{"id":58966,"date":"2024-06-25T11:38:05","date_gmt":"2024-06-25T11:38:05","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=58966"},"modified":"2024-07-03T16:55:12","modified_gmt":"2024-07-03T16:55:12","slug":"phytochemical-analysis-of-anthocyanins-extracted-from-the-flowers-of-two-dahlia-cultivars-and-their-cytotoxic-properties","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no2\/phytochemical-analysis-of-anthocyanins-extracted-from-the-flowers-of-two-dahlia-cultivars-and-their-cytotoxic-properties\/","title":{"rendered":"Phytochemical Analysis of Anthocyanins Extracted from the Flowers of Two Dahlia Cultivars and their Cytotoxic Properties"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Anthocyanins are part of the\nflavonoid group of phenolic substances and are synthesized from anthocyanidins1,2<\/sup>. They play a crucial role in determining the color of various plant\norgans, including flowers, fruits, tubers, and leaves. Anthocyanins are essential for plant survival, as\nthey act as optical filters and protect plant tissues from excessive\nultraviolet radiation. Additionally, anthocyanins are effective in inhibiting\nreactive oxygen species3,4<\/sup>. These compounds are\nstored in plant cells in the form of glycosides, and their color (rose, red-purple,\ndark violet, or blue) depends on various factors such as the pH of the medium,\ntemperature, and the structure of the molecule. The color also depends on the\nnumber and position of OH groups, methylation, the nature of the sugar, the\namounts of molecules and their location.<\/p>\n\n\n\n

There are over 1000 natural anthocyanins obtained from the\nanthocyanidin aglycones with different glycosylations and acylations5<\/sup>. The most common\nanthocyanidins in nature are cyanidin,\npelargonidin, peonidin, delphinidin, petunidin, and malvidin6<\/sup>.<\/p>\n\n\n\n

Anthocyanins are\ncompounds that have a wide range of pharmacological effects and are used in\nmedicines and as food additives7<\/sup>. The anthocyanins that have a hydroxyl group at position 3\nof the C ring possess high antioxidant properties, which are further enhanced\nby the acylation of sugar residues8,9<\/sup>. The ability of\nanthocyanins to enhance the oxidative function of mitochondria in muscle cells\nand brown adipose tissue affects the increase in the rate of energy metabolism\nin the body. Anthocyanins, by suppressing the activity of hydrolytic enzymes,\nslow down the hydrolysis of food, and as a result, after eating, the level of\nglucose in the blood rises evenly8<\/sup>. Among the delphinidin and cyanidin groups, the representatives exhibit\nthe highest antioxidant activity and can inhibit the inflammatory process8-10<\/sup>.\nAnthocyanins are also known to restore the integrity of genomic DNA, stabilize\ncollagen molecules, and reduce capillary permeability8,11,12<\/sup>.\nNumerous studies have demonstrated the antidiabetic, hypocholesterolemic,\nanticancer, cardioprotective, and hypotensive effects of anthocyanins13-17<\/sup>.\nAnthocyanins reduce markers of inflammation, therefore they are promising in\nthe treatment of obesity and concomitant or independent diseases such as\ndiabetes, cancer, and dysbiosis. Cardiovascular diseases are directly related to oxidative stress,\nmetabolic syndrome, and chronic inflammation in the human body. Consuming natural\nproducts containing anthocyanins on a regular basis helps to reduce the lipid\nprofile and blood sugar levels, which is important in the prevention of\ndiseases of the cardiovascular system8,11,13,14,17<\/sup>. Katsube\nN, et al conducted a study on blueberry extract and found that among the\nisolated anthocyanins, delphinidin or malvidin as an aglycone inhibited the\ngrowth of HL60 cells by inducing apoptosis, and pure delphinidin and glycoside\ninhibited the growth of HCT116 cells16<\/sup>. It was established their ability to\nsuppress the growth of Gram-positive (Staphylococcus\naureus, Streptococcus faecalis, <\/em>and Bacillus\ncereus<\/em>) and Gram-negative (Pseudomonas\naeruginosa,<\/em> <\/em>and Escherichia\ncoli<\/em>) bacteria as well as phytopathogenic (Penicillium spinulosum<\/em> and Rhizopus\nstolonifer<\/em>) and mold (Aspergillus\nflavus, Aspergillus niger<\/em>) fungi18<\/sup>. <\/p>\n\n\n\n

Curtain plant species are currently being used as an industrial source of anthocyanins. Among them fruits of grapes (Vitis vinifera<\/em> L.), blackberries (Morus alba<\/em> L.), blueberries (Vaccinium corymbosum<\/em> L.), jambul (Syzygium cumini<\/em> (L.) Skeels), strawberries (Fragaria x ananassa<\/em> Duchesne ex Rosier) ad purple corn (Zea mays<\/em> L.) as well as leaves of red cabbage (Brassica oleracea<\/em> L. var. capitata<\/em> f. rubra), taproots of carrot (Daucus carota<\/em> L.) and radish (Raphanus sativus<\/em> L.) or tubers of purple sweet potato (Ipomoea batatas<\/em> (L.) Lam.)19<\/sup>. Adding anthocyanins to food products not only imparts different colors, making them visually appealing, but also acts as an antioxidant, protecting the food to which they were added20<\/sup>.<\/p>\n\n\n\n

