and the sample respectively. The results were represented as IC50 values which were calculated from a plotted graph of percentage of inhibition against the concentrations of the samples (mg/mL) (Table 2).

Table 2.

DPPH radical scavenging activity of essential oil Mentha citrata

Concentration (mg/mL)	1	10	50	150	300	IC50 (mg/mL)
I (%)	19.62 ± 1.49	31.37 ± 0.49	39.1 ± 0.92	55.97 ± 2.62	69.52 ± 0.21	114.58

Results and discussion

Chemical composition

Table 1 summarized the chemical compositions of Southern Algeria Mentha citrata essential oils, the yield of EOs was 1.8, 1.55 and 0.88% (v/w) for Hydrodistillation (HD), steam distillation (SD) and also by microwave-assisted distillation (MAD), respectively. The results showed that 39 components were identified, and it was noticed that linalool [38.28 (HD), 31.74 (SD) and 34.69% (MAD)] and linalyl acetate [37.36 (HD), 46.24 (SD) and 35.75 (MAD)] were the major constituents followed by 1,8-cineole [4.28 (HD), 4.39 (SD) and 4.38% (MAD)], and α-terpineol [4.7 (HD), 1.81 (SD) and 1.85% (MAD)]. The EOs were found to be rich in oxygenated monoterpenes [48.46 (HD), 38.92 (SD) and 47.54% (MAD)] followed by sesquiterpenes hydrocarbons [4.57 (HD), 5.99 (SD) and 5.30% (MAD)]. It is worthy to mention that the (EOs) obtained by microwave-assisted distillation showed the presence of the oxygenated hydrocarbon; methyl chavicol which might be the cause of the superior odour. These findings are in agreement with those found earlier on the same species where oxygenated monoterpenes, followed by monoterpenes hydrocarbons, sesquiterpenes hydrocarbons and oxygenated sesquiterpenes are the predominant secondary metabolites (Verma et al. 2016).

Table 1.

The chemical composition of essential oil Mentha citrata Ehrh.

Compoundsa	RIb	RIc	HD %	SD %	MAD %
α-Pinene	932	925	0.17	0.16	0.17
Sabinene	969	969	0.19	0.20	0.24
β-Pinene	974	972	0.55	0.49	0.56
1-Octen-3-ol	974	978	–	0.04	–
β-Myrcene	989	989	1.24	1.18	0.58
3-Octanol	988	995	–	0.11	–
α-Terpinene	1014	1015	0.05	0.06	0.05
p-Cymene	1020	1024	0.03	0.02	0.07
Limonene	1024	1028	0.57	0.49	0.40
1.8-Cineole	1026	1030	4.28	4.39	4.38
(Z)-β-Ocimene	1032	1037	1.36	1.22	0.64
(E)-β-Ocimene	1044	1048	0.90	0.77	0.51
γ-Terpinene	1054	1058	0.51	0.54	0.30
Terpinolene	1086	1089	0.29	0.25	0.18
Linalool	1095	1103	38.28	31.74	34.69
2-Methyl butyl isovalerate	1103	1108	0.38	0.41	–
1-Octen-3-yl acetate	1110	1112	0.19	0.21	–
3-Octanol acetate	1120	1124	0.12	0.12	0.12
δ-Terpineol	1162	1169	0.11	0.15	0.23
α-Terpineol	1186	1192	4.70	1.81	1.85
Methyl chavicol	1195	1195	–	–	5.94
Nerol	1227	1230	0.40	0.09	0.33
Linalyl acetate	1254	1259	37.36	46.25	35.75
Lavandulyl acetate	1288	1293	0.80	0.89	0.96
Neryl acetat	1359	1365	2.04	0.66	0.43
Geranyl acetate	1379	1369	–	–	0.91
α-Duprezianene	1387	1391	0.19	0.11	–
β-Elemene	1389	1394	0.11	0.12	–
α-Gurjunene	1409	1414	0.08	0.11	–
(E)-Caryophyllene	1417	1425	2.10	2.88	1.71
(E)-β-Farnesene	1454	1458	0.17	0.20	0.07
β-Santalene	1457	1460	0.20	0.31	–
Germacrene D	1480	1489	0.88	1.40	1.21
δ-Cadinene	1522	1529	–	0.13	0.08
Elemol	1548	1556	0.84	0.73	2.23
Guaiol	1600	1600	0.28	0.35	–
γ-Eudesmol	1630	1641	0.21	0.26	0.16
Lyral	1665	1661	0.17	0.22	–
Bulnesol	1670	1664	0.24	0.38	–
Total identified	–	–	99.99	99.45	94.75
Yield of essential oil (v/w) %	–	–	1.80	1.55	0.88
Monoterpene hydrocarbons	–	–	5.86	5.38	3.70
Oxygenated monoterpenes	–	–	48.46	38.92	47.54
Sesquiterpene hydrocarbons	–	–	4.57	5.99	5.30
Oxygenated sesquiterpene	–	–	0.73	0.99	0.16
Other compounds	–	–	40.37	48.17	38.05

