Chemical characterization and biological activity of apolar fraction from Melissa officinalis L. leaves

Haydelba D’Armas1*; Carmita Jaramillo-Jaramillo2; Ligia Llovera3; Liz Cubillán3; Mayra D’Armas4

1. Universidad Estatal de Milagro, Milagro 091050, Provincia de Guayas, Ecuador. Universidad de Oriente, Cumaná, Sucre 6101, Venezuela., Universidad Estatal de Milagro, Universidad Estatal de Milagro,

<city>Milagro</city>
<postal-code>091050</postal-code>
, Ecuador , 2. Universidad Técnica de Machala, Machala, Provincia del Oro, Ecuador., Universidad Técnica de Machala, Universidad Técnica de Machala,
<city>Machala</city>
, Ecuador ,
3. Instituto Venezolano de Investigación Científica, Centro de Química, Miranda 1204, Venezuela., Instituto Venezolano de Investigación Científica, Centro de Química,
<state>Miranda</state>
<postal-code>1204</postal-code>
, Venezuela ,
4. Universidad Estatal de Milagro, Milagro 091050, Provincia de Guayas, Ecuador., Universidad Estatal de Milagro, Universidad Estatal de Milagro,
<city>Milagro</city>
<postal-code>091050</postal-code>
, Ecuador

Correspondence: *. Corresponding author: Haydelba D’Armas, Universidad Estatal de Milagro, Milagro, Provincia de Guayas, Ecuador. 00-(593)-967682942. http://www.unemi.edu.ec/ E-mail: E-mail:


Abstract

Medicinal plants have been traditionally used in the treatment of many diseases and their pharmacological and therapeutic properties have been attributed to various chemical constituents isolated from their crude extracts. Melissa officinalis L. (Lamiaceae), or lemon balm, is widely used as herbal tea to treat or relieve nervous sleep disturbance and functional gastrointestinal disorders. For studying the plant, the hexane extract of the leaves of M. officinalis collected in the city of Machala, El Oro Province, Ecuador, was analyzed. The antibacterial, antifungal and toxic properties of the crude extract were evaluated, revealing a strong antibacterial activity against bacteria S. aureus and P. aeruginosa, as well as a significant lethality (LC50 of 78.82 μg/ml) against Artemia salina. B and C fractions obtained using column chromatography eluted with hexane: dichloromethane in different proportions exhibited a significant lethality (LC50 between 29-32 μg/ml). All the chromatographic fractions were bioactive against S. aureus bacteria, with the exception of E. In addition, fraction B showed moderate antibacterial activity against B. cereus strain and strong antifungal activity against the strains C. albicans and Mucor sp. Through analysis of fraction B by GC/MS some compounds were identified: 7,9- di-tertbutyl-1- oxaspiro [4,5] deca-6,9-diene-2,8-dione, docosanol as majority compound, a mixture of cholestane type sterols and fatty acid ester of a sterol.

Received: 2017 October 27; Accepted: 2018 March 14

revbio. 2020 May 31; 5: e385
doi: 10.15741/revbio.05.e385

Keywords: Keywords: Bioactivity, campesterol, GC/MS, Melissa officinalis, phytochemistry.

Introduction

Medicinal plants have traditionally been used in the treatment of several human diseases and their pharmacological and therapeutic properties have been attributed to different chemical constituents isolated from their crude extracts. Particularly important, chemical components with antioxidant activity are found in high concentrations in plants and may be responsible for their preventive effects in various degenerative diseases, such as cancer, neurological and cardiovascular diseases (Picada et al., 2009).

Melissa officinalis L. (Lamiaceae) or lemon balm is a perennial plant related to the mint family and native from Europe, an edible herb for the Mediterranean region. Its use as a medicinal herb dates back to the Middle Ages, is widely used as herbal tea, and is very well known for its ability to reduce stress and anxiety, stimulate sleep, improve appetite, relieve pain or antispasmodic properties and the discomfort associated with digestion (functional gastrointestinal disorders). Some studies that have demonstrated antitumor and neuroprotective effects of M. officinalis are of great importance (De Sousa, 2004; Marongiu, 2004).