Over the past few years, researchers have taken a keen interest in studying various species of flowers (such as Clitoria ternatea<\/em> L., Hibiscus sabdariffa<\/em> L., Dahlia<\/em>, Viola<\/em> L., and others) for their anthocyanin content21-25<\/sup>. These anthocyanins can be used in medical treatments, as well as in the food industry. Mishra and his team obtained a purple dye from dahlia flowers that they suggested could be used as an eco-friendly alternative to phenolphthalein26<\/sup>. The anthocyanins in dahlia flowers have also been proposed as an ingredient in yogurt production. In Mexican cuisine, dahlias are used in salads, desserts, and other dishes27<\/sup>.<\/p>\n\n\n\n

Scientists are\nsearching for plants with a high concentration of anthocyanins for the\ndevelopment of herbal medicines. Plants of the Dahlia genus, which belong to\nthe Asteraceae family, are of particular interest in this field because they\ncan accumulate anthocyanins in their flower petals. This genus is native to\nMexico to Colombia (as shown in Fig. 1), but it is now widely distributed\naround the world due to its easy cultivation. There are a total of 41 species\nand 15,000 varieties and cultivars in this genus28<\/sup>.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: T<\/strong>he geographic distribution of the different species that belong to<\/strong> the Dahlia<\/em> genus all around the world<\/strong> (according to ‘Plants of the World online’28<\/sup>).<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The Dahlia species\nis known to contain inulin, which could be beneficial for patients with type 2\ndiabetes by preventing high blood glucose levels29<\/sup>. Additionally, these plants can accumulate valuable phenolic\nsubstances, amino acids, and organic acids30<\/sup>. The various species and cultivars of Dahlia plants allow\nfor the selection of the most promising ones based on their chemical\ncomposition and pharmacological effects30-33<\/sup>. The color of dahlia flowers ranges\nfrom white-rose to dark purple, which is related to the level of accumulation\nof anthocyanins. Due to the diversity of species and cultivars, it is crucial\nto conduct a phytochemical analysis of plant raw materials for the quality assessment and\nefficacy of herbal medicines34<\/sup>.<\/p>\n\n\n\n

This study aimed to\nconduct a phytochemical analysis of anthocyanins in the flowers of \u2018La Baron\u2019\nand \u2018Colorado Classic\u2019 Dahlia\ncultivars and to determine the in vitro<\/em>\ncytotoxic activity of their extracts.<\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

Plant raw\nmaterial<\/strong> <\/strong><\/p>\n\n\n\n

The study focused on the flowers\nof the \u2018La Baron\u2019 and \u2018Colorado Classic\u2019 Dahlia cultivars (Fig. 2) that belong to a group of \u2018Decorative (ornamental) dahlias\u2019 (according to the\nclassification of The International Dahlia Register35<\/sup>). From 2016, we have been conducting research on inulin in\nunderground tubers and phenolic substances in above-ground organs of various\ndahlias 29, 30<\/sup>. The research objects were selected from the botanical garden\nat Kharkiv Karazin University in Ukraine, taking into account the tuber weight\nand flower color. The ‘Colorado Classic’ flowers (Fig. 2, A) are light pink at the base and\nbright lilac at the tips, up to 15.0 cm long and\n0.6-1.3 cm wide, with\nan elongated oval shape and smooth, pointed, concave edges. The ‘La Baron’ cultivar flowers (Fig. 2,\nB) are dark pink to purple, 7.0-10.0 cm long and 0.6-1.0 cm wide, with a flat\nor slightly concave shape, elongated lanceolate form, and smooth edges with a\nblunt top. <\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2: Inflorescences of Dahlia cultivars: \u2018Colorado Classic\u2019 (<\/strong>\u0410<\/strong>) and \u2018La Baron\u2019 (<\/strong>\u0412<\/strong>)<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The plants\u2019 raw materials were\ncollected during the flowering stage in August\n2021 at the collection site of the botanical garden named after V. N. Karazin (Kharkiv,\nUkraine). Flowers were air-dried to an air-dry state by the requirements of the\nState Pharmacopoeia of Ukraine36<\/sup>. The identity of dahlia cultivars has\nbeen confirmed by Yuri Gamulya, PhD in Biological Sciences, the curator of the\nherbarium fund of V. N. Karazin Kharkiv National University.<\/p>\n\n\n\n

Spectrophotometric analysis<\/strong><\/p>\n\n\n\n

The total content of anthocyanins was measured using a spectrophotometric method with UV-VIS\nspectrophotometer Specord 200 (Analytik Jena, Germany)36<\/sup>. Five series of raw materials of each dahlia cultivar were\nused to prepare the test solution. 5.00 g of raw material was crushed and\nsifted through a sieve with a hole diameter of 1-2 mm. Then the raw material\nwas placed in a 100 mL conical flask and 95 mL of methanol was added, and\nstirred evenly for 30 min. The extract was filtered through a paper filter into\na 100 mL volumetric flask. The filter was rinsed and the extraction volume was\nbrought to the mark of 100 mL with the same solvent. A 50-fold dilution of\nthe resulting solution was prepared in 0.1% hydrochloric acid in methanol30<\/sup>.\n<\/p>\n\n\n\n

