19 P H A R M A c y ISSN 2413-6077. IJMMR 2016 Vol. 2 Issue 1 DOI 10.11603/ijmmr.2413-6077.2015.2.6374 QUALITATIVE cOmPOsITION ANd ORGANIc AcIds cONTENT IN ThE ABOVEGROUNd PART Of PLANTs fROm fAmILIEs Lamiaceae, asteraceae, apiaceae ANd chenopodiaceae S. M. Marchyshyn, M. I. Shanayda, I. Z. Kernychna, O. L. Demydiak, I. S. Dahym, T. S. Berdey, I. M. Potishnyj I. HORBACHEVSKY TERNOPIL STATE MEDICAL UNIVERSITY, TERNOPIL, UKRAINE Background. Organic acids are the compounds of aliphatic or aromatic orders, which are widespread in flora and have a wide range of biological activity. We studied the qualitative composition and quantitative contents of organic acids in the aboveground part of some unofficial medicinal plants from families Lamiaceae, Asteraceae, Apiaceae and Chenopodiaceae is relevant. Objective. The objects of the research are the aboveground part of unofficial medicinal plants from families Lamiaceae, Asteraceae, Apiaceae and Chenopodiaceae. Methods. Identification of organic acids was performed by means of thin-layer and paper chromatography, their content was determined by means of gas chromatography, the quantitative amount of organic acids was defined by titrimetric analysis. Results. In the studied raw plants the quality of organic acids and their total contents were determined (in terms of malic acid). It is established that the maximum content of organic acids is accumulated in the grass Hyssopus officinalis L. (Lamiaceae), and the minimal is in the leaves of Chrysánthemum xhortorum L. variety Apro (Asteraceae). In all studied raw plants the dominance of aliphatic acids (citric, malic, oxalic and malonic) was determined by means of gas chromatography. Benzoic is predominant among the aromatic acids. Conclusions. In the studied raw plants the quality of organic acids and their total content were determined. The following results can be used in developing the methods of quality control of the studied raw plants and during the study of new bioactive substances. KEY WORDS: organic acids, Lamiaceae, Asteraceae, Apiaceae, Сhenopodiaceae, grass, leaves, thin- layer chromatography, gas chromatography, paper chromatography. Introduction Organic acids are the biologically active substances which are in plants in the free state, in the form of salts, esters, dimers and com- pounds with other substances. They are inter- mediate products of plants’ metabolism: in- volved in the oxidation of carbohydrates, fats, amino acids and proteins; used in the synthesis of amino acids, alkaloids, steroids, etc. [1, 2]. Organic acids have a wide range of biological effects. They enhance the secretory and motor activity of the digestive tract, improving diges- tion; help to reduce nitration processes in the organism and to reduce chemical carcinogenesis; raise the protective strength and vitality of the organism. The antioxidant, antiallergic, anti- inflammatory, antiseptic properties of these compounds are established [2]. According to the information above, we consider that the study of the qualitative com- position and quantitative contents of organic acids in the aboveground part of some unofficial medicinal plants from families Lamiaceae, Asteraceae, Apiaceae and Chenopodiaceae is relevant and is of significant scientific and practical interest [3–5]. This will make it possible to justify the use of these plants in the future pharmaceutical researches. The aim of our research is to define the qua- litative composition and quantitative con tents of free organic acids in the aboveground part of medicinal plants from families Lamiaceae (Hyssopus officinalis L., Lophanthus anisatus L.), Asteraceae (Bellis perennis L. — cultivated, Ta getes tenuifolia Cav., Chrysánthemum xhor torum L. variety Apro), Apiaceae (Angelica syl vestris L.) and Chenopodiaceae (Chenopodium album L.). The leaves of Chrysánthemum xhortorum and Angelica sylvestris and the grass of the rest species were used for phytochemical analysis. Raw plants for research were harvested during their mass flowering. S. M. Marchyshyn et al. Corresponding author: Svitlana Marchyshyn, Department of Pharmacognosy and Medical Botany, I, Horbachevsky Ternopil State Medical University, 1 Maidan Voli, Ternopil, Ukraine, 46001 Tel.: +3800352520518 E-mail: marchyshyn@tdmu.edu.ua International Journal of Medicine and Medical Research 2016, Volume 2, Issue 1, p. 19–22 copyright © 2016, TSMU, All Rights Reserved 20 P H A R M A c y ISSN 2413-6077. IJMMR 2016 Vol. 2 Issue 1 Materials and Methods Identification of free organic acids in raw