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https://doi.org/10.29013/AJT-20-1.2-52-55
Usmanov D. A., Ramazonov N. Sh., Institute of the Chemistry of Plant Substances, Academy of Sciences, Tashkent, Uzbekistan E-mail: [email protected]
NEW IRIDOID GLYCOSIDE FROM PHLOMIS SEVERTZOVII
Abstract. Structure of the novel iridoid glycosides, Diasetogin was isolated from the aerial parts of Phlomis severtzovii (Lamiaceae) growing in Uzbekistan and characterised as a new iridoid with a novel structure which has never before been reported in literature from Phlomis or any other source. Its structure has been established as (lS,4aS,6S,7S,7aS)-4-(methoxycarbonyl)-7-methyl-1-((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yloxy)-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-6,7-diyl diacetate. Its structure has been established on the basis of NMR, mass and IR spectral characteristics and chemical transformations.
Keywords: iridoid constituent, Diasetogin, iridoid glycosides, Phlomis severtzovii (Lamiaceae).
Introduction
Phlomis is a large genus of the Lamiaceae family with over 100 species mainly distributed throughout Asia, Europe and North Africa with great prevalence in the Central Asia and Iran (Limem-Ben Amor I. et al. [7]; Li M. X. et al. [6]). The first one includes South and East Anatolia and North-Western Iran, and the second expands from the Central Asian parts of the former Soviet Union to Eastern China (A zizian D. et al. [1]). In the flora of Uzbekistan, the genus Phlomis is represented by about 20 perennial species and 4 spaces are endemic (Vve-denskiy [12]).
Phytochemical studies on various species of genus Phlomis have resulted in the isolation of terpenoids, iridoids, flavonoids, lipids and lignans [Çali§ I. et al. [2]; Ding, M. M. et al. [4]; Usmanov, D. A., et al., [10; 11]]. The various species of this genus generally used as herbal tea against gastrointestinal troubles and to promote health by protecting liver, kidneys, bones and cardiovascular system [Carmo-na, M. D. et al. [3]].
Among the various low-molecular biologically active substances synthesized by plants, iridoids occupy a prominent place. At present time, it is clear, that this type of compounds are widespread in plant world [Galves M., et al., [5]]. Presence of important biological activities (antitumor activity, antimicrobial activity, etc.) of these compounds is main perspective practical reason for further investigation. Iridoid gly-cosides have great theoretical importance as in terms of chemistry and due to their participation as precursors in alkaloid biosynthesis. Therefore search of novel iridoid containing plant sources, development of rational scheme of isolation of these compounds, determination their chemical structure, revealing of physical, chemical parameters and useful properties of novel compounds are actual problems of the modern bioorganic chemistry [Yang, L., et al., [13]].
At this point, new studies on uninvestigated Phlomis species could provide valuable information's in this pool for the genus Phlomis.
In the present work, for the first time were studied the chemical compounds of butanol extract ob-
tained from the aerial part (flowers, leaves, thorns, and stems) of Phlomis severtzovii (Lamiaceae) which grows on fine-earth slopes in the lower belt of the mountains of the Tashkent region of Uzbekistan.
Materials and methods
Plant material
The aerial parts of Phlomis severtzovii was collected from the Tashkent regions of Uzbekistan in 2016 and and identified by Dr. O. A. Nigmatullaev. The authenticated voucher specimen of the specie (Accession no. 1520) is kept in the Department of Herbal Plants (Institute of the Chemistry of Plant Substances, Uzbekistan).
High resolution mass spectra (HR MS) analysis
High resolution mass spectra (HR MS) were measured on a Bruker micrOTOF II instrument using elec-trospray ionization (ESI). The measurements were done in a positive ion mode (interface capillary voltage -4500V) or in a negative ion mode (3200V); mass range from m/z 50 to m/z 3000; external or internal calibration was done with ESI Tuning Mix, Agilent (Tsedilin A. M. et al. [8]). A syringe injection was used for solutions in acetonitrile, methanol, or water (flow rate 3 ^L/min). Nitrogen was applied as a dry gas; interface temperature was set at 180 °C.
Nuclear magnetic resonance spectroscopy.
1H and 13C NMR spectra were obtained on a Bruker AVANCE-II 600 spectrometer with an operating frequency of 600 (1H) and 150 MHz (13C) equipped with a broadband sensor with a Z-gradi-ent coil. Samples were dissolved in pyridine-d5 and placed in NMR tubes (diameter 5 mm). The spectra were recorded at 298 K, chemical shifts were calibrated with respect to the internal standard Me4Si (dH 0.0 ppm, dC0.0 ppm). All one-dimensional and two-dimensional experiments were recorded and processed using the Bruker Topspin software (version 3.2); the experimental parameters were borrowed from the standard TOPSPIN libraries.
Results and discussion
We have previously (Usmanov, D., et al., [9]) reported the presence in the aerial parts of Phlomis
severtzovii of 6^-hydroxypolamid (1), loganin (2), pulchelloside (3), shanshiside methyl ester (4) and phlorigidoside C (5).
By rechromatography on a column of silica gel of the mother solutions obtained in the isolation of the above substances we have now isolated new iridoid glycosides -Diasetogin (6).