Measurements were carried out at a wavelength of 528\u00b12 nm in terms of\ncyanidin-3-O<\/em>-glucoside chloride. A\nsolution of 0.1% hydrochloric acid in methanol was used as a compensation\nsolution.<\/p>\n\n\n\n

This formula was used for the calculations of the total content of anthocyanins:<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

A \u2013 optical density of the test solution;<\/p>\n\n\n\n

m\n\u2013 weight of the sample of raw material;<\/p>\n\n\n\n

718\n– specific absorption rate of cyanidin-3-O-glucoside chloride.<\/p>\n\n\n\n

Chromatographic\nanalysis using high-performance liquid chromatography (HPLC)<\/strong><\/p>\n\n\n\n

The chromatographic analysis of\nanthocyanins was carried out in the acidified aqueous extracts of the studied\nraw materials using reverse-phase HPLC. We used a Shimadzu LC-20 Prominence\nliquid chromatograph (Japan), equipped with a four-channel pump LC-20AD, a\ncolumn thermostat STO-20A, an automatic sampler SIL-20A, a diode matrix\ndetector SPDM20A and a ChemStation LC20. An Agilent Technologies liquid\nchromatograph (model 1200) was also used. The chromatograph is equipped with a\nG1379A vacuum degasser, a G1313A automatic injector, a G13111A four-channel\nlow-pressure gradient pump, a G1316A column thermostat, and a G1316A diode\narray detector. The results were calculated using ChemStation LC20 software.<\/p>\n\n\n\n

To separate anthocyanins, a Luna C18\n(2) chromatographic column (250 mm x 4.6 mm, sorbent size 5 \u03bcm); the Phenomenex column was used. The flow rate of the mobile phase during analysis is 1\nml\/min. The temperature of the column thermostat is 34\u00ba C. The volume of the\ninjected sample is 10 \u00b5l. Detection was performed at a wavelength of 520 nm. A mixture of mobile phases was used for the gradient elution of anthocyanins. Mobile phase A consisted of acetonitrile (for HPLC). Mobile phase B comprised\na mixture of glacial acetic acid, acetonitrile, trifluoroacetic acid, and\nhighly purified water (MilliQ) (10:0.2:87.8). The resulting solutions were\nfiltered through a membrane filter with a pore diameter of 0.2 \u03bcm. The elution mode is presented in Table 1.<\/p>\n\n\n\n

Table 1: The elution mode in the HPLC analysis of dahlia\u2019s anthocyanins<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
\n

Time (min)<\/strong><\/p>\n<\/td>\n

\n

Mobile phase A (%)<\/strong><\/p>\n<\/td>\n

\n

Mobile phase \u0411 (%)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

100<\/p>\n<\/td>\n<\/tr>\n

\n

5<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

100<\/p>\n<\/td>\n<\/tr>\n

\n

20<\/p>\n<\/td>\n

\n

20<\/p>\n<\/td>\n

\n

80<\/p>\n<\/td>\n<\/tr>\n

\n

25<\/p>\n<\/td>\n

\n

40<\/p>\n<\/td>\n

\n

60<\/p>\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

100<\/p>\n<\/td>\n<\/tr>\n

\n

35<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

100<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

The acidified\naqueous extracts were processed to obtain the anthocyanins from plant raw material. 2.00 g of dried and\ncrushed raw material was sifted through a sieve with a hole diameter of 1-2 mm,\nplaced in a 100 mL round-bottom flask, poured with 50 mL of a 4% phosphoric\nacid solution, and kept in a boiling water bath for 30 minutes using a reflux\ncondenser. The resulting solutions were cooled to room temperature and filtered\nthrough a red ribbon paper filter. Before the chromatographic analysis, the\ntest samples were filtered through a membrane filter with a diameter of 0.45 \u03bcm37<\/sup>.<\/p>\n\n\n\n

The absorption spectra were recorded during HPLC analysis. The spectral measurements were carried out in the wavelength\nrange 200-600 nm and the volume of the injected sample\nwas 10 \u03bcL. To determine the total content of anthocyanins,\nthe normalization method was used.<\/p>\n\n\n\n

The contents of identified anthocyanins were determined by the area of the peaks. The content of each compound was calculated based on  the total amount of anthocyanins36<\/sup> using the formula:<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

where: \u0410i<\/sub> \u2013 peak area calculated from chromatograms of the test solution;<\/p>\n\n\n\n

\u0410n<\/sub> \u2013 the sum of the pigment peak areas, calculated from the chromatograms of the test solution.<\/p>\n\n\n\n

Pharmacological <\/strong>in vitro<\/em><\/strong> <\/strong>studies<\/strong><\/p>\n\n\n\n

To obtain a dry extract for pharmacological study, the\nflowers of both Dahlia cultivars were crushed to a particle size of 2-3 mm. 50%\nethanol acidified with hydrochloric acid was used as an extractant. The\nextraction was carried out three times. The raw material-extractant ratio was\n1:9 and the time of each extraction was 1 h. The obtained extracts were\nfiltered, combined, and then evaporated in a vacuum evaporator at a temperature\nof 50\u00ba C. The yield of the dry extract from the flowers of the \u2018La Baron\u2019 cultivar\nwas 12.06%, and from \u2018Colorado Classic\u2019 it was 11.34%.<\/p>\n\n\n\n

