Резюме
Попова Н.В. Гідроксикоричні кислоти видів суховершків.
Проведено аналіз та ідентифіковані гідроксикоричні кислоти у сировині суховершків звичайних, с. ланцетних та с. великоквіткових. Вміст розмаринової кислоти коливалося від 1,0 до 2,9 %, а рівень кофейної кислоти знаходиться в близьких межах 0,012-0,016 %. Перспективним видом за рівнем розмаринової кислоти є трава суховершків великоквіткових.
Ключові слова: види суховершків, аналіз та вміст розмаринової, кофейної та хлорогенової кислот, хроматографія.

Резюме
Попова Н.В. Гидроксикоричные кислоты видов черноголовки.
Проведен анализ и идентифицированы гидроксикоричные кислоты в сырье черноголовки обыкновенной, ч. ланцетной и ч. крупноцветковой. Содержание розмариновой кислоты колебалось от 1,0 до 2,9 %, а уровень кофейной кислоты находится в близких пределах 0,012-0,016 %. Перспективным видом по уровню розмариновой кислоты является трава черноголовки крупноцветковой.
Ключевые слова: виды черноголовки, анализ и содержание розмариновой, кофейной и хлорогеновой кислот, хроматография.

Summary
Popova N.V. Hydroxycinnamic acids of Prunella species.
It was carried out analysis and assay of hydroxycinnamic acids in the herb of Prunella species: P.vulgaris, P.lanceolata, P.grandiflora. The content of rosmarinic acid ranged from 1.0 to 2.9 %, and caffeic acid is in close range 0,012-0,016 %. Perspective species by the level of rosmarinic acid was established Prunella grandiflora herb.
Key words: Prunella species, analysis and assay of rosemaric, caffeic and chlorogenic acids, chromatography.

Рецензент: д.фарм.н., проф. В.М. Ковальов

УДК 547.587.52:582.949.2

Національний фармацевтичний університет (Харків)

Национальный фармацевтический университет (Харьков)

61002, г. Харьков, ул. Пушкинская, 53

National University of Pharmacy (Kharkov)

pharmsy1@rambler.ru

Introduction. There has been interest in a caffeic acid derivatives, among which should be mentioned rosemarinic acid. Previously it was believed that rosemarinic acid, together with its derivatives related to tannic compounds described as a depsid of caffeic acid. Before the isolation and determination of structures of rosemarinic acid and its derivatives were known as "Labiatengerbstoffe" - tanin-like compounds. In 1958 Italian scientists M. Scarpati and G. Oriente extracted from the rosemary leaves (Rosmarinus officinalis L) ether of a caffeic acid and 3 - (3,4-dihydroxyphenil) lactic acid, which later was named authors as rosemarinic acid. (Figure 1) [1, 14].

Figure 1 Rosemarinic acid

Using the results of biogenetic research found that two amino acids - phenylalanine and tyrosine are involved in the synthesis of this acid. So caffeic acid was synthesized from phenylalanine and 3,4-dihydroxyphenillactic acid from tyrosine. Established set of enzymes and genes involved in various stages of biosynthesis [1, 18]. Phenylpropane acid and rosemarinic acid is particularly of interest to pharmacy and medicine as substances with high antioxidant, antiviral (antiherpes), anti-allergic, anti-inflammatory activity with low toxicity, it shows high activity in the treatment of Alzheimer's disease. It is responsible for antitumor (inhibits protein synthesis in cancer cells) and thyroid-regulatory activity types. In laboratory tests it was found that extracts of rosemarinic acid containing medicinal plants are effective in the treatment of Graves' disease. They block the action of antibodies and thyroid-stimulating hormone, thereby preventing an increase in thyroid gland [1, 8, 13, 14, 18].

Among the plants that are rich in rosemarinic acid, most typical representatives families are: Lamiaceae, Boraginaceae and Apiaceae [1, 6, 13, 18].

Representatives of these plants are species of the genus Prunella that grow in Ukraine: P. vulgaris, P. grandiflora and P. lanceolata. Analysis of known data showed that the above objects are used as haemostatic, wound healing, anti-inflammatory, antimicrobial, antipyretic, expectorant and tonic agent. Herbal drug of these medicinal plant are recommended to treat: the thyroid gland cancer,  limphogranulomas, lymphoma, bronchitis, respiratory diseases,  hemoptysis, empitigo, psoriasis,  seborrhea, exudative diathesis, laryngitis, nephritis, hemorrhoids, diarrhea, tuberculosis (throat, skin, lungs), diphtheria, dysentery, hypertension, arthritis, rheumatic arthritis, lymphadenitis, hyperthyroidism, thyrotoxicosis, stomachalgia, epilepsy, scurvy, mastitis, obesity, bruises, dislocations [2-7,12, 16-19].

