INTRODUCTION:
The herb of Monarda fistulosa contains essential oil, which includes about 40 components. Phenolic chemotypes of Monarda fistulosa are most common, in which predominate thymol and carvacrol caused the antibacterial activity.4,7-9 The component of the Monarda fistulosa essential oils, thymoquinone, in studies on mice with leukemia induced a regression of leukemia.9 It is known, that thymoquinone is effective in the prevention and treatment of cancer of the pancreas, prostate and colon, while reducing the side effects of chemotherapeutic drugs and increasing their antitumor properties.7,9 The anti-inflammatory activity of the Monarda fistulosa was detected in animal experiments.2,3,10 Other valuable biologically active compounds have been described for the plant, in particular flavonoids, which contribute to the pharmacological activity of the plant raw material. The quercetin, rutin, luteolin, luteolin-7-glucoside, kaempferol, hyperoside, naringenin, hesperidin, diosmin, 3,5-diglucoside pelargonidine acylated with coumaric and malonic acids have been identified by various researchers in the monarda herbs.11,12 In our opinion, it is advisable to standardize the herb of Monarda fistulosa not only for essential oil, but also for the content of flavonoids.13 However, despite the attention of scientists to the problems of the standardization of Monarda fistulosa herbs, inconsistency is observed in approaches to the method of analysis of flavonoids in this plant raw materials. The methods of analysis of Monarda fistulosa herbs used by researchers vary multiplicity extraction, the concentration of the ethyl alcohol as the extracting solvent, the analytical wavelength, reference standards.
Thus, the aim of our research is the scientific evidence of the approaches to the standardization of Monarda fistulosa L. herbs.
MATERIAL AND METHODS:
Plant material:
The herb of the Monarda fistulosa L. was collected on the territory of the Samara region, in the Botanical Garden of Samara University in the month of July 2018. The plant was identified by Director of Botanical Garden of Samara University PhD Svetlana Rozno. The voucher specimen (2018/75) was deposited at the Herbarium of Department of Pharmacognosy, Botany and Phytotherapy of Samara State Medical University (Ulitsa Gagarina 18, Samara, 443079, Russian Federation).
Extraction of Plant Materials:
The extraction of the herb of Monarda fistulosa L. (100 g) was carried out with the using of 70% EtOH in ratio 1:10, combining the maceration method with subsequent thermal extraction in a boiling water bath. The obtained water-alcohol extract was filtered and then evaporated by using a rotary evaporator at low temperature (40-50°C) and reduced pressure to thick residue (about 50 ml).
Isolation of Compounds from Plant Materials:
The isolation of flavonoids 1 and 2 from the obtained thick extract of the Monarda fistulosa L. herbs was carried out with the using of the method of adsorption column chromatography. For this the obtained concentrated extract was dried on L 40/100 silica gel and the resulting powder was applied to a silica gel layer formed in chloroform. The chromatographic column was eluted with chloroform and a mixture of chloroform-ethanol in various ratios (99: 1, 97: 3, 95: 5, 93: 7, 90:10, 85:15, 80:20, 75:25, 70:30, 60: 40). As results, we isolated flavonoids 1 and 2. The additional purification of the isolated flavonoids was carried out by recrystallization from the mixture of alcohol and water. The elution of the compounds was monitored by TLC analysis on Sorbfil PTLC-AF-A-UV plates in a system of chloroform-ethanol-water (26: 16: 3) and n-butanol-glacial acetic acid-water (4: 1: 2). The spots on the plate were detected by luminescence in UV light at a wavelength of 254 and 366 nm and by color after processing the chromatograms with an alkaline solution of diazobenzenesulfonic acid.
Methods of Structural Elucidation of Flavonoids:
UV spectra were recorded using a spectrophotometer Specord 40 (Analytik Jena, Germany) in cuvettes with a layer thickness of 10 mm in the wavelength range from 190nm to 700nm. 1H-NMR spectra were obtained on a spectrometer Bruker AM 300 (Bruker, Germany) at a frequency 300 MHz, 13C-NMR spectra were obtained on a spectrometer Bruker DRX 500 (Bruker, Germany) at a frequency 126.76 MHz., mass spectra were recorded on a mass spectrometer Kratos MS-30 (Kratos, United Kingdom. The acidic hydrolysis of flavonoid glycosides was carried out with the using of 2% HCl at heating on the boiling water bath during of 2 h.