materials was performed out by means of thin- layer chromatography (TLC), paper chroma- tography (PC) and gas chromatography (GC) according to [6, 7]. For TLC and PC the aqueous extracts of raw plants were prepared. As stan- dard samples for PC and GC benzoic, oxalic, malic, tartaric, succinic, salicylic, citric acids and the following solvent systems: n-butanol-formic acid-water (10:1:4), 95% ethanol-chloroform- concentrated solution of ammonia-purified water (70:40:20:2); 95% ethanol-concentrated solution of ammonia (16:4.5) and ethyl acetate- formic acid-water (3:1:1) were used. The chromatograms were developed after drying in 0.05% alcohol solution bromothymol blue and 0.1% solution of 2,6-dichlorophe nolin- dophenol sodium salt hydrate. The action of ammonia vapours on chromatograms after a few seconds improved the contrast of spots. The quantitative contents of organic acids in aqueous extracts of raw plants were deter- mined according to [7] by titrimetric method. The contents of free organic acids (X) in terms of malic acid in absolutely dry raw materials in percentage were calculated by the formula: , )W100(10m 1001002500067,0V X −×× ×××× = where: V — volume of 0.1 M sodium hydroxide solution consumed on titration, ml; 0.0067 — the amount of malic acid corres- ponding to 1 ml of 0.1 M sodium hydr oxide solution; m — mass of raw material, g; W — loss in weight because of drying, %. The quantitative contents of individual or- ganic acids were defined by modified methods for determining the fatty acids in raw plants with further detection of organic acids. It is based on getting acid methyl esters (fatty, orga- nic, phenolic) by methylating agent and their removal for further chromatographing by means of the gas chromatograph Agilent Tech- nologies 6890 N with mass spectrometric de- tector 5973 N. Methyl esters of organic acids were obtained by a modified method of A. Carrapiso [8]. Results 3–5 organic acids were identified in the raw plants of every studied species by TLC and PC methods (Table 1). According to Table 1, all studied species contain citric, oxalic and malic organic acids; tartaric, salicylic, benzoic and succinic acids were found only in some representatives. The results of quantitative contents of organic acids determination (in terms of malic acid) are shown in Table 2. According to Table 2, the maximum contents of organic acids accu- mulate in the grass Hyssopus officinalis, the lowest is in the leaves of Chrysánthemum xhor- torum. The component composition and quanti- tative contents of individual organic acids in the aboveground organs of some studied species was defined by means of GS method (Table 3). 13 organic acids in raw plants of Hyssopus offi- cinalis, 19 — in Lophanthus anisatus, 5 — in Bellis perennis, 5 — in Chrysánthemum xhortorum, 18 — in Chenopodium album, 15 — in Angelica sylvestris were determined. Discussion According to the results, aliphatic organic acids (citric, oxalic, malonic and malic) were defined in all studied raw plants by means of the methods of thin-layer, gas and paper chroma- Table 1. Results of Organic Acids Identification in Raw Plants from Families Lamiaceae, Asteraceae, Apiaceae and Chenopodiaceae Acid Types of plants Hyssopus officinalis (grass) Lophanthus anisatus (grass) Bellis perennis (grass) Tagetes tenuifolia (grass) Chrysán- themum xhortorum (leaves) Chenopo- dium album (grass) Angelica sylvestris (leaves) succinic + + – + – + traces tartaric – – traces – – – – citric + + + + + + + salicylic – traces + – – – traces oxalic + + + + + + + malic + + traces + + + + benzoic + + – – – + – Note: “+” — labels identified compounds, “–” — labels unidentified compounds. S. M. Marchyshyn et al. 21 P H A R M A c y ISSN 2413-6077. IJMMR 2016 Vol. 2 Issue 1 tography. The detection of organic acids is a topical issue of phytochemical researches. The study on composition and contents of these compounds in raw plants was not the urgent matter before [2, 9, 10]. Citric acid is very widespread in nature and is used in medicine as a part of drugs to improve energy metabolism; malic and oxalic acids are used in food industry [2, 9]. We consider that among all investigated objects the grass Chenopodium album is the most promising source of oxalic acid, and the grass Hyssopus officinalis — of citric and malic. Malonic acid is the predominant organic acid in all investigated raw plants except the grass Chenopodium album. This dicarboxylic acid is an important component of biochemical reactions in a plant organism and a precursor in the