The essential oil of M. officinalis is recommended for its demonstrated antimicrobial activity (Mimica-Dukic et al., 2004) and the aqueous extracts of this species due to their antiviral and antioxidant properties (Hussain et al., 2011). Additionally, it was reported that M. officinalis contains substances that inhibit protein biosynthesis in cancer cells. These biological activities have been attributed to the essential oil (where terpenoids are the major constituents , such as citral or mixture of neral and geranial isomers, citronellal, geraniol, nerol and linalool), tannins (WHO, 2002), flavonoids (Patora & Klimek, 2002), anthocyanins (Hossain et al., 2009) and hydroxycinnamic phenolic acids (Caniova & Brandsteterova, 2001).

Qualitative variations of its essential oil have been demonstrated by genetic factors and qualitative changes due to environmental conditions, day length and soil composition. Studies on this essential oil have been extensive, but information on its nonvolatile components is scarce (Herodez et al., 2003). Mencherini et al., (2007) isolated and characterized five new triterpenes ursin and oleanene type and a new ursin glycoside from the polar extract of the leaves and stems of M. officinalis collected in Italy; as well as the known compounds quadranoside III, luteolin and rosmarinic and salviic acids A. Furthermore, they demonstrated the antimicrobial effect of the extracts and compounds. Several studies suggest that lemon balm is beneficial for a wide variety of human disorders such as cancer, HIV-1, Alzheimer’s disease, attention deficit hyperactivity disorder, indigestion, gas, insomnia, and hyperthyroidism (Geuenich et al., 2008; Kennedy et al., 2006; Muller & Klement, 2006).

Materials and methods

Sampling

The specimens of the Melissa officinalis plant were collected in the city of Machala, Del Oro Province, southwest region of Ecuador, between 3° 16› 0” S and 79° 59› 0» W, during April 2014. The sample was taxonomically identified by botanist Jesus Inca from the National Herbarium of Quito and filed in the Pilot Plant of Pharmacy of the Universidad Técnica de Machala (Ecuador) with the registration PPFMO016.

Extraction

The apolar extract was obtained in the following way: The leaves were washed with distilled water, dried in the shade at room temperature in the laboratory, and then in the oven at 40o for 24 hours. Subsequently, the grinding of the dried leaves was carried out in an electric mill to be macerated with hexane for 48 hours. After filtering the solvent, the residue was again reextracted with hexane to ensure complete extraction of the secondary metabolites. The total filtering of the two macerations was evaporated and concentrated under reduced pressure in a rota-evaporator to obtain the hexane extract. Afterward, bioactivity and chromatographic separation tests were carried out.

Chromatographic fractionation

The compounds separation of the hexane extract was carried out by gravity Column Chromatography (CC) with silica gel 35-70 mesh (0.2-0.5 mm), in a column of 2.5 cm in diameter and 70 cm in length. 3.8960 g of the extract and 116.8800 g of silica were used in a ratio sample: silica 1:30. Hexane, mixtures of increasing polarity of hexane-dichloromethane, dichloromethane-acetone, acetone-methanol, and 100 % methanol were used as eluents. Thin Layer Chromatographic (TLC) on glass chromatographic plates of 20 x 20 cm2 was performed to each one of the obtained eluates. The plates were coated with 60 mesh silica-gel type GF, with a thickness of 0.5 mm, using mixtures of hexane-acetone in proportions of 4:1, 1:1 and 100 % acetone, obtaining six fractions (A-F) by comparing their Rf. Ultraviolet (UV) light of short wavelength (100-280 nm) and a solution of 5 % ammonium molybdate in 10% H2SO4 were used as developers.

Structural characterization of fraction B

Gas chromatography and mass spectrums by electronic impact (70eV) were performed in the Mass Spectrometry Laboratory of the Instituto Venezolano de Investigación Científica (IVIC), which were registered in VARIAN GC/MS equipment Saturn 2000 model, with an electronic impact ionization source and an ion trap detector. A column of CP-SIL-8CB-MS of 30 m x 0.25 I.D and helium (He) was used as the entraining gas. The injector temperature was 280°C, the initial temperature of the oven was 100° C with a speed of heating of 5°C per minute, until reaching a final temperature of 295°C. Subsequently, the identification of the compounds with 95% confidence was made by computerized comparison with the WILEY and NIST libraries.

