Hydroxycinnamic acids in the raw material of hybrid bearded iris

Results. Hydroxycinnamic acids were identified in the iris raw materials by paper chromatography. The spectrophotometric method established of the hydroxycinnamic acid content in the recalculation on chlorogenic acid – was from 0.79 % to 2.83 %. The dry extracts of the leaves and rhizomes of I. hybrida “Little Dream” at a 1 % concentration have shown moderate inhibitory activity for Gram-positive bacteria and fungi. For the first time from the rhizomes of Iris hybrida “Indian Pow Waw” were isolated caffeic, chlorogenic, neochlorogenic, ferulic acids by the column chromatography and their structures were determined by spectral methods.

The genus Iris (Iridaceae) has long been known as ornamental, honey-bearing and medicinal plants, the number of which covers more than 300 species [1]. Iris count more than 4.000 hybrid varieties, distributed not only in Ukraine but also in Europe, Central, and Eastern Asia, North and South America [2]. According to the literature, iris accumulate secondary metabolites -flavonoids, isoflavonoids, xanthones, coumarins, hydroxycinnamic acids, saponins, tannins, vitamins, organic acids [3]. In folk medicine, the rhizomes of iris have been used as an anti-inflammatory, antibacterial, laxative, analgesic agent [4]. Based on these data, the phytochemical study of iris has a practical interest in the modern pharmacy.
As a result of the previous study, the hydroxycinnamic acids were identified in the leaves and rhizomes of Iris hungarica, and their quantitative content was established 2.6 % in the rhizomes and 4.7 % -in the leaves [5].
The hydroxycinnamic acids belong to the class of phenolic compounds, were found in almost all plants in the free acids form, their dimers, and esters. The pharmacological activity of this group of biologically active substances is being studied, there is information about choleretic, antimicrobial, tuberculostatic, antioxidant, hepatoprotective activity [6,7]. The caffeic acid and its derivatives (chlorogenic and isomers), which have an anti-inflammatory and choleretic effect, are most common in nature [8]. Ferulic acid is found in plant cells. Together, chlorogenic, ferulic, caffeic, coumaric acids have a hypoazotemic effect, enhance kidney function and stimulate the anti-toxic function of the liver [9].

Aim
Continuing the study of Iris genus plants the aim was to establish the qualitative and quantitative composition of hydroxycinnamic acids in the raw materials of iris hybrid varieties, as well as to establish the antimicrobial activity of dry extracts and isolation individual compounds.