According to its PMR spectrum, iridoid (6) contained ester groupings. This was shown by the presence in its PMR spectrum of a six-proton singlet at 2.03 ppm from the methyls of two acetyl groups.
On comparing the PMR spectra of (4) and of the iridoid (6), a considerable paramagnetic shift 1.58 ppm of the signals of the CH3-10 methyl group was observed in the latter. This fact permitted the assumption that in iridoid (6) one of the acetyl groups was present at the C-8 hydroxyl. A paramagnetic shift of the sigwhals of the H-9 and H-1 protons also confirmed the addition of an acetyl group to the hydroxyl at C-8.
In addition, on the acetylation of OH-7 the signals of the H-7 proton underwent a downfield shift. This could have taken place under the influence of an acetyl group located on the same carbon atom.
The acid hydrolysis of iridoid (6) led to D-glu-cose and a black decomposition product of the agly-
^ 1' OH
1: R1 = OH, R2 = OH, R = H, R4 = OH 2: R1 = H, R2 = H, R3 = OH, R4 = H 3: R1 = OH, R2 = OH, R = OH, r4 = H 4: R = H, R2 = OH, R =3 H, R4 = OH 5: R = H, R2 = OH, R + R4 = O
6. R = H, R2 = H, R3 = OCOCH3, R4 = OCOCH3
The anomeric proton, H-1', of the carbohydrate moiety of the iridoid resonated at 5.43 ppm in the form of a doublet with J = 7.76 Hz, which showed the ^-configuration of the glycosidic center.
Using the 1H - 1H NOESY and 1H - 13C HMBC methods, signals corresponding to the gly-cosylated C (1) atom of the aglycone were detected in the spectrum, the chemical shift ofwhich (d 93.82 ppm) indicated its attachment to two oxygen atoms. Then, the assignment of those spectral signals that provide information on the location of sp2-hybrid-ized carbon atoms belonging to ester and olefin fragments was carried out.
When decoding the spectra, the signals corresponding to the carbon atoms of carboxyl groups in the region d 167.77 ppm and acetyl groups in the region d 169.55-169.92 ppm were taken as the initial (reference) points The correlation peaks corresponding to the H(5) proton (at the angular carbon atom), H(3) proton (at the carbon - carbon double bond), and also H(12) protons (as part of the methyl group of the ester fragment) made it possible to establish the structure of the aglycon.
In compound 6, the chemical shifts of C(7) and C(8) atoms, as well as the signal of one proton bound to C(7), according to the HSQC spectrum, indicate the presence of an oxygen-containing substituent at C(7) and C(8) atoms. In compound 6, the hydroxyl groups are acetylated, as evidenced by four signals of carbon atoms in the spectrum and characteristic correlation peaks in the NOESY spectrum between the protons of the methyl groups.
Thus, Diasetogin (6) is 7,8-di-O-acetylloganin.
Diasetogin (6). Compound 6 was isolated as pale yellow crystal, C21H30O13, m.p. 114 °C, UV -(C2H5OH) \ax 235.5 nm. IR (KBr,) v, sm-1: 3388 (OH), 1639 (C=O), 1551 (C=C). Yeild 0.024%.
1H NMR (Bruker 500 MHz, C5D5N): 6.23 (1H, d, /=4.96, H-1), 7.71 (1H, s, H-3), 3.70 (1H, m, H-5), 1.80 (1H, m, Ha-6), 2.63 (1H, m, Hb-6), 5.46 (1H, dd, /=7.53, 6.63, H-7), 3.04 (1H, m, H-9)
1.58 (3H, c, H-10), 3.60 (3H, c, H-12), 2.03 (3H, c, H-14), 1.80 (3H, c, H-16), 5.43 (1H, a, J = 7.76, H-1'), 4.10 (1H, t, H-2'), 4.27 (1H, t, H-3'), 4.27 (1H, t, H-4'), 4.03 (1H, ddd, H-5'), 4.50 (1H, dd,, Ha-6'), 4.34 (1H, dd, Hb-6');
13C NMR (126 MHz, C5D5N): 93.82 (C-1), 152.17 (C-3), 110.16 (C-4), 29.67 (C-5), 35.33 (C-6), 76.78 (C-7), 88.78 (C-8), 49.14 (C-9), 19.59 (C-10), 167.77 (C-11), 51.47 (C-12), 169.55 (C-13), 21.07 (C-14), 169.92 (C-15), 21.82 (C-16), 100.66 (C-1'), 74.71 (C-2'), 78.45 (C-3'), 71.65 (C-4'), 78.35 (c-5'), 62.86 (C-6').
Experimental Part
For general observations, see (Usmanov, D., et al., [9]). For column chromatography we used KSK and L 40/100/zm (Czech Republic) silica gels and the solvent systems chloroform-methanol 50:1 (1) and 100:1 (2). The mother solutions obtained in the isolation of the iridoids described previously from 2000 g of P. se-vertzovii (Usmanov, D., et al., [9]) were combined and chromatographed on a column of silica gel. Elution with system 1 yielded 68 mg of a mixture of two iridoids. Rechromatography ofthis mixture on a column ofsilica gel with the same solvent system led to the isolation of 48 mg (0.024% on the weight of the air-dry raw material) of Diasetogin (6), C21H30O13 (crystals).
Acid Hydrolysis of Diasetogin (6). A solution of 10 mg of the glycoside in 5