The quantitative\nassessment of the basic in vitro<\/em>\ncytotoxicity of the test substances was performed using microscopy under SOP\n\/T\/001.538<\/sup>,<\/sup>39<\/sup>. The rat BMC were isolated from the diaphysis of the femoral bones\nof animals according to the generally accepted method37<\/sup>. The\nresulting suspension contained single cells in an amount of 2.0-2.1\u2219106 cells\/mL.\nCells were incubated at 37\u00b12\u00b0C (without CO2<\/sub>). To obtain the initial\nsolutions of the studied substances, the dry extracts were dissolved in a physiological\nsolution (the concentration of the initial solutions was 2%). Then the initial\nsolutions were titrated in an immunological plate using the rolling method. The\nfollowing concentrations of the test substances were studied: 1%, 0.5%, 0.25%,\n0.125% and 0.0625%.<\/p>\n\n\n\n

Equal volumes of\nrat MBC suspension were added to each well of the immunological plate with the studied\nextracts. The native rat MBC in suspension with saline was used as a negative\ncontrol. A quantitative assessment of the basic cytotoxic effect of the samples\nwas carried out after 15, 45and 90 min of incubation.<\/p>\n\n\n\n

The quantitative assessment of included cytotoxicity was conducted by counting\nthe number of viable\/non-viable cells in the Goryaev chamber. The results were\nexpressed as the percentage of non-viable cells comparatively to their total\nnumber. The 0.1% trypan blue staining method was used to determine cell\nviability. Trypan blue is an acidic aniline dye that is unable to penetrate\ncells through an intact cell membrane; it selectively stains dead cells with\ndamaged cell membranes37<\/sup>,<\/sup>38<\/sup>.<\/p>\n\n\n\n

Statistical analysis<\/strong><\/p>\n\n\n\n

The statistical processing of the results of quantitative determinations\nwas carried out by the method of dispersion analysis following the requirements\nof the State Pharmacopoeia of Ukraine under the Microsoft Excel 7.0 program\nusing the “Statistica” application program package36<\/sup>.<\/p>\n\n\n\n

Statistical processing of the obtained pharmacological data was\nprocessed using the Statistica 11 program using analysis of the variance of the\ndata at a significance level of p<0.05.<\/p>\n\n\n\n

Results and Discussion<\/strong><\/p>\n\n\n\n

The profile and content of anthocyanins in the studied\ndahlia cultivars<\/strong><\/p>\n\n\n\n

According to the results of\nspectrophotometric analysis (Table 2), the total amount of anthocyanins was slightly\nhigher in the flowers of the \u2018La Baron\u2019 cultivar (1.250%) compared to the \u2018Colorado\nClassic\u2019 one (1.138%).<\/p>\n\n\n\n

Table 2: Quantitative content of anthocyanins in the flowers of \u2018Colorado Classic\u2019 and \u2018La Baron\u2019 cultivars of dahlia (n=5)<\/strong><\/p>\n\n\n\n\n\n\n\n
\n

\u0421ultivar<\/strong><\/p>\n<\/td>\n

\n

Content, %<\/strong><\/p>\n<\/td>\n

\n

\u00b1\u03b5, %<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

\u2018Colorado Classic\u2019<\/p>\n<\/td>\n

\n

1.<\/strong>138<\/strong> \u00b1 0.002<\/strong><\/p>\n

\u0425\u0441\u0440<\/sub> = 1.138<\/p>\n

S2<\/sup> = 0.0000050<\/p>\n

Scp<\/sub> = 0.00100<\/p>\n

P = 0.95<\/p>\n<\/td>\n

\n

0.2442<\/p>\n<\/td>\n<\/tr>\n

\n

\u2018La Baron\u2019<\/p>\n<\/td>\n

\n

1.<\/strong>250<\/strong> \u00b1 0.002<\/strong><\/p>\n

\u0425\u0441\u0440<\/sub> = 1.250<\/p>\n

S2<\/sup> = 0.0000053<\/p>\n

Scp<\/sub> = 0.00102<\/p>\n

P = 0.95<\/p>\n<\/td>\n

\n

0.2289<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

The UV spectra of anthocyanins in raw material of both studied cultivars are presented in Fig. 3,4.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 3: UV<\/strong>–<\/strong>spectrum of anthocyanins from the flowers of the \u2018La Baron\u2019 cultivar<\/strong><\/p>\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure 4: UV<\/strong>–<\/strong>spectrum of anthocyanins from the flowers of the \u2018Colorado Classic\u2019 cultivar<\/strong><\/p>\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The chromatographic profiles of anthocyanins were analyzed by the HPLC method (Fig. 5,6). The revealed anthocyanins were the derivatives of the delphinidin, cyanidin, petunidin, peonidin, and malvidin groups (Table 3). It was found that the predominant groups of anthocyanins for both studied cultivars were cyanidin and delphinidin derivatives. The content of the petunidin, peonidin, and malvidin groups was significantly lower.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 5: HPLC chromatogram of anthocyanins extracted from the flowers of the \u2018La Baron\u2019 cultivar<\/strong><\/p>\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure 6: HPLC-chromatogram of anthocyanins extracted from the flowers of the \u2018Colorado Classic\u2019 cultivar<\/strong><\/p>\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 3: Composition of anthocyanins in the flowers of \u2018La Baron\u2019 and \u2018Colorado Classic\u2019 dahlia cultivars<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Compound<\/strong><\/p>\n<\/td>\n