Materials and methods
Experimental. "Little Dream". These varieties belong to the group of standard dwarf bearded irises, which are widely cultivated and valuable for their high decorative properties. The objects of research were identified by one of the authors (Yu. V. Buidin) Plant material was dried to air-dry condition, stored in a cool well-ventilated place. The raw materials were crushed to a particle size of 2-3 mm for qualitative, quantitative analysis and for the isolation of substances.
Qualitative analysis. Analytical samples of leaves and rhizomes of varieties iris were crushed to 2-3 mm particles, an extracting agent (70 % ethanol) was added in a ratio of 1 : 10, a reflux condenser was added and heated in a water bath at 90 °C for 1 hour to obtain extracts. After cooling, the extracts were filtered through a paper folded filter. Qualitative analysis was performed by paper and thin-layer chromatography in the solvent systems of 15 % acetic acid, 2 % acetic acid. On the chromatograms, the substances were detected by the characteristic fluorescence in UV light at a wavelength of 365 nm and 254 nm. Ammonia vapors, 2 % alcohol solution of aluminum chloride, 10 % sodium/ potassium hydroxide solution, 5 % alcohol solution of diazotized sulfanilic acid (diazo reactive) were used as developers, which allows obtaining zones with brighter and more characteristic fluorescence in UV light. The Rf of spots was also determined for the identification of substances and compared with standard samples of phenol carboxylic acids -chlorogenic, neoclorogenic, coffee, ferulic, cinnamic, hydroxycinnamic, p -coumaric.
Quantitative analysis. To establish the quantitative content of the amount of hydroxycinnamic acids in the leaves and rhizomes of varieties irises the spectrophotometry method was used ("Herb Egeronum Canadian" (TPM 42-U-6/37-323-96) [10]: about 1.0 gram (an exact weight) of the crushed raw material was placed in a 200 ml flask, 50 ml of 40 % ethyl alcohol was added, a reflux condenser was added and heated in a water bath for 1 hour, maintaining a gentle boil. After cooling, the extract was filtered through a paper filter. 1 ml of the obtained extract was transferred to a 50 ml volumetric flask and the volume was adjusted to the mark with 20 % ethyl alcohol. 20 % ethyl alcohol was used as a compensation solution. Spectra were measured at a wavelength of 327 nm. The content of hydroxycinnamic acids in terms of chlorogenic acid and absolutely dry raw materials was calculated by the formula, using the specific absorption rate of chlorogenic acid: A * 50 * 50 * 100 , 531 * m n * 1 * (100 -W) where А: the optical density of the test solution; m n : sample weight, g; W: weight loss on drying, %; 531: specific absorption rate of chlorogenic acid.
Isolation of individual compounds. Iris hybrida "Indian Pow Wow" raw material was used for the experiment, collected in the M. M. Gryshko National Botanical Garden in 2017. Air -dry raw materials (rhizomes) in the total mass of 1 kg were extracted with 70 % ethyl alcohol (10 L). The extract (8.5 L) concentrated to a volume of 800-900 ml and left for 24 hours to precipitate chlorophylls and gums (5-10 °С). The tar precipitate was filtered and washed by hot water (0.5 L). The resulting extract was evaporated and treated sequentially with chloroform, ethyl acetate, butanol. The fractions were evaporated until the solvents were removed.
The qualitative composition of the fractions was identified by paper and thin-layer chromatography. Chloroform and ethyl acetate extraction had a similar component composition, so they were combined. For this, the chloroform extraction was evaporated to a minimum amount, washed with 96% ethyl alcohol, and then treated with ethyl acetate.
The combined fraction was evaporated, mixed with 5 g of silica gel and applied to the column (d = 4.5; h = 80). The substances were eluted with chloroform and a mixture of chloroform: ethanol with an increasing concentration of alcohol. 120 fractions were obtained.
Extraction procedure of plant for bioassay. To determine the antibacterial activity previously was obtained dry extracts from the leaves and rhizomes of irises. 100.0 g of crushed raw materials were placed in a round-bottom flask, poured 1000 ml of solvent (distilled water), heated in a boiling water bath for 2 hours. The first portion of the extract was taken, after which 500 ml of distilled water was added to the raw material and heated in a water bath for another 2 hours. The combined extract was filtered through a Buchner funnel and evaporated to dryness.
Antibacterial activity. According to the WHO recommendations, antibacterial activity was determined by agar well diffusion method in vitro. The inoculum suspension was prepared using a Densi-La-Meter apparatus (PLIVA-Lachema, 540 nm). The cultures were synchronized using low temperature (4 о С). The microbial load was 107 cells per 1 ml of the medium and was determined according to the McFarland standard [11]. The 18-24 -hour culture of microorganisms was used for the test. Mueller-Hinton agar was used ("HIMedia Laboratories Pvt. Ltd India", India) for bacteria. The strains of Candida albicans were cultivated using Sabouraud agar ("HIMedia Laboratories Pvt. Ltd India", India). The standard medium was prepared according to the requirements of the manufacturer. When determining microorganisms' sensitivity to the samples of iris take into account zones of growth of bacteria strains. An alcoholic solution of chlorophyllipt at a concentration of 10 mg/ml was used as a reference drug (GNZLS "OZ").
Statistical data processing was carried out according to the requirements of the State Pharmacopoeia of Ukraine using software (Microsoft Office Excel 7.0) [12].