Biological assays

The biological activity of M. officinalis extract and fractions of the leaves was evaluated by using the following bioassays:

Antibacterial activity. The presence of antibacterial compounds was detected using strains of certified bacteria: Staphylococcus aureus and Bacillus cereus (Gram positive); Escherichia coli and Pseudomonas aeruginosa (Gram negative), following the agar diffusion technique or antibiogram method (Bauer et al., 1966). The antibiosis was verified by measuring the diameter (mm) of the inhibition zones around the 5 mm diameter discs. The diameters of the inhibition zones were evaluated, taking the methodology of crossings and the criteria exposed for extracts by some researchers of natural products area as a reference (Monks et al., 2002, Ríos et al., 2009).

Antifungal activity. Likewise, this biological activity was measured by inoculating with the strains of pathogenic fungi Candida albicans, Penicillum sp and Mucor sp, following the procedure described by Madubunyi (1995). The appearance of inhibition zones around the disc (5 mm) was indicative of the fungal activity of the extract or fraction, which were measured considering the diameter in mm.

Lethal activity. The degree of lethality or toxicity of the extracts and chromatographic fractions obtained was evaluated in larvae of Artemia salina, following the technique described by Meyer et al. (1982). Three replicas and one control with the same number of replicas were made for each concentration. The quantification of the nauplii mortality was carried out 24 hours after having assembled the bioassay. The results obtained of nauplii mortality were analyzed to calculate the median lethal concentration, through the application of LC50 V2.5 software designed by Stephan (1977) for this purpose, which includes computerized statistical analysis methods (Probit, Binomial, Logit and Moving Average) with 95 % confidence limits.

Results and Discussion

The bioassays allowed to evaluate the effects of the hexane crude extract and some fractions obtained from chromatographic separation of M. officinalis extract on the microorganisms tested. The results obtained provided information about the bioactive fractions, in which the secondary metabolites causing the observed biological activity were found, and allowed to select the most bioactive fractions to be chemically characterized.

Antimicrobial activity

The hexane crude extract showed a strong antibacterial effect against strains S. aureus (12 mm) and P. aeruginosa (14 mm) in the bioassay conducted against various microbial strains (fungi and bacteria); conversely, the bacteria E. coli and B. cereus did not show sensitivity to the hexane extract. Fractions B and C showed high antibacterial activity against S. aureus (13 and 11 mm respectively), while fraction B exhibited a moderate antibiosis against B. cereus (9 mm, antagonistic effect). Additionally, P. aeruginosa bacteria showed a slight sensitivity (7 mm) to fraction C; however, it showed a very strong bacterial sensitivity (15 mm) when being tested with fraction E (Table 1).

Table 1.

Antimicrobial activity shown by the hexane extract and fractions of M. officinalis leaves against the strains used.


Microorganism ECH FA FB FC FD FE
Bacteria:
Staphylococcus aureus +++ ++ +++ +++ +++ -
Escherichia coli - - - - - -
Pseudomonas aeruginosa +++ - - + - > +++
Bacillus cereus - - ++ - - -
Fungus:
Mucor sp - - +++ - - -
Penicillum sp - - - - - -
Candida albicans - - +++ - - -

Diameters of the inhibition zones: (-) no antimicrobial activity (diameter <6 mm); (+) mild antimicrobial activity (diameter between 6-8 mm); (++) moderate antimicrobial activity (diameter between 8-10 mm); (+++) high or strong antimicrobial activity (diameter between 10-14 mm). ECH: crude extract in hexane. F: chromatographic fractions.

None of the fractions tested showed bactericidal action against E. coli bacteria; however, all fractions except fraction E, exhibited moderate to strong antibacterial activity against S aureus. In addition, it was observed that both the hexane extract and the fractions did not present a fungicidal effect against some of the fungi used, with the exception of fraction B that exhibited a high antifungal activity against Mucor sp. (12 mm) and C. albicans (11 mm) fungal strains (Table 1).

These results indicate the existence of secondary metabolites with antibacterial and antifungal potential in the apolar extract of M. officinalis, and the biological activity observed may be due to the high concentration of those components in the fraction, as well as the presence of more than one bioactive compound in the extract. The apolar extract of M. officinalis showed an antibacterial effect against the Gram positive S aureus and P. aeruginosa strains, and antifungal activity against Mucor sp and C. albicans; a similar result was obtained in a study carried out with polar extracts of stems and leaves of this plant, where these exhibited a bacteriostatic effect (zone of diffuse inhibition) against S. aureus and S. epidermitis bacteria, and B. spizizenii fungus (Mencherini et al., 2007).

