IDENTIFICATION OF BIOACTIVE COMPOUNDS OF ALCHEMILLA CAUCASICA USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY

IDENTIFICATION OF BIOACTIVE COMPOUNDS OF ALCHEMILLA CAUCASICA USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY

Esen Sezen Karaoglan ^{* 1} and Bilal Yilmaz ²

Department of Pharmaceutical Botany ¹, Department of Analytical Chemistry ², Faculty of Pharmacy, Ataturk University, 25240, Erzurum, Turkey.

ABSTRACT: Alchemilla species (Rosaceae) are locally known as "aslanpençesi" in Turkey. Alchemilla plants (lady's mantle) are used in traditional medicine for different indications. In this study, the methanol extract of Alchemilla caucasica was analyzed using gas chromatography-Mass Spectrometry, while the mass spectra of the compounds found in the extract were matched with the National Institute of Standards and Technology Library. The study revealed the presence of fourteen phyto-components. The most abundant compounds in the methanol extract were 13-docosenamide (Z)-,  5- octadecenoic acid 2,3-dihydroxy-propyl ester and hexadecanoic acid 2-hydroxy-1-(hydroxyl-methyl) ethyl ester which had concentrations of 59.77%, 15.34%, and 7.59% respectively. Results indicated that polarity of the solvents used in extraction influenced the relative abundance and type of compounds extracted. Various compounds identified are known to have varying bioactivities such as being anticancer and antioxidant. Therefore, the presence of these compounds in Alchemilla caucasica could be useful in the preparation of functional foods and neutraceuticals in preventing and treating debilitating and chronic human diseases.

Alchemilla caucasica, Rosaceae, Bioactive compounds, Gas chromatography-mass spectrometry

INTRODUCTION: Plants play a major role in primary health care as therapeutic remedies in developing countries ¹. They serve as a source of medicine and an important component in health care system ². This stems from the fact that aside from providing vitamins and minerals, plants contain phytochemicals which are secondary metabolites. These phytochemicals have various bioactivities such as being antioxidants, anti-nutritional or cytotoxic.

Antioxidants are substances used by the body to protect itself from damage caused by free radicals, whose accumulation has been linked to several diseases such as heart diseases ³, liver diseases and cancers ⁴.

The anti-nutritional activity of a bioactive compound is based on its ability to inhibit the absorption and utilization of nutrients. Currently, there is upsurge interest in the knowledge of bioactive compounds present in plants and hence their beneficial potentials as nutraceuticals and functional foods; hence, having positive health-promoting effects. Herbal medicine, as a major part of traditional medicine, has been used in medical practice since antiquity and is a common element of Ayurvedic, homeopathic, and naturopathic medicine.

World health organization notes that 74% of the plant-derived medicines are used in modern medicine, in a way that their modern application directly correlates with their traditional use as herbal medicines by native cultures ^{5, 6}.

Alchemilla species belonging to Rosaceae family are locally known as "aslanpençesi" in Turkey. These species include fatty acids, esters, aldehydes, terpenes, hydrocarbons, mainly phenolic compounds, including flavonoids and tannins ^{7 - 10}. Alchemilla plants (lady's mantle) are used in traditional medicine for different indications which are minimizing the symptoms of sore throat, promoting wound healing, stopping bleeding, gynecological diseases, alleviating nausea and vomiting ¹¹. Different studies showed that the phenolic compounds (tannins, flavonoids, etc.) present in the plant are responsible for the pharmacological activity of Lady’s mantle ^12-14. Alchemilla caucasica is a member of this genus. This plant has short, erect flowering stems, wide and reniform leaves ¹⁵ Fig. 1.

FIG. 1: ALCHEMILLA CAUCASICA PLANT

Knowledge of the chemical constituents of Alchemilla caucasica is desirable not only for the discovery of therapeutic agents but also because such information may be of great value in disclosing new sources of economic phyto-compounds for the synthesis of complex chemical substances and for discovering the actual significance of folkloric remedies. Hence, a thorough validation of the herbal drugs has emerged as a new branch of science emphasizing and prioritizing the standardization of the natural drugs and products because several of the phytochemical have the complementary and over-lapping mechanism of action. Mass spectrometry coupled with chromatographic separations such as gas chromatography (GC-MS) is normally used for direct analysis of components existing in traditional medicines and medicinal plants. In previous studies, it was reported that the seeds contained mainly palmitic, stearic, oleic and linoleic fatty acids ¹⁶. In this study, the fourteen chemical content of the aerial part of the plant was tried to be determined by GS-MS method.