Spectrophotometry Analysis of Plant Materials:
The quantitative estimation of total flavonoids in the herb of Monarda fistulosa L. there was carried out by the using of differential spectrophotometry at an analytical wavelength of 394nm calculated on isorhoifolin. The raw material was crushed so that its particles passed through a sieve with holes 1mm in diameter. An accurate weighed sample of ground material (about 1g) was placed in a 100mL flask and 50mL of 60% ethyl alcohol was added. The flask was closed with a stopper and weighed on a balance accurate to 0.01g. The flask was attached to a reflux condenser and heated in a boiling water bath for 60 min. After boiling, the flask was cooled for 30 min, closed with the same stopper, weighed again, and the extractant was added to its original weight. The resulting aqueous-alcoholic extract was filtered through a paper filter. The resulting extract (1mL) was poured into a 50mL volumetric flask, 2mL of a 3% alcohol solution of aluminum chloride was added, then the solution volume was adjusted to the mark with 96% ethyl alcohol (test solution). The optical density of the test solution was determined 40 min after preparation on a spectrophotometer at a wavelength of 394nm (to calculate the content of flavonoids). The comparison solution was a solution containing 1 mL of an aqueous-alcoholic extract (1:50) and 96% ethyl alcohol, which was adjusted to the mark in a 50 mL flask.
The content of total flavonoids in percent (X) in terms of isorhoifolin and absolutely dry raw materials was calculated by the formula:
(A*50*50*100)
Х= ----------------------------
(m*195*(100-W)
where:
A optical density of the test solution;
m mass of raw materials, g;
195 specific absorbance (E
) of isorhoifolin at 394 nm;
W mass loss on drying, %.
RESULTS AND DISCUSSION:
In the course of our research were there first for the time were isolated from Monarda fistulosa L. isorhoifolin (1) and linarin (2) (Fig. 1), identified with using of UV, 1H-NMR (Fig. 2 and 3), 13C-NMR spectroscopy, mass spectrometry, and chemical transformations.
Figure 1: The chemical structures of flavonoids isolated from Monarda fistulosa herbs:
R = H: isorhoifolin; R = CH3: linarin.
Isorhoifolin (7-O-rutinoside of apigenin) (1). The crystalline substance is a light-yellow color composition C27H30O14; m.p. 257260°C (water alcohol). Λmаx EtOH 270, 340 nm; + NaOAc 270, 340 nm; + NaOAc + H3BO3 270, 405 nm; + АlCl3 278, 308, 345, 384 nm; + АlCl3 + HCl 278, 308, 345, 384 nm; + NaOMe 254, 269, 400 nm.
1H-NMR spectrum (300 MHz, DMSO-d6, δ, ppm, J/Hz): 1.07 (3H, d, J = 6, CH3 of rhamnose), 3.05.2 (10H of rutinose), 4.56 (1H, br. s, H-1 of rhamnopyranose), 5.07 (1H, d, J = 7, H-1 of glucopyranose), 6.46 (1H, d, J = 2, H-6), 6.77 (1H, d, J = 2, H-8), 6.87 (1H, s, H-3), 6.93 (2H, d, J = 8.5, H-3 and H-5), 7.94 (2H, d, J = 8.5, H-2 and H-6), 12.95 (1H, s, 5-OH group).
13C-NMR spectrum (126.76 MHz, DMSO-d6, δC, ppm): C-2 (162.87), C-3 (103.11), C-4 (181.97), C-5 (161.17), C-6 (99.54), C-7 (164.37), C-8 (94.79), C-9 (156.92), C-10 (105.38), C-1 (121.03), C-2 and C-6 (128.62), C-3 and C-5 (116.06), C-4 (161.32), C-1 of glucose (99.91), C-2 (72.06), C-3 (75.62), C-4 (70.74), C-5 (76.27), C-6 (66.05), C-1 of rhamnose (100.52), C-2 (70.32), C-3 (69.57), C-4 (73.08), C-5 (68.31), C-6 (CH3 of rhamnose) (17.79).
Маss-spectrum (ESI-MS, 180°C, m/z): m/z 579.1739 [M+H]+, m/z 601.1554 [M+Nа]+, m/z 617.1285 [M+K]+.
Linarin (7-O-rutinoside of acacetin) (2). Crystalline substance of white color composition C28H32O14; m.p. 260°C (dec.) (water alcohol). λmаx EtOH 272, 330 nm; + NaOAc 272, 330 nm; + NaOAc + H3BO3 272, 330 nm; + АlCl3 280, 384 nm; + АlCl3 + HCl 280, 384 nm; + NaOMe 287, 372 (sh.) nm.