RI retention index

^aCompounds are identified based on the comparison of their mass fragmentation pattern and their retention indexes and the compounds were listed in the order of elution from Rtx-5MS gas chromatography column

^bRetention index from literature (Adams 2007)

^cExperimentally determined retention index (RI)

– not found

In addition, the richness of the (SD-EOs) with linalyl acetate 46.25%, and linalool 31.74% is in a good agreement with that found by (Lawrence 2007) who distinguished two chemotypes of Mentha citrata Ehrh. The first chemotype is rich in linalyl acetate and the other was rather rich in linalool. Similar the results were reported by Malizia et al. (1996). However small variation could be noticed with those published in Cuba, which might be attributed to the collection time, the distillation mode and the geographic and climatic factors (Pino et al. 1999).

The biological assessment

DPPH radical scavenging activity test

The EOs produced by HD exhibited a Weak radical scavenging effect with an IC50 of 114.58 mg/mL (Table 2) when comparing with IC50 values for standard compounds BHT, α-Tocopherol and BHA 180, 15, 22.5 µg/mL respectively (Table 3). The results are in agreement with data previously reported about other Mentha sp. (Mimica-Dukic et al. 2004; Mimica-Dukic and Bozin 2008). The essential oil of the Mentha citrata antioxidant activity may be attributed to the high percentage of the oxygenated mono- and sesquiterpenes (El-Ahmady et al. 2013). To our knowledge, there are no issued studies focusing on antioxidant activity of Mentha citrata essential oil.

Table 3.

DPPH radical scavenging activity of standard solution

	Concentration (µg/mL)	5	10	25	50	100	200	500	IC50 (µg/mL)
BHT		0.5 ± 0.001	1 ± 0.003	3.4 ± 0.002	7.8 ± 0.008	18.5 ± 0.002	51.3 ± 0.004	67 ± 0.007	180
α-Tocopherol (Vitamin E)	I(%)	15.4 ± 0.016	31 ± 0.002	79.5 ± 0.008	89.9 ± 0.0014	93.1 ± 0.0014	91.1 ± 0.008	100	15
BHA		13.9 ± 0.28	32 ± 2.98	53 ± 0.92	78.1 ± 1.42	83.7 ± 1.49	84.4 ± 0.28	100	22.5

Cytotoxic activity test

Cytotoxic activity of the essential oil Mentha citrata was tested against both HCT116 [colon cancer (ATCC^® CCL-247™)] and HepG2 (Human hepatocellular carcinoma [ATCC^® HB-8065™)]. The results (Table 4) revealed that the essential oil possesses cytotoxic activity on HCT116 with IC50 and IC90 values of 80.6 and 119.1 μg/mL, respectively. The inhibition percentage of this essential oil against HCT116 colon cancer cell line and is 71.83% for 100 μg/mL.

Table 4.

Cytotoxic activity of Mentha citrata essential oil against human cell lines (% of inhibition cells ± SEM)

Sample code	100 (μg/mL)	50 (μg/mL)	25 (μg/mL)	12.5 (μg/mL)	LC50 (μg/mL)	LC90 (μg/mL)	Doxorubicin LC50 (μg/mL)	DMSO at 100 ppm (%)	Negative control (%)
HCT116	71.83 ± 4.14	18.77 ± 1.55	4.83 ± 1.52	0	80.6	119.1	37.6	1	0
HePG2	19.3	0	0	0	0	0	21.6	1	0

Every value represents the mean percentage of inhibition cells of three replicates ± SEM (mean of standard error)

On the other hand; the oil was inactive against the HepG2 cell line within the tested range (19.3% for 100 μg/mL). These results appear with the agreement of previously reported data on other same species (Bardaweel et al. 2018; Nedel et al. 2012). All results could be explained the results obtained earlier that indicate the promising cytotoxic activity of Mentha citrata leaf extract on the similar cell line.