synthesis of cinnamic acids and flavonoids; its significant contents indicates the level of metabolism in plants during the preparation of raw plants (in the period of their flowering). Its accumulation in plants depends on the intensity of photosynthetic activity, enzymic reactions, temperature, etc. Studying the role of this acid in plants and its effect on the biological activity of phytosubstances of plants is a promising area for scientific investigations [11]. Benzoic acid is quantitatively dominant among the aromatic acids in the grass family Lamiaceae (Table 3), which is rather useful in pharmacy because it has anti-inflammatory, antibacterial and immunotropic properties [2]. the grass Hyssopus officinalis is the most promising for benzoic acid among the studied species. Conclusions The quantitative content of organic acids was studies and determined in the aboveground parts (grass or leaves) of the plants from fami- lies Lamiaceae, Asteraceae, Apiaceae and Cheno- podiaceae. The component composition of free organic acids in raw plants from families Lamiaceae (Hyssopus officinalis, Lophanthus anisatus), Asteraceae (Bellis perennis — cultivated, Tagetes tenuifolia, Chrysánthemum xhortorum variety Apro), Apiaceae (Angelica sylvestris) and Chenopo- diaceae (Chenopodium album) were analysed by means of gas chromatography for the first time. The dominance of aliphatic acids (citric, malic, oxalic and malonic) was determined. The follo- wing results can be used to develop the me- thods of quality control of the studied raw plants and during the study of new bioactive sub stances. Table 2. Quantitative Contents of Organic Acids in Raw Plants from Families Lamiaceae, Asteraceae, Apiaceae and Chenopodiaceae Types of plants Hyssopus officinalis (grass) Lophanthus anisatus (grass) Bellis perennis (grass) Tagetes tenuifolia (grass) Chrysán- themum xhortorum (leaves) Chenopo- dium album (grass) Angelica sylvestris (leaves) Contents of acids, % 3,26±0,03 2,49±0,02 0,69±0,01 2,78±0,16 0,34±0,05 2,37±0,03 0,69±0,29 Table 3. Contents of Main Organic Acids in Raw Plants from Families Lamiaceae, Asteraceae, Apiaceae and Chenopodiaceae Acid Component Content, mg/kg Hyssopus officinalis (grass) Lophanthus anisatus (grass) Bellis perennis (grass) Chrysánthemum xhortorum (leaves) Chenopodium album (grass) Angelica sylvestris (leaves) oxalic 84 370 70 310 20257 0,69±0,29 malonic 3731 3578 1069 557 963 1066 succinic 82 214 – – 727 37 benzoic 466 101 – – 81 – malic 946 590 – 2797 297 1101 citric 3063 1500 438 1562 792 1122 S. M. Marchyshyn et al. 22 P H A R M A c y ISSN 2413-6077. IJMMR 2016 Vol. 2 Issue 1 References 1. Dibner J, Butin Р. Use of organic acids as a model to study the impact of gut microflora on nutrition and metabolism. J. Appl. Poultry Research 2002; 11 (№ 4): 453–463. 2. Kurkin VA. Pharmacognosy. Handbook for students of pharmaceutical specialties. – Samara, 2004: 202-214. (in Russian) 3. Venkateshappa SM, Sreenath KP. Potential medicinal plants of Lamiaceae. American Int. J. of Research in Formal, Applied & Natural Sciences 2013; 3(1): 82-87. 4. Kokanova-Nedialkova Z, Nedialkov P, Nikolov S. The genus Chenopodium: phytochemistry, ethno- pharmacology and pharmacology. Pharmacognosy Review 2009; (3): 280-306. 5. Ramya R, Mahna S, Bhamunathi S, Bhat S. Analysis of phytochemical composition and bacte- riostatic activity of Tagetes sp. Int. Research J. of Pharmacy 2012; 3(11): 114-115. 6. Benzel’ IL, Darmohrai RL, Benzel’ LV. Inves- tigation of content of ascorbic acid and free organic acids in herbal substances of Bergenia crassifolia. Pharm. journal 2010; 1: 98–101. (in Ukrainian) 7. Emelyanova IV, Kovaliov VS, Kovalev SV, Zuravel IO. Investigation of qualitative composition and dynamics of the accumulation of free organic acids in vegetative and generative organs of Grindelia squarrosa. Pharm. journal 2009; 1: 80–84. (in Ukrainian) 8. Carrapiso A, García C. Development in lipid analysis: some new extraction techniques and in situ transesterification. Lipids 2000; 35 (11): 1167–1177. 9. Brul S, Coote P. Preservative agents in foods, mode of action and microbial resistance mechnismes. Intl. J. Food Microbiology 1999; 50 (1–2): 1–17. 10. Chirikova NK., Olennikov DN., Rokhin AV. Organic acids from medicinal plants: Scutellaria baicalensis (Lamiaceae). Chem. of Nat. Compounds 2008; 44 (1): 84-86. 11. Kenneth EK. Kurt WF., Schatz FP. A one-step synthesis of cinnamic acids using malonic acid. Journal of Chem. Educ. 1990; 67 (12): 304-308. Received: 2015-11-12 S. M. Marchyshyn et al.