MATERIALS AND METHODS:

Chemicals and Reagents: Glycolaldehyde dimethyl acetal, furfural, phthalic acid dibutyl ester, n-hexadecanoic acid, linoleic acid, linolenic acid, octodecanoic acid, hexadecanoic acid 2-hydroxy- 1- (hydroxymethyl) ethyl ester, dicyclohexyl phthalate, octadecanoic acid, 2,3-dihydroxypropyl ester, 13- docosenamide (Z)-, octadecanamide, 24, 25 dihydroxycholecalciferol and β-sitosterol were purchased from Sigma-Aldrich (St. Louis, MO, USA).

GC-MS System: Chromatographic analysis were carried out on an Agilent 7820A gas chromatography system equipped with 5977 series mass selective detector, 7673 series autosampler and chemstation (Agilent Technologies, Palo Alto, CA). HP-5 MS column with 0.25 μm film thickness (30 m × 0.25 mm I.D., USA) was used for separation. The temperatures of the inlet, transfer line, and detector were 250, 250 and 300 °C, respectively.

GC-MS Conditions: Different temperature programs were investigated for GC-MS method. The end of this investigation, the temperature program of the GC/MS was as follows: initial temperature was 50 °C, held for 1 min, increased to 100 °C at a rate of 20 °C/min held for 1 min, increased to 180 °C at a rate of 10 °C/min held for 1 min, increased to 220 °C at a rate of 5 °C/min held for 5 min, and finally to 300 °C at a rate of 10°C/min and held for 5.5 min.

Extraction Procedure: Alchemilla caucasica were collected from Konaklı Mountain (Erzurum province, Turkey) in June 2016. A voucher specimen was deposited in the Herbarium of Ataturk University, Faculty of Pharmacy (AUEF1023). The air-dried and powdered aerial parts of the plant were extracted three times with methanol in the mantle heater at 40 °C (3 × 1 L). After filtration, 1 μl of this solution was employed for GC-MS analysis.

Identification of Components: Identification was based on the molecular structure, molecular mass, and calculated fragments. Interpretation on mass spectrum GC-MS was conducted using the database on National Institute Standard and Technology having more than 62,000 patterns. The name, molecular weight, and structure of the components of the test materials were ascertained. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. The spectrum of the unknown component was compared with the spectrum of the component stored in the National Institute of Standards and Technology Library Version (2005), Software, Turbomass 5.2.

RESULTS AND DISCUSSIONS:

Method Development and Optimization: The method development for the assay of phyto-components was based on their chemical properties. In this study, the capillary column coated with 5% phenyl, 95% dimethylpolysiloxane is a good choice for separation of these analytes since they elute as symmetrical peaks at a wide range of concentrations. Different temperature programs were investigated for the GC oven. The end of this investigation, the best temperature program was selected for good separation. The temperature programs of the GC oven were as follows: initial temperature 50 ºC, held for 1 min, increased to 100 ºC at a rate of 20 ºC/min held for 1 min, increased to 180 ºC at a rate of 10 ºC/min held for 1 min, increased to 220 ºC at a rate of 5 ºC/min held for 5 min, and finally to 300 ºC at a rate of 10 ºC/min and held for 5.5 min. The splitless injection mode was chosen. The injector volume was 1 µl in splitless mode, and the carrier gas was helium at a flow rate of 1 ml/min.

GC-MS Analysis: The more precise information in qualitative analysis can be obtained by GC-MS. For quantitative determination, gas chromatography with flame ionization detector (GC-FID) and GC-MS are preferred. GC-MS is one of the best techniques to identify the constituents of volatile matter, long chain, branched chain hydrocarbons, alcohols, acids, esthers etc.

The GC-MS analysis of Alchemilla caucasica extracts revealed the presence of fourteen compounds (phytochemical constituents) that could contribute to the medicinal quality of the plant. The identification of the phytochemical compounds was confirmed based on the peak area, retention time and molecular formula. The major phytochemical constituent’s present in methanolic extract of Alchemilla caucasica is presented as compound chromatogram is in Fig. 2.

FIG. 2: GC-MS CHROMATOGRAM OF METHANOLIC EXTRACT OF ALCHEMILLA CAUCASICA

GC-MS analysis revealed the presence of fourteen compounds in methanol extracts of Alchemilla caucasica. Peak number, retention time, compound name, molecular weight, and molecular formula are stated in Table 1. It shows compounds identified in methanol extract of Alchemilla caucasica which represented the hydrophobic and hydrophilic bioactive fraction. Three compounds were identified as the major bioactive compounds. They are 13-docosenamide (Z)- and it had a relative abundance of 59.77% and eluted as represented at peak 11, octadecenoic acid 2, 3-dihydroxy-propyl ester ester had a relative abundance of 15.34% and eluted as represented at peak 10 and hexadecanoic acid 2-hydroxy-1-(hydroxymethyl)ethyl ester had a relative abundance of 7.59% and eluted as represented at peak 8. Major fatty acids have biological activities such as being anti-inflammatory, anti-androgenic, cancer preventing, dermatigenic, hypocholesterolemic, anaemiagenic, insectifuge as well as being a 5-alpha-reductase inhibitor ¹⁷. n- hexadecanoic acid has been reported to show selective toxicity to human leukemic cells as well as antitumor activity and is suggested to be a lead compound in anti-cancer drugs ¹⁸. Other fatty acids and fatty acid esters identified were linoleic acid (0.69%), linolenic acid (1.96%), octadecanoic acid (0.56%) and n-hexadecanoic acid (1.81%).