1H-NMR spectrum (300 MHz, DMSO-d6, δ, ppm, J/Hz): 1.07 (3H, d, J = 6, CH3 of rhamnose), 3.05.3 (10H of rutinose), 3.83 (s, 3H, CH3O), 4.54 (1H, br. s, H-1 of rhamnopyranose), 5.07 (1H, d, J = 7, H-1 of glucopyranose), 6.45 (1H, d, J = 2, H-6), 6.78 (1H, d, J = 2, H-8), 6.92 (1H, s, H-3), 7.14 (2H, d, J = 8.5, H-3 and H-5), 8.04 (2H, d, J = 8.5, H-2 and H-6), 12.90 (1H, s, 5-OH group).
13C-NMR spectrum (126.76 MHz, DMSO-d6, δC, ppm): C-2 (163.94), C-3 (103.80), C-4 (182.01), C-5 (161.13), C-6 (99.65), C-7 (165.11), C-8 (96.45), C-9 (156.96), C-10 (105.45), C-1 (122.66), C-2 and C-6 (128.44), C-3 and C-5 (114.70), C-4 (162.42), C-1 of glucose (99.94), C-2 (73.06), C-3 (75.66), C-4 (70.34), C-5 (76.24), C-6 (68.31), C-1 of rhamnose (100.51), C-2 (70.74), C-3 (70.25), C-4 (72.05), C-5 (69.60), C-6 (CH3 of rhamnose) (17.79), CH3O (55.55).
Mass-spectrum (ESI-MS, 180°C, m/z): m/z 593.1888 [M+H]+, m/z 615.1710 [M+Nа]+.
Figure 2: 1H-NMR spectrum of isorhoifolin (1) in DMSO-d6.
Figure 3: 1H-NMR spectrum of linarin (2) in DMSO-d6.
The results of the study of the electronic spectra of water-alcohol extraction of Monarda fistulosa herbs indicate that raw materials are characterized by absorption maxima at wavelengths of 270 ± 2 and 330 ± 2nm (Fig. 4). A study of the UV spectrum of water-alcohol extraction (differential version) showed that the main absorption maximum is observed at a wavelength of 394 nm (Fig. 5). Moreover, the specificity of the absorption of this spectrum correlates with the UV spectrum of isorhoifolin. The bathochromic shift of the maximum at 330 nm to the region of 394 nm is observed in the presence of AlCl3 (Fig. 6 and 7). It was determined, that linarin has similar spectral characteristics. This suggests, that in the presence of AlCl3, the specificity of the absorption curve of the UV spectrum of the wateralcohol extraction of the Monarda fistulosa herbs is mainly due to isorhoifolin and linarin. On this basis, a methodology has been developed for determination of the total of flavonoids using an analytical wavelength of 394 nm in a variant of differential spectrophotometry.
Figure 4: The UV spectra of water-alcohol extraction of Monarda fistulosa herbs.
Designations: 1-extraction; 2-extraction with the addition of aluminum chloride.
Figure 5: The differential UV spectrum of water-alcohol extraction of Monarda fistulosa herbs.
Figure 6: The UV spectra of alcohol solutions of isorhoifolin. Designations: 1 - initial solution; 2 the solution with the addition of aluminum chloride.
Figure 7: The differential UV spectrum of alcohol solution of isorhoifolin.
The specific absorbance of the isolated isorhoifolin in 70% ethanol was determined at an analytical wavelength of 394 nm for standardization purposes. It amounted to 195. The obtained value of the specific absorbance was used in the development of the method for quantitative determination of the total flavonoids in the Monarda fistulosa herbs.
During the development of the method of quantitative determination of the total flavonoids, we used previously determined optimal parameters for the extraction of flavonoids from the herb of Monarda fistulosa: extraction solvent60% ethyl alcohol; the ratio of raw material - extraction solvent1:50; extraction time extraction in a boiling water bath for 60 minutes13
The results of statistical processing of the analytical data indicate that the error of a single determination of the total flavonoids in the Monarda fistulosa herbs with a confidence probability of 95% is ± 4.65%. It was also determined that the content of flavonoids in the Monarda fistulosa herb varies from 5.96±0.08% to 7.68±0.12%.
