THE RESEARCH OF ORGANIC ACIDS IN BLACK ALDER (ALNUS GLUTINOSA (L.) GAERTN.) BUDS AND LEAVES

By the means of the chromatography-mass-spectrometry method the comparative analysis of the qualitative composition and the quantitative content of organic acids in black alder buds and leaves have been carried out. Twelve organic acids have been identified in black alder buds, their total content is 1353.7 mg/kg, and there are 11 organic acids in black alder leaves with the content twice as high as in buds – 3007.9 mg/kg. One monocarboxylic, 6 dicarboxylic and 1 tricarboxylic, 4 aliphatic and aromatic organic acids have been found in buds. Two monocarboxylic, 5 dicarboxylic and 1 tricarboxylic, 5 aromatic organic acids have been found in leaves. The qualitative composition of organic acids is different in leaves and buds of black elder: 9 compounds have been found in both types of the plant material, namely capronic, oxalic, malonic, fumaric, succinic, malic, citric, vanillic and ferulic acids. Azelaic, benzoic and cinnamic acids have been found only in black alder buds, and black alder leaves contain only 2-hexenic and α-hydroxyphenylpropionic acids. The dominating component in buds is citric acid (625.8 mg/kg), the lowest content has been detected for capronic (10.3 mg/kg) and fumaric (4.5 mg/kg) acids. The highest content in leaves has been detected for oxalic acid (1403.9 mg/kg), in small amounts fumaric and succinic (8.3 mg/kg and 13.8 mg/kg, respectively) acids are present in this plant material. The results obtained will be used in the further study of the black alder plant material.

A black alder -Alnus glutinosa (L.) Gaertn. from the birch (Betulaceae) family is a deciduous tree of the countries with a moderate climate. It grows on the coasts of the ponds, arm-holes of the rivers, swamps, often forms swamp forests [3]. The plant is officinal, the raw material (stem) is inscribed in the State Pharmacopeia USSR, edition XI [2]. The scientific medicine recommends the stem (cones) as an astringent, styptic, antimicrobial agent [9,11]. The Ukrainian pharmaceutical industry produces a gastroprotective and the antiulcer medicine "Altan" and a cardioprotective drug "Elgatsin" made of this raw material [1,6]. Buds and leaves of alder are applied by folk medicine as antimicrobic, astringent, styptic, antirheumatoid, antineoplastic agent in tumours of the breast, pancreas, stomach, esophagus, duodenum and rectum, throat, uterus [4]. The cytotoxic, antioxidant and antibacterial action of leaves is also known in literature [5,7,8,10]. The chemical composition of this plant is rather studied, but there is no detailed information about organic acids or comparative analysis of the content of these compounds in buds and leaves of black alder. Therefore, comparison of the qualitative composition of organic acids of buds and leaves of this plant, as well as comparison of the quantitative content of the components are relevant.
The aim of our research is comparative analysis of the qualitative composition and the quantitative content of organic acids in buds and leaves of black alder.

Materials and Methods
The objects of the research were buds (the swelling phase), leaves (the phase of full leaf expansion) collected in 2013 in Vilshansky district of the Kharkiv region from 10 trees of one bunch for more reliable comparison of results.
The research was performed by the method of chromatography-mass-spectrometry on an Agilent Technology 6890N chromatograph with a mass-spectrometry detector 5973N. For this purpose the internal standard (the solution of 50 mkg tridecane in hexane) and 1 ml of the methyl agent (14% BCl 3 in methanol solution, Supelco 3-3033) was added to 50 mg of a weighed quantity of the raw material in a 2 ml vial. The mixture was kept in the hermetically closed vial for 8 hours at 65°C. The plant material was filtered and the filtrate was diluted in 1 ml of the distilled water. Methyl ethers were extracted in 0.2 ml of chloride methylene shaken several times within an hour, then the extract obtained was chromatographed. The sample introduction (2 mkl) to the chromatograph column was carried out in a splitless mode -without dividing the flow. The rate of the sample introduction was 1.2 ml/min for 0.2 min.
The chromatograph column was capillary INNOWAX with the internal diameter of 0.25 mm and 30 m in length. The carrier gas velocity (helium) was 1.2 ml/min. The temperature of the sample injection heater was 250°C. The thermostat temperature was programmed from 50°C to 250°C with the rate of 4 deg/min.
For identification of the components the data of the library of mass spectra of NIST05 and WILEY 2007 with the total number of spectra of more than 470000 were used with the help of the programme for AMDIS and NIST identification [12].

Results and Discussion
The results are given in Table. Twelve organic acids in buds and 11 ones in leaves have been found.
Azelaic, benzoic and cinnamic acids have been found only in black alder buds, and black alder leaves contain only 2-hexenic and α-hydroxyphenylpropionic acids.
The total content of organic acids in buds was 1353.7 mg/kg, in leaves twice as high -3007.9 mg/kg. In buds citric acid (625.8 mg/kg) was in the highest content, which was comparable with the content of this compound in leaves (620.9 mg/kg). In leaves the highest content was determined for oxalic acid -1403.9 mg/kg, it was more than 3.5 times higher the content of this acid in buds. It can be associated with accumulation of crystals and druses of calcium oxalate in leaves.
The content of dicarboxylic malonic and malic acids in buds was 44.7 mg/kg and 60.2 mg/kg, respectively. In leaves the content of these acids increased six times and more than four times and was 267.9 mg/kg and 266.8 mg/kg, respectively. Besides, the ring A of the molecules of flavonoids is synthesized from 3 active molecules of malonic acid.
There were 4.5 mg/kg of fumaric acid in buds and 8.3 mg/kg in leaves. This compound is found in the plants of Fumaraceae, Papaveraceae family, in some lichens and fungi; it is an intermediate product in the synthesis of asparagenic acid.
There were 111.2 mg/kg of hydroxicinnamic acidpherulic acid in buds and 157.0 mg/kg in leaves. This compound is an effective antioxidant, antitumoral agent, has the stress-protective action, reducing lesions of the gastric mucosa and myocardial damage caused by the immobilization-pain stress. It is caused most likely by the ability of pherulic acid to inhibit the processes of lipid peroxidation and to strengthen oxidant processes in the cardiac muscle.
Thus, buds of black alder contained 39.7 mg/kg of azelaic acid known as a keratolytic, antibacterial and antiinflammatory agent. In the plants azeloinic acid is 'a flash distress', which takes part in defense reactions after infection. Under the effect of this compound the accumulation of salicylic acid -an important component of the defense reaction of plants begins. Azelaic acid is contained in the plants of Poaceae family. Medicines containing azelaic acid are active in relation to Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus, etc.
The results obtained will be used in the further study of the black alder plant material. CONCLUSIONS 1. Twelve organic acids have been identified in black alder buds, and there are 11 mono-, di-, tri-carboxylic aliphatic and aromatic organic acids in black alder leaves.
2. The qualitative composition of organic acids is different, 9 compounds have been found in both types of the plant material.
3. The dominating component in buds is citric acid (625.8 mg/kg), in leaves it is oxalic acid (1403.9 mg/kg).