Results
Qualitative analysis. According to the results of qualitative reactions with alcoholic solutions of sodium hydroxide, aluminum chloride and diazoreactant on chromatograms with extracts Iris hybrida hort. "Bright white", Iris hybrida hort. "Indian Pow Waw", Iri hybrida hort. "Galleon Gold", Iris hybrida hort. "Mini Dynamo", Iris hybrida hort. "Little Dream" was found more than 8-21 substances of phenolic nature. At the same time, absorption zones with blue, violet, and blue-violet fluorescence characteristic of phenol carboxylic acids were identified.
Chromatograms differed in the number of spots, their chromatographic behavior, the color of the spots before and after their treatment in the UV light, the values of Rf.
According to the chromatographic behavior and comparison with the standards in the all iris varieties were identified caffeic, cinnamic, hydroxycinnamic, p-coumaric, fumaric, chlorogenic, and neoclorogenic acids ( Table 1).
Quantitative analysis. The quantitative content of hydroxycinnamic acids in the leaves and rhizomes of varieties

Оригинальные исследования
irises was established by the spectrophotometric method, the data are presented in Table 2. Antibacterial activity. Hydroxycinnamic acids exhibit many pharmacological activities, including antibacterial. Based on the data obtained, in the leaves and rhizomes of I. hybrida "Little Dream" contains the maximum amount of hydroxycinnamic acids, antimicrobial activity was established by the method of diffusion into agar ( Table 3).

Discussion
Fumaric acid was found in the leaves and rhizomes of I. hybrida "Galleon Gold", I. hybrida "Little Dream", I. hybrida "Indian Pow Waw"; n-coumaric -in the leaves and rhizomes of I. hybrida "Bright white", I.hybrida "Little Dream"; caffeicin the leaves I. hybrida "Bright white", I.hybrida "Indian Pow Waw", and in the rhizomes of I. hybrida "Mini Dynamo", I.hybrida "Little Dream". Chlorogenic, neochlorogenic, cinnamic and hydroxycinnamic acids were found in all objects. The content of hydroxycinnamic acids in the samples varies from 0.79 % to 2.83 %. The smallest content in the rhizomes is from 0.79 % to 2.49 %, which is due to the peculiarities of the metabolism of herbaceous plants. However, it should be noted that in the rhizomes of I. hybrida "Little Dream" the content of hydroxycinnamic acids is relatively high -2.49 %. The largest amount of hydroxycinnamic acids is observed in the leaves of I. hybrida "Little Dream" (2.83 %), I. hybrida "Galleon Gold" (2.82 %), I. hybrida "Mini Dynamo" (2.55 %); the smallest is in the leaves of I. hybrida "Indian Pow Waw".
The data in Table 3 indicate that dry extracts of I. hybrida "Little Dream" leaves and rhizomes exhibit antibacterial activity against Staphylococcus aureus, Escherichia coli, Basillus subtilis, Candida albicans and a weak effect on Proteus vulgaris and Pseudomonas aeruginosa.

Conclusions
1. In the raw materials of 5 dwarf bearded irises varieties, hydroxycinnamic acids were identified.
2. The quantitative content of hydroxycinnamic acids in terms of chlorogenic acid was established. It is noted that hydroxycinnamic acids accumulate in larger amounts in the leaves of irises.
3. In I. hybrida "Little Dream" raw materials the content of hydroxycinnamic acids is the highest: 2.49 % -in rhizomes and 2.83 % -in leaves. 4. The antimicrobial activity of the dry extracts of I. hybrida "Little Dream" leaves and rhizomes was established: the preparations are highly sensitive to Staphylococcus aureus, Escherichia coli, Basillus subtilis, Candida albicans; weak activity on Proteus vulgaris and Pseudomonas aeruginosa.
5. The method of column chromatography was used to isolate 4 substances, derivatives of phenol carboxylic acids: caffeic, chlorogenic, neochlorogenic, ferulic acids, the structure of which was proved by spectral methods of analysis.
6. The results of the study show the prospects for the use of cultivated varieties of irises as a source of hydroxycinnamic acids.  2.55 ± 0.13 1.77 ± 0.08 I. hybrida "Mini Dynamo"