TABLE 1: CHEMICAL COMPOSITION OF THE BIOACTIVE COMPOUNDS OF ALCHEMILLA CAUCASICA

Peak	Retention time (minute)	Compound	Molecular formula	Chemical formulation	% of total
1	3.252	Glycolaldehyde dimethyl acetal	C4H10O3		0.319
2	3.459	Furfural	C5H4O2		0.319
3	17.187	Phthalic acid dibutyl ester	C16H22O4		0.980
4	18.675	n-Hexadecanoic acid	C16H32O2		1.811
5	21.581	Linoleic acid	C18H32O2		0.688
6	21.712	Linolenic acid	C18H30O2		1.963
7	22.070	Octodecanoic acid	C18H36O2		0.555
8	29.728	Hexadecanoic acid 2-hydroxy-1-(hydroxymethyl) ethyl ester	C19H38O4		7.594
9	29.838	Dicyclohexyl phthalate	C20H26O4		2.031
10	32.396	Octadecanoic acid 2,3-dihydroxypropyl ester	C21H42O4		15.335
11	33.097	13-Docosenamide, (Z)-	C22H43NO		59.771
12	13.246	Octadecanamide	C18H37NO		1.728
13	34.867	24, 25 dihydroxycholecalciferol	C27H44O3		0.711
14	38.534	β- sitosterol	C29H50O		1.561

Phthalic acid and phthalate derivatives are major industrial materials used to manufacture plastic products like toys and bottles, also being widely used as plasticizers, adhesives, films, polymers, etc. However, some studies revealed that these compounds could affect the male reproductive systems producing testicular atrophy ¹⁹ and alter the normal development of fetuses in pregnant rats ²⁰. In view of these adverse effects on living beings, the US Environmental Protection Agency (EPA) classified the phthalic acid and some industrial phthalates as priority pollutants ²¹ and so, for example, the maximum admissible content in water for a common phthalic acid derivative such as the phthalic acid dibutyl ester was established in 6 g/dm.

Sterols are important constituents of all eukaryotes and play a vital role in plant cell membranes. Plant sterols possess valuable physiological activities; they are biogenetic precursors of many hormones and oviposition stimulants of some insects ²². Among all phytochemicals, β-sitosterol is the main phytosterol found in many plants. It has been reported to show anti-inflammatory, antineoplastic, antipyretic, and immunomodulating activity ²³.

The chemical composition of the total methyl esthers of fatty acids from the extracts of Alchemilla caucasica showed to have a very similar profile. Fatty acid methyl esthers are an alternative diesel fuel (namely, biodiesel) derived from vegetable oils or animal fats ²⁴. The main components of vegetable oils and animal fats are triglycerides or also known as esthers of fatty acids attached to a glycerol. The most widely used industrial method for the commercial production of fatty acid methyl esthers from vegetable oils/fats is a base catalyzed trans-esterification process using sodium hydroxide or potassium hydroxide as the homogeneous catalyst and methanol as the lower alcohol ²⁵.

CONCLUSION: The source of many plants can often be identified from the peak pattern of the chromatograms obtained directly from GC-MS analysis. Similarly, unique qualitative and quantitative patterns from a GC analysis will often help identify the source of many alcoholic beverages. The technique of fingerprint could identify false herbal products. The construction of chromatographic fingerprints aims at evaluating the quality of herbal medicines. The fundamental reason of quality control of herbal medicines is based on the concept of phytoequivalence of herbs, and then to use this conception to identify the real herbal medicine and the false one, and further to do quality control. The importance of the study is due to the biological activity of some of these compounds. The present study, which reveals the presence of components in Alchemilla caucasica suggest that the contribution of these compounds on the pharmacological activity should be evaluated.

ACKNOWLEDGEMENT: Nil

CONFLICT OF INTEREST: Nil

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    How to cite this article:

    Karaoglan ES and Yilmaz B: Identification of bioactive compounds of Alchemilla caucasica using gas chromatography-mass spectrometry. Int J Pharmacognosy 2018; 5(5): 287-93. doi link: http://dx.doi.org/10.13040/IJPSR.0975-8232.IJP.5(5).287-93.

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