Hancianu et al. (2008) reported on the cytotoxic activity and strong antibacterial (against Gram-positive strains) and antifungal activity against Candida albicans fungus, shown by M. officinalis; as well as Mimica-Dukic et al. (2004) reported the high antimicrobial activity of the essential oil of this species. These results are similar to those obtained in this research, in relation to the strong bacterial sensitivity exhibited by S. aureus (Gram-positive) when being tested with the hexane extract and chromatographic fractions. However, the hexane extract showed no antifungal activity and fraction B showed an antagonistic effect when presenting bioactivity against C. albicans, in accordance with that reported by Hancianu et al (2008) in the literature.

Lethal activity

The in vivo lethality of Artemia salina can be used as a mean for discrimination and fractionation in the discovery of new bioactive natural products; consequently, it is commonly used for the evaluation of natural products and pharmacological activity (Pino & Lazo, 2010). The presence of anticancer and antitumor compounds isolated from marine organisms has been reported, which previously showed toxic activity in the A. salina test. This is a simple bioassay that in some way correlates with cytotoxicity (Pino & Lazo, 2010).

The toxicity bioassay showed LC50 values of 78.82 μg/ml for the hexane extract; 31.16; 29.06, and 731.08 μg/ml for B, C, and D fractions (obtained by chromatographic separation), respectively (Table 2). The results obtained showed a very significant lethal activity for the hexane extract and B and C fractions, which shows a significant toxicity (Meyer et al., 1982) for this species and leads to presume the existence of compounds or secondary metabolites with a high cytotoxic activity. Probably, the observed mortality is due to the fact that the extracts increase the rate of oxygen consumption; as a result, the physiological processes act to compensate for the toxic stress, when the animal is exposed to a toxic agent, causing alterations that can lead to death of the organism.

Table 2.

Lethal or toxic activity of the hexane extract and fractions of M. officinalis leaves against Artemia salina.


Extract or fraction Statistical method LC50 (μg/ml) (24 h)
ECH Binomial 78.82
FA - -
FB Binomial 31.16
FC Binomial 29.06
FD Logit 731.08
FE - -

TFN1(LC50): median lethal concentration; (-): high concentration (>1000 μg/ml); ECH: crude extract in hexane. F: chromatographic fractions.


According to reports in the literature, values of LC50 <1000 μg/ml are considered toxic and there is a good correlation with anti-cancer cell lines of type 9KB (human nasopharyngeal carcinoma) and 9SP (leukemia) when LC50 shows values less than or equal to 30 μg/ml, so that the extract or bioactive compounds that constitute a particular species can exhibit an important cytotoxic potential (Carballo et al., 2002); which makes M. officinalis a source of chemical compounds with possible antitumor activity.

This study provides interesting results, since investigations of acute toxicity against Artemia in the botanical parts of M. officinalis are almost nonexistent. Nonetheless, there is research on the chemical composition and bioactivity of the essential oil of M. officinalis leaves, where its significant cytotoxic activity is reported (Hancianu et al., 2008).

Characterization of fraction B

Fraction B was obtained as a beige solid in very little quantity, and was considered the most bioactive fraction. It was decided to analyze this fraction by GC/MS since it showed strong and moderate antibacterial activity against S. aureus and B. cereus, respectively, displayed strong antifungal activity against Mucor sp and C. albicans strains, exhibited a very significant LC50 of 31.16 μg/ml, and did not show a good separation of its constituents on TLC. The gas chromatography of this fraction (Figure 1) showed the presence of several compounds, the following being identified: 7,9-di-tert-butyl-1-oxaspiro [4,5] deca-6,9-diene-2,8-dione (I, RT= 12.43 min), docosanol (II, RT= 13.75 min) as the major compound, a mixture of sterols (III, RT= 22.81 and V, RT= 29. 77 min), and a fatty acid ester of a sterol (IV, RT= 28.03 min).


[Figure ID: f1] Figure 1.

Gas chromatogram of fraction B.


The mass spectrum of 7,9-di-tert-butyl-1-oxaspiro [4,5] deca-6,9-diene-2,8-dione (I, RT=12.43 min) displayed a molecular ion at m/z 276 [M+], according to the molecular formula C17H24O3, and the corresponding fragments to the most abundant ion peaks at m/z 261, 206, 175, 135, 109, 91, and 57 (base peak). This ketone has not been reported in species of the Lamiaceae family; this is the first report of this compound from a plant belonging to that family. There is no information in the literature about some type of pharmacological activity related to this compound; however, it was identified in the apolar extract of rice by GC/MS, in an investigation of allelopathic activity of this cereal (Murillo, 2006).