\n

Cultivars<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

\u2018La Baron\u2019<\/strong><\/p>\n<\/td>\n

\n

\u2018\u0421olorado Classic\u2019<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Retention time, min<\/strong><\/p>\n<\/td>\n

\n

Content, %<\/strong><\/p>\n<\/td>\n

\n

Retention time, min<\/strong><\/p>\n<\/td>\n

\n

Content, %<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Delphinidin-3-O<\/em>-galactoside<\/p>\n<\/td>\n

\n

11.12<\/p>\n<\/td>\n

\n

7.23<\/p>\n<\/td>\n

\n

11.11<\/p>\n<\/td>\n

\n

23.15<\/p>\n<\/td>\n<\/tr>\n

\n

Delphinidin-3-O<\/em>-glucoside<\/p>\n<\/td>\n

\n

12.54<\/p>\n<\/td>\n

\n

0.10<\/p>\n<\/td>\n

\n

12.36<\/p>\n<\/td>\n

\n

0.16<\/p>\n<\/td>\n<\/tr>\n

\n

Cyanidin-3-O<\/em>-galactoside<\/p>\n<\/td>\n

\n

13.46<\/p>\n<\/td>\n

\n

23.01<\/p>\n<\/td>\n

\n

13.46<\/p>\n<\/td>\n

\n

8.42<\/p>\n<\/td>\n<\/tr>\n

\n

Delphinidin-3-O<\/em>-arabinoside<\/p>\n<\/td>\n

\n

14.05<\/p>\n<\/td>\n

\n

12.05<\/p>\n<\/td>\n

\n

14.06<\/p>\n<\/td>\n

\n

39.80<\/p>\n<\/td>\n<\/tr>\n

\n

Cyanidin-3-O<\/em>-glucoside<\/p>\n<\/td>\n

\n

14.50<\/p>\n<\/td>\n

\n

0.73<\/p>\n<\/td>\n

\n

14.50<\/p>\n<\/td>\n

\n

1.26<\/p>\n<\/td>\n<\/tr>\n

\n

Petunidin-3-O<\/em>-glucoside<\/p>\n<\/td>\n

\n

15.07<\/p>\n<\/td>\n

\n

4.44<\/p>\n<\/td>\n

\n

15.12<\/p>\n<\/td>\n

\n

1.70<\/p>\n<\/td>\n<\/tr>\n

\n

Cyanidin-3-O<\/em>-rutinoside<\/p>\n<\/td>\n

\n

15.32<\/p>\n<\/td>\n

\n

1.96<\/p>\n<\/td>\n

\n

15.32<\/p>\n<\/td>\n

\n

2.80<\/p>\n<\/td>\n<\/tr>\n

\n

Cyanidin-3-O-<\/em> arabinoside<\/p>\n<\/td>\n

\n

15.83<\/p>\n<\/td>\n

\n

31.85<\/p>\n<\/td>\n

\n

15.84<\/p>\n<\/td>\n

\n

11.41<\/p>\n<\/td>\n<\/tr>\n

\n

Petunidin-3-O<\/em>-rutinoside<\/p>\n<\/td>\n

\n

16.33<\/p>\n<\/td>\n

\n

0.21<\/p>\n<\/td>\n

\n

16.33<\/p>\n<\/td>\n

\n

0.18<\/p>\n<\/td>\n<\/tr>\n

\n

Peonidin-3-O<\/em>-galactoside<\/p>\n<\/td>\n

\n

16.58<\/p>\n<\/td>\n

\n

3.80<\/p>\n<\/td>\n

\n

16.59<\/p>\n<\/td>\n

\n

1.28<\/p>\n<\/td>\n<\/tr>\n

\n

Petunidin-3-O<\/em>-arabinoside<\/p>\n<\/td>\n

\n

16.96<\/p>\n<\/td>\n

\n

3.50<\/p>\n<\/td>\n

\n

16.96<\/p>\n<\/td>\n

\n

0.57<\/p>\n<\/td>\n<\/tr>\n

\n

Peonidin-3-O<\/em>-glucoside<\/p>\n<\/td>\n

\n

17.48<\/p>\n<\/td>\n

\n

0.67<\/p>\n<\/td>\n

\n

17.50<\/p>\n<\/td>\n

\n

0.11<\/p>\n<\/td>\n<\/tr>\n

\n

Delphinidin-3-O<\/em>-xyloside<\/p>\n<\/td>\n

\n

18.08<\/p>\n<\/td>\n

\n

5.29<\/p>\n<\/td>\n

\n

18.09<\/p>\n<\/td>\n

\n

0.45<\/p>\n<\/td>\n<\/tr>\n

\n

Malvidin-3-O<\/em>-galactoside<\/p>\n<\/td>\n

\n

18.30<\/p>\n<\/td>\n

\n

0.55<\/p>\n<\/td>\n

\n

18.16<\/p>\n<\/td>\n

\n

1.86<\/p>\n<\/td>\n<\/tr>\n

\n

Peonidin-3-O<\/em>-arabinoside<\/p>\n<\/td>\n

\n

19.00<\/p>\n<\/td>\n

\n

0.14<\/p>\n<\/td>\n

\n

19.01<\/p>\n<\/td>\n

\n

0.03<\/p>\n<\/td>\n<\/tr>\n

\n

Cyanidin-3-O-<\/em>xyloside<\/p>\n<\/td>\n

\n

19.60<\/p>\n<\/td>\n

\n

0.16<\/p>\n<\/td>\n

\n

19.60<\/p>\n<\/td>\n

\n

0.24<\/p>\n<\/td>\n<\/tr>\n

\n

Malvidin-3-O<\/em>-glucoside<\/p>\n<\/td>\n

\n

19.99<\/p>\n<\/td>\n

\n

2.36<\/p>\n<\/td>\n

\n

20.04<\/p>\n<\/td>\n

\n

0.47<\/p>\n<\/td>\n<\/tr>\n

\n

Malvidin-3-O<\/em>-arabinoside<\/p>\n<\/td>\n

\n

20.80<\/p>\n<\/td>\n

\n

0.26<\/p>\n<\/td>\n

\n

20.82<\/p>\n<\/td>\n

\n