The goal of this work is to find sources of rosemarinic acid among genus Prunella of family Lamiaceae and for further development on the their basis herbal remedies with a wide range of pharmacological effects.

Methods and materials of research

For investigation we used the herb of three species of Prunella (Lamiaceae) - P. vulgaris, P.lanceolata and P.grandiflora. Samples of herb were collected in the Kharkiv region, in the Botanical Garden of the National University of Pharmacy and Botanical Garden of Kharkiv National University  named by V.N. Karazin during flowering period of plants (June-July  2009-2011).

To identify and analyze the contents of hydroxycinnamic acids in herb it was made extracts by vacuum-filtration extraction of 50% ethanol at a ratio of 1:5 - 1:6.

To identify hydroxycinnamic acids in the objects it was used 50% alcoholic extract, which was subjected to chromatographic analysis using paper and thin-layer chromatography (paper mark "Filtrak" of different numbers and  chromatographic plate "Silufol", "Sorbfil", "Merck" ).To the chromatogram plate was applied by micropipette 0.01 ml of water-alcohol extract samples of plant material. Analysis was carried out using the following solvent system: chloroform-methanol-water (24:14:3), toluene - ethylformiat - formic acid (50:40:10), butanol-acetic acid - water (4:1:2), 2% and 15% acetic acid. Chromatograms examined under UV-light before and after treatment with specific reagents. Hydroxycinnamic acids were determined by specific fluorescence in ultraviolet light (365 nm) using appropriate reagents and reference solutions [9-11, 15].

The content of hydroxycinnamic acids in samples of plant material were determined by HPLC chromatography on chromatograph Shimadzu LC 20 Prominence. The package chromatograph was: flow degasser, pump station with a low pressure gradient module, Autosampler, column thermostat and a diode-array detector. For the analysis of the firm using column Macherey-Nagel 150 mm long and 3 mm in diameter, filled with drawn-phase sorbent Nucleosil C18 AB, 3 micron grain size and porosity of 100 Å. 2 ml sample volume, detection at wave l=280 nm, 330 nm, 360 nm with a scan rate of 3 Hz. Produced in gradient elution mode share growth in solution (a mixture AsSN: MeOH: H2O + HClO₄ in the ratio of 40:40:20, pH 2.5) in a mixture of solution A (aqueous HClO₄ pH 1.8) from 0 to 100% % for 80 minutes at 30 ° C.

Identification of peaks produced by UV-spectrum compared with spectrum from the database and at retention times according to reference compounds. Mass concentration was determined by the calibration characteristic of reference compounds using the new LC Solutions (Shimadzu).

Results and Discussion

Using chromatography analysis it was determined about 20-24 phenolic compounds in herbal samples. Chromatographic features of the identified compounds are shown in Table 1.

Table 1

 Chromatographic features of hydroxycinnamic acids of Prunella species

Comparative analysis shows that the largest area of rosemarinic acid has the following sequence: Prunella grandiflora herb  > Prunella vulgaris herb >  Prunella lnceolata herb.

The results of HPLC analysis are shown in Table 2 and in Figure 2, 3, 4.

Figure. 2 HPLC chromatogram of alcohol extract of Prunella grandiflora herb (1- caffeic acid, 2- rosemarinic acid).

Figure. 3 HPLC chromatogram of alcohol extract of Prunella lanceolata herb ( 1- caffeic acid, 2- rosemarinic acid).

Figure. 4 HPLC chromatogram of alcohol extract of Prunella vulgaris herb ( 1- caffeic acid, 2- rosemarinic acid).

Table 2

Content of rosemarinic and caffeic acids in Prunella species

European Pharmacopoeia analyzes several types of herbal drugs by content of rosemarinic acid (melissa leaf - not less 1.0%; peppermint dry extract - not less than 0.5%) or the amount of hydroxycinnamic acids with the reference intoof rosemarinic acid (rosemary leaf - not less than 3.0%) [6]. Experimental data about the level of rosemarinic acid (1.2 to 2.9%) in the Prunella species show perpective herbal sample in the direction of phytomedications with wide range of biological effects.

Conclusions

1. For the first time content analysis and rosemariinic and caffeic acids in Prunella species was determined.

2. In a different species of Prunella herb rosemarinic acid content ranged from 1.0 to 2.9%, while the caffeic acid is close range 0,012-0,015%.