The mass spectrum of docosanol (II, RT= 13.75 min) exhibited a molecular ion at m/z 308 [M+], which corresponds to the molecular formula C22H46O and the fragments of the most abundant peaks at m/z 97 and 43 (base peak). This saturated fatty alcohol, used mainly as an anti-viral agent, is the active ingredient of topical creams used for the treatment of diseases caused by the herpes simplex virus and works by inhibiting the fusion of the human host cell with the viral envelope of the herpes virus, preventing replications. Docosanol was reported as one of the fatty acids of the Nicotiana tabacum L plant in a study conducted by Sacks et al. (2001), and, in addition, the allelopathic activity of this species was reported.

The mass spectrum of sterol 4,4,10,13,17-pentamethyl-17-(1,5-dimethylhexyl) -colest-8,14-dienyl (III, RT = 22.81 min) showed a molecular ion at m/z 468 [M+], corresponding to the molecular formula C32H52O2, and the peaks of the most abundant fragments were observed at m/z 393, 355, 295, 239, 159, 119 and 43 (base peak). The mass spectrum of the other compound identified as 4, 4,17-trimethyl cholest-8,14-dien-3-yl dodecanoate (IV, RT= 28.03) exhibited a molecular ion at m/z 608 [M+], which corresponds to the molecular formula C42H72O2, and the peaks of the most abundant fragments were observed at m/z 564, 489, 409, 393, 375 (base peak), 355, 269, 230, 207, 145 and 55. While that of 4, 4-dimethyl-cholesta-8, 14, 24-trien-3-ol (V, RT = 29.77 min), displayed a molecular ion at m/z 410 [M+] in accordance with the molecular formula C29H46O and the peaks of the most abundant mass fragments were observed at m/z 396, 382 (base peak), 355, 330, 304, 270, 228, 145 and 41.

The presence of 4,4,17-trimethyl cholest-8,14-dien-3-yl dodecanoate in M. officinalis leaves is of medicinal importance since it has been shown that the fatty acids esters of plant sterols decrease the levels of LDL-cholesterol and triacylglycerols in the blood (Arnqvist, 2007); as well as the efficacy of phytosterols as medium reduction agents in low density lipoproteins was reported (Eldin & Moazami, 2006).

Although no specific biological activities of natural sterols or phytosterols are known, besides the biological functions mentioned above, it has been shown that they also have immune-modulatory, antiinflammatory, antitumor, bactericidal and fungicidal properties (Muñoz et al., 2011). Research has shown that aromatic plants (including those of the Lamiaceae family) respond differently to the environment, biosynthesizing chemical families with diverse biological activities in different proportions for their ecological interaction (Ordaz et al., 2011).

Conclusions

The apolar extract of M. officinalis L. exhibited a strong antibacterial activity against strains of S. aureus and P. aeruginosa, as well as a significant lethality (LC50 of 78.82 μg/ml) against Artemia salina. Meanwhile, the chromatographic fraction B showed strong and moderate antibacterial activity against S. aureus and B. cereus, respectively, strong antifungal activity against Mucor sp and C. albicans fungi, and a very significant toxic activity (LC50 = 31.16 μg/ml).

The leaves of M. officinalis L. cultivated in Ecuador are a promising source of bioactive compounds with pharmacological activity (antimicrobial and cytotoxic or antitumor).


fn1Cite this paper: Haydelba D’Armas, Carmita Jaramillo-Jaramillo, Ligia Llovera, Liz Cubillán, Mayra D’Armas. (2018). Chemical characterization and biological activity of apolar fraction from Melissa officinalis L. leaves. Revista Bio Ciencias 5, e385. doi: https://doi.org/10.15741/revbio.05.e385

Acknowledgment

The authors wish to express their gratitude to the Prometheus Project of Secretaría Nacional de Educación Superior, Ciencia y Tecnología of the Republic of Ecuador (SENESCYT) for their research funding and Instituto Venezolano de Investigaciones Científicas (IVIC) for the gas chromatography analysis with mass detector.

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