0.03<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Table 3 shows data on the percentage of each component\namong all identified. As can be seen from Table 3, a larger total\namount of anthocyanins was identified in the flowers of the \u2018La\nBaron\u2019 cultivar than in the \u2018Colorado\nClassic\u2019. Among the representatives of the delphinidin group, four glycosides were identified in the flowers of both cultivars. It should be\nnoted that\nanthocyanins of the delphinidin group accumulated in a larger\namount in the \u2018Colorado Classic\u2019 cultivar (67.7%) compared to \u2018La Baron\u2019 one (25.1%). Delphinidin-3-O-arabinoside\nand delphinidin-3-O-galactoside accumulated in the largest quantities in both cultivars. Delphinidin-3-O<\/em>-rutinoside, delphinidin-3-O-xyloside and delphinidin-3-O-glucoside also accumulated in insignificant quantities.<\/p>\n\n\n\n

In the flowers of both cultivars, five substances of\nthe cyanidin group were identified. The total content of cyanidins in the\nflowers of the \u2018La Baron cultivar was 58.7%, and in the \u2018Colorado Classic\u2019\n25.7%. Among the identified cyanidins, cyanidin-3-O-arabinoside and\ncyanidin-3-O-galactoside accumulated in large quantities in the flowers of both\ncultivars compared to the other three cyanidin glycosides. The total content\nof petunidin glycosides was higher in the flowers of the \u2018La Baron cultivar\n(8.3% of the total anthocyanins) compared to the \u2018Colorado Classic one (2.6%).\nThe total content of peonidin was 4.7% in the \u2018La Baron\u2019 cultivar and 1.5% in\nthe \u2018Colorado Classic\u2019 Malvidin anthocyanins were represented\nby a higher content in the flowers of the \u2018La\nBaron\u2019 cultivar (3.2%) compared to the \u2018Colorado Classic\u2019 (1.5%). <\/p>\n\n\n\n

Pharmacological studies<\/strong><\/p>\n\n\n\n

As it is known, the in vitro<\/em> cytotoxicity assays are used for the initial assessment of the toxic potential of chemical or natural substances. It allows to evaluation the effects of plant extracts on cells\u2019 viability, growth, and membrane permeability. <\/p>\n\n\n\n

The results of the conducted in vitro<\/em> studies of the effect of the extracts obtained from \u2018La Baron\u2019 and \u2018Colorado Classic\u2019 cultivars of dahlia on the viability of rat BMC are presented in Fig. 7,8. It was revealed that both dry extracts showed a cytoprotective effect on cell viability at concentrations of 0.0625 and 0.125 for all three exposures of 15, 45, and 90 min.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Fig<\/strong>ure 7<\/strong>:<\/strong> Determination of the basic cytotoxicity of a dry extract from dahlia flowers of the <\/strong>\u2018<\/strong>La Baron<\/strong>\u2019<\/strong> cultivar (<\/strong>n=3<\/strong>).<\/strong><\/p>\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure <\/strong>8<\/strong>: Determination of the basic cytotoxicity of a dry extract from dahlia flowers of the <\/strong>\u2018<\/strong>Colorado Classic<\/strong>\u2019<\/strong> cultivar<\/strong> (<\/strong>n=3<\/strong>).<\/strong><\/p>\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

It was concluded that the dry extract from the flowers of the \u2018La Baron\u2019 cultivar had a significant cytoprotective effect at a concentration of 0.0625%. Under the influence of such a minimum concentration, the number of viable cells increased by 8.0\u20135.0% after exposure for 45 and 90 min. With an increase in concentration of 0.125% in test samples using the \u2018Colorado Classic\u2019 extract, the number of stained cells increased to 23% (Fig. 8). The extract from the \u2018La Baron\u2019 flowers showed cytoprotective activity at all exposures at this concentration.<\/p>\n\n\n\n