CONCLUSION:
From the herb of the Monarda fistulosa L. there were isolated for the first time isorhoifolin (apigenin-7-O-rutinoside) and linarin (acacetin-7-O-rutinoside) chemical structures of which there were elucidated by means of 1H-NMR-, 13C-NMR-, UV-spectroscopy, mass spectrometry and results of chemical transformations. It was determined, that the isolated flavonoids cause the spectral characteristics in the UV spectra of the water-ethanolic extracts from the Monarda fistulosa L. herbs. It was substantiated the expediency of the estimation of the total flavonoids in the herbs of Monarda fistulosa L. calculated on isorhoifolin by the using of the differential spectrophotometry at an analytical wavelength of 394 nm. It was determined that the content of the total of flavonoids in the raw materials of the studied plant varies from 4.23±0.08% to 7.68± 0.12%.
CONFLICTS OF INTEREST:
The authors declare no conflicts of interest.
REFERENCES:
1. Kurkin VA Farmakognoziya: uchebnik dlya studentov farmatsevticheskikh vuzov (fakul'tetov) [Pharmacognosy: a textbook for students of pharmaceutical universities (faculties)]. Samara: Ofort Publ.; 2019; pp. 1278.
2. Wild bergamot (Monarda fistulosa L.). United States Department of Agriculture Natural Resourced Conswevation Service. URL: https://plants.usda.gov/plantguide/pdf/cs_mofi.pdf 17.08.2020.
3. Naumenko EN, Zhilyakova ET, Novikov OO et al. Research of anti-inflammatory activity of medicine "Monavitol" in vivo. Scientific statements of Belgorod State University. Series: Medicine. Pharmacy. 2012; 22-1(141): 195-198.
4. Oparin RV, Pokrovsky LM, Vysochina GI, Tkachev AV. The study of the chemical composition of essential oils Monarda fistulosa L. and Monarda didyma L., cultivated in Western Siberia Chemistry of Plant Raw Materials. 2000; 3: 19-24.
5. Krasyuk EV, Makarova NN, Petrova IV, Pupykina KA, Valeeva LA. Pharmacological activity of monarda types, introduced in the Republic of Bashkortostan. Medical Bulletin of Bashkortostan. 2015; 10(5): 67-70.
6. Fedotov SV. Monarda essential oils of Monarda fistulosa L., Monarda didyma L., Monarda citriodora Cervantes ex Lag., their chemotypes and biological activity. Collection of scientific papers of the State Nikitsky Botanical Garden. 2015; 141: 131-147.
7. Nikolaevsky VV. Aromatherapy: A Guide. M.: Medicine Publ.; 2000; pp. 336 p.
8. Nikitina AS, Aliev AM, Feskov SA, Nikitina NV. Component composition of essential oil Monarda fistulosa L. herbs from the collection of Nikitsky Botanical Garden Chemistry of Plant Raw Materials. 2018; 2: 55-62.
9. Pang J, Shen N, Yan F, Zhao N, Dou L, Wu LC., Seiler CL, Yu L, Yang K, Bachanova V, Weaver E, Tretyakova NY, Liu S. Thymoquinone exerts potent growth-suppressive activity on leukemia through DNA hypermethylation reversal in leukemia cells. Oncotarget. 2017; 8(21): 34453-34467.
10. Zhilyakova ET, Novikov OO, Naumenko EN et al. Antimicrobial and anti-inflammatory activity of new formulation with monarda oil. Scientific reports of Belgorod State University. Series: Medicine. Pharmacy. 2013; 25-1(168): 198-201.
11. Dmitrienko SG, Stepanova AV, Kudrinskaya VA. Apyari VV. Specifics of separation of flavonoids by reverse phase high performance liquid chromatography on the Luna 5u C18(2) column. Moscow University Chemistry Bulletin. 2012; 53(6): 369-373.
12. Krasyuk EV, Pupykina KA. Qualitative analysis and development of methods of quantification of flavonoids in Monarda species introduced in the Republic of Bashkortostan. Medical Bulletin of Bashkortostan. 2016; 11(5): 73-77.
13. Lapina AS, Kurkin VA. The development of approaches to standardization of the Monarda fistulosa herbs. Pharmacy. 2019; 68(4): 11-16.
Received on 19.08.2020 Modified on 13.10.2020
Accepted on 17.11.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(7):3916-3920.
DOI: 10.52711/0974-360X.2021.00680