Comparison of our data with litterateur

Data of Mentha citrata Ehrh., OEs (Algeria), were compared with the results reported in the literature (India, Cuba, Croatia and Argentina data) (Verma et al. 2016; Pino et al. 1999; Mastelić et al. 2000; Malizia et al. 1996), is given in Table 5.

Table 5.

Comparison of our data with other references

Compounds	Our data (Algeria)			Data of India (Verma et al. 2016)		Data of Cuba (Pino et al. 1999)	Data of Croatia (Mastelić et al. 2000)	Data of Argentina (Malizia et al. 1996)
	HD %	SD %	MAD %	SD %	HD %	HD %	HD %	SD %
β-Myrcene	1.24	1.18	0.80	1.90	1.40	1.70	8.10	0.50
1.8-Cineole	4.28	4.39	4.80	1.10	1.80	0.20	12.51	0.72
(Z)-β-Ocimene	1.36	1.22	0.64	1.20	1.30	0.90	2.02	–
(E)-β-Ocimene	0.90	0.77	0.51	0.70	0.30	0.80	5.27	–
Linalool	38.28	31.74	34.69	48.10	40.40	28.60	13.68	23.8
α-Terpineol	4.70	1.81	1.85	3.20	3.70	0.60	7.38	0.09
Methyl chavicol	–	–	5.94	–	–	–	–	–
Nerol	0.40	0.09	0.33	–	–	1.90	0.98	0.10
Geraniol	–	–	–	–	–	5.40	3.18	0.12
Linalyl acetate	37.36	46.25	35.75	27.80	34.70	15.90	21.46	60.9
Neryl acetat	2.04	0.66	0.43	1.00	0.30	3.30	3.56	0.70
(E)-Caryophyllene	2.10	2.88	1.71	–	–	–	–	–
β -Caryophyllene	–	–	–	2.70	2.60	4.10	–	–
Germacrene D	0.88	1.4	1.21				–	–
Elemol	0.84	0.73	2.23	0.20	0.60	7.40	–	–
viridiflorol	–	–	–	–	–	5.00	–	–
γ-Eudesmol	0.21	0.26	0.16	–	–	–	0.47	–
10-epi-γ-Eudesmol	–	–	–	–	–	1.10	–	–
Yield of EOs
(v/w) %	1.80	1.55	0.88	1.02	1.48	2.10	–	–
(w/w) %							1.56	1.10

HD hydro-distillation, SD steam distillation, MAD microwave-assisted distillation, EOs essential oil

The composition determined from Essential oils of Algeria Mentha citrata Ehrh., was dominated by linalool, linalyl acetate and 1.8-Cineole. This composition was qualitatively the same as the oils from India (Verma et al. 2016). However, β-Caryophyllene was a presente component in the samples from India, but absente in Algeria Mentha EOs.

The linalool and linalyl acetate are used as a sedative and inflammatory medicine and are beneficial to mental and physical health as well as its widespread use in cosmetic and sterilization industries (Jentzsch et al. 2015; Ghoreishi et al. 2012). In all data shown in Table 5, the linolool and linalyl acetate are the predominant components of Mentha citrata Ehrh., OEs. However, there is a disparity in its ratio from one research to on other, also it was shown that the yield was close in all the studied research. These differences could probably be Iink to plant nutrition, climate, water and soil condition.

Conclusion

The present work has allowed us to point out the chemical composition of the essential oil of Mentha citrata Ehrh., three methods of extraction; namely hydrodistillation, steam distillation and microwave distillation using GC–FID and GC–MS analyses. The present study shows that the essential oil of Mentha citrata is rich in linalool and linalyl acetate, and also many other compounds were identified and characterized, in addition the essential oil of Mentha citrata has shown a significant antioxidant effect as well as a notable anti-cancer activity with respect to the HCT116 colon cancer cell line.