A noticeable increase in cell death by\n40% was revealed after the influence of both tested extracts at a concentration\nof extract 0.25% and higher (with an exposure of 90 min). A reliable\ncytotoxic effect of extracts from both cultivars was recorded at a concentration of 0.5%. Moreover, for the \u2018La Baron\u2019 cultivar (with an exposure of 90 min) the death of BMC corresponded to 67%, while for the Colorado Classic\nvariety, it was 50%. At the maximum concentration (1.0%) of extracts, a\ncytotoxic effect was observed at all exposures for both cultivars. The\npercentage of cell death in assays using extracts of \u2018Colorado Classic\u2019 and \u2018La\nBaron\u2019 did not differ significantly from each other.<\/p>\n\n\n\n

Generally, the conducted studies\nmade it possible to establish the in vitro <\/em>cytoprotective and cytotoxic\nactivities of dry extracts from the flowers of \u2018La Baron\u2019 and \u2018Colorado Classic\u2019\ncultivars on rats\u2019 BMC. The obtained results indicate that the vital activity of BMC was\ninfluenced by both the concentration of the dry extracts studied as well as the\ntime of exposure to the cells. At the lower concentrations of 0.065-0.25%, the \u2018La\nBaron\u2019 and \u2018Colorado Classic\u2019 extracts did not have a cytotoxic effect on BMC. On\nthe contrary, they demonstrated cytoprotective activity. Thus, more than 50% of\nBMC died when it used the concentrations of 0.5-1.0% which revealed the\npresence of a cytotoxic effect.<\/p>\n\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

The most common anthocyanins found in plants are cyanidin, delphinidin,\nmalvidin, petunidin and pelargonidin4<\/sup>,20<\/sup>. In fruits and vegetables, cyanidin\naccounts for 50% of the distribution, while delphinidin and peonidin account\nfor 12%, and malvidin and petunidin each account for 7%20<\/sup>. Study of black flowers of dahlia cultivars revealed that the\ndark color of the petals is influenced by a significant amount of compounds\nfrom the cyanidin group40<\/sup>.\nSpecifically, the\nflowers of the ‘Dandy’ cultivar contained cyanidin-3-glucoside-5-arabinoside,\nwhile the flowers of Dahlia variabilis<\/em> accumulated cyanidin-3-monoside,\npelargonidin, and delphinidin4<\/sup>2<\/sup>.<\/p>\n\n\n\n

Pelargonidin glycosides, including diglucoside pelargonin and 3-monoside pelargonin, have been found in Dahlia coecinea<\/em> and Dahlia<\/em> coronata<\/em>43<\/sup>. Granados-Balbuena and co-authors investigated the anthocyanins profile of Dahlia pinnata<\/em> flowers using UPLC-MS\/MS. They identified four major substances, namely delphinidin-3-glucoside, delphinidin-3-rutinoside, pelargonidin-3-sambubioside-5-glucoside, and peonidin-3-sambubioside-5-glucoside21<\/sup>. <\/p>\n\n\n\n

The color of\nanthocyanins and anthocyanidins is determined\nby how they absorb light in the UV and visible parts of the spectrum. When\nthe pH is low, anthocyanins\nare in the form of flavylium cations, and at neutral pH, uncharged quinones\nare formed. Changes in pH can destroy anthocyanins and cause\nthem to lose their color. Generally, adding hydroxyl groups to anthocyanins\nmakes them bluer but less stable, while adding methyl groups makes them redder\nand more stable45<\/sup>. Berries that are red, blue, violet, or purple are the richest sources of\nanthocyanins, containing up to 5 mg\/g46<\/sup>.<\/p>\n\n\n\n

Recent studies have shown that delphinidin and its\nglycosides represent a promising group of substances for the treatment of lung,\nskin, liver, prostate, breast, and ovarian cancer47<\/sup>, they have\nantioxidant and anti-inflammatory properties5,4<\/sup>8-51<\/sup>. Cyanidin-3-O-galactoside is a monoglycosylated anthocyanin found in\nfruit of chokeberries, red-skinned apples, blueberry, elderberry, and\nblackberry and cranberries which are an important part of the human diet. The amount of Cy3Gal varies depending on the species.\nThe accumulation of Cy3Gal and other anthocyanins is significantly influenced\nby temperature, light, and air humidity 5<\/sup>2<\/sup>.\nThe presence of this substance is linked to the dark red or dark purple coloration of the fruits5<\/sup>3<\/sup>. <\/p>\n\n\n\n

Rupasinghe and the co-authors discovered\nthat the concentration of cyanidin-3-O-galactoside in apple juice can reach 39 mg\/L. This juice exhibited antioxidant activity in in vitro<\/em> ferric ion reduction\nantioxidant capacity and oxygen\nradical absorbance capacity assays.\nThese studies suggested\nthat cyanidin-3-O-galactoside could be used as an effective therapeutic additive5<\/sup>4<\/sup>.\nThere is evidence that cyanidin-3-O-galactoside improves human cognitive\nfunction5<\/sup>5<\/sup>. <\/p>\n\n\n\n