3. The most perspective species by the level of rosemarinic acid herb is Prunella grandiflora.

4. The possibility of further phytochemical investigation of the genus Prunella, as well as promising pharmacological screening of potential drugs based on them are actual.

Література

1. Буданцев А.Л. Розмариновая кислота: источники и биологическая активность / А.Л. Буданцев, Е.Е. Лесиовская // Раст. ресурсы – 2012. – Т.48, вып. 3. – С. 453- 468.
2. Дикорастущие полезные растения России / Под. ред. А.Л. Буданцева, Е.Е. Лесиовской. – СПб.: Изд-во СПХФА, 2001. – 663 с.
3. Дмитрук С.И. Антипирическое действие сухого экстракта черноголовки / С.И. Дмитрук, Т.П. Прищеп // Решение актуальных задач фармации на современном этапе: тез. докл. науч. конф., посвящ. 50-летию НИИ фармации. – 1994. – С. 240.
4. Дмитрук С.И. Действие экстракта черноголовки на экспериментальный тонзиллит / С.И. Дмитрук, С.Е. Дмитрук // Природные ресурсы Сибири: тез. докл. 4-й Межд. науч. практ. конф. - Томск, 1998. – С. 210.
5. Дмитрук С.И. Противовоспалительные свойства, антибактериальная и антифунгальная активности экстракта из надземной части Prunella vulgaris L. / C.И. Дмитрук // Растит. ресурсы. – 2001. – Вып. 4. – С. 92-96.
6. Попова Н.В. Лекарственные растения мировой флоры / Н.В. Попова, В.И. Литвиненко. – Харьков: СПДФО Мосякин В.Н., 2008. – 510 с.
7. Репаративные свойства фенольного комплекса черноголовки обыкновенной / С.Т. Дмитрук [и др.] // Современные изыскания в области фармации. – Ярославль, 1996. – С. 122.
8. Фармакологическая активность розмариновой кислоты / О.В. Азарова, В.М. Брюханов, Я.Ф. Зверев [и др.] // Вопросы биол., мед. и фармац. химии. – 2010. – № 6 – С. 3-8.
9. Хроматография на бумаге / Под ред. И.М. Хайса, К. Мацека. – М.: Иностр. лит-ра, 1962. - 851 с.
10. Шаршунова М. Тонкослойная хроматография в фармации и клинической биохимии : в 2–х ч. / М. Шаршунова, В. Шварц, Ч. Михалец. - М. : Мир, 1980. - 622 с.
11. Куликов А.Ю. Тонкошарова хроматографія: теоретичні основи та практичне використання / А.Ю. Куликов. – Харків: ХНУ імені В. Н. Каразіна, 2011. – 260 с.
12. Du D.A. A new unusual delta11(12)-oleane triterpene and anti-complementary triterpenes from Prunella vulgaris spikes / D.A. Du, Z. Cheng, D. Chen // Nat. Prod. Commun. – 2012. – Vol.7, № 4. – P. 501-505.
13. Modnicki D. Potential sources of rosmarinic acid. / D. Modnicki, I. Matławska // Herba Polonica. – 2007. – Vol. 53, № 2. – P. 165-166.
14. Petersen M. Rosmarinic acid / M. Petersen, M.S.J. Simmonds // Phytochemistry. – 2003. – Vol. 62. – P. 121–125.
15. Wagner H. Plant drugs analysis: a thin layer chromatography atlas / H. Wagner, S. Bladt. - [2–nd ed.] - Berlin : Springer–Verlag, 1995. – 384 р.
16. Marková H. Prunella vulgaris L.-a rediscovered medicinal plant / H. Marková, J. Sousek, J. Ulrichová // Ceska Slov Farm. – 1997. – Vol. 46, № 2. – P. 58-63.
17. Murakami M. Considerations for the treatment of chronic fatigue syndrome // M. Murakami // Nihon Rinsho. – 2007. – Vol. 65, № 6. – P. 1089-1092.
18. Parnham M.J. Rosmarinic acid / M.J. Parnham, K. Kesselring // Drugs Future. - 1985. – № 10. – Р. 756-757.
19. Permeability of rosmarinic acid in Prunella vulgaris and ursolic acid in Salvia officinalis extracts across Caco-2 cell monolayers / Z. Qiang [et al.] // J. Ethnopharmacol. – 2011. – Vol. 137, № 3. – P.1107-1112.