Lim and the co-authors conducted experiments on mice and found\nthat an extract from the fruits of Aronia\nmelanocarpa<\/em>, which was rich in cyanidin-3-O-galactoside, exhibited anti-obesity properties56<\/sup>.\nThe supplementation of this extract was linked\nwith a decrease in insulin resistance, serum triglyceride levels, low-density\nlipoproteins, and total cholesterol. It also resulted\nin a reduction in the weight of white adipose tissue. Furthermore,\nstudies have demonstrated that anthocyanins\nfrom the purple tubers of Ipomoea batatas<\/em>\nare effective in protecting the kidneys against\nuric acid-related disorders57<\/sup>.<\/p>\n\n\n\n

Certain substances act as effective\nantioxidants, specifically petunidin-3-O-arabinoside and\npetunidin-3-O-glucoside. These substances are found in high concentrations in blueberries, dark\ngrapes, raspberries, and strawberries56<\/sup>. Additionally, peonidin-3-galactoside was found in red\nraspberry, mango, blackberry, and blueberry, while peonidin-3-O-glucoside was\nfound in red wine, purple onions, and red corn58<\/sup>. <\/p>\n\n\n\n

Malvidin-3-O-galactoside is a\ncompound found in various blueberry varieties and is known for its antitumor\nactivity. Studies have shown that it can inhibit the proliferative\nability of Huh-7 cells19<\/sup><\/a>. Furthermore, it can\npromote cell apoptosis and inhibit cell proliferation through the regulation of\np38\/JNK\/ERK MAPK and Akt\/PTEN signaling pathways59<\/sup><\/a>. Additionally, malvidin and its glycosides have properties\nthat can help regulate blood sugar, prevent cardiovascular diseases, and\nimprove brain function due to their antioxidant and anti-inflammatory effects.<\/p>\n\n\n\n

After analyzing\ndata from scientific sources on the content of anthocyanins in the fruits and\nseeds of various plants, it was found that the\nblack soybean varieties contained anthocyanin content ranging from 1.89-26.33\nmg\/g60<\/sup>. The anthocyanin content of flesh berries varies significantly, depending\non the species. Thus, the amounts of anthocyanins vary from\n75.0 mg\/g fresh fruit in Ribes rubum<\/em>\nup to 46000 mg\/g fresh fruit in Aronia\nmelanocarpa<\/em>61<\/sup>. <\/p>\n\n\n\n

There is scientific data that the properties and reactivity of substances are\naffected by their spatial structure. Stetsenko and the co-authors utilized\nquantum-chemical methods with modern computer software to investigate how the\nspatial arrangement of substances impacts their properties and reactivity60<\/sup>. Their research revealed that\nanthocyanins have a significant ability to counteract free radicals when proton\nlevels are elevated. Anthocyanins are electrophilic compounds, enabling them to\nreadily capture unpaired electrons from free radicals in chemical reactions,\nwhich explains their strong antioxidant properties. By scavenging oxygen\nradicals within cells, anthocyanins can prevent oxidative stress and protect\ncell membranes from damage. This suggests that the membrane-stabilizing and\nantioxidant effects of anthocyanins can be anticipated and potentially\nharnessed in certain doses.<\/p>\n\n\n\n

Therefore,\nderivatives of cyanidin, delphinidin, petunidin, peonidin, and malvidin from\nplant sources could be considered very\npromising substances for the prevention of chronic non-infectious diseases such\nas cardiovascular, metabolic, and neurodegenerative disorders.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

In this\nstudy, we used HPLC and spectrophotometric methods to examine the anthocyanin\nprofiles of two dahlia cultivars: ‘La Baron’ and ‘Colorado Classic’. The revealed\nanthocyanins\nwere classified into delphinidins, cyanidins, petunidins, peonidins, and malvidins groups. A\ntotal of 18 components were identified and measured. The main coloring\nsubstances in ‘Colorado Classic’ flowers were the anthocyanins of the\ndelphinidin group, while the ‘La Baron’ cultivar had a higher concentration of\ncyanidins. Both cultivars\ncontained small amounts of malvidins,\npeonidins, and petunidins. Additionally, the study assessed the toxicity of dry\nextracts from the flowers of the studied cultivars using the BCM in vitro<\/em> model. The results showed that\nthe cytoprotective and cytotoxic effects of the extracts varied with the dose\nadministered. However, further research is required to fully evaluate the\npharmacological properties of the extracts from these two cultivars.<\/p>\n\n\n\n

Acknowledgment<\/strong><\/p>\n\n\n\n

We would like to express our gratitude to the employees of the Botanical Garden at Kharkiv National University named after V.N. Karazin (Kharkiv, Ukraine) for providing us with samples of raw materials.<\/p>\n\n\n\n

Conflict of\ninterest<\/strong><\/p>\n\n\n\n

The authors\ndeclare no conflict of interest <\/p>\n\n\n\n

Funding\nSource<\/strong><\/p>\n\n\n\n

The authors declare that no grants or other support were\nreceived during the preparation of the manuscript..<\/p>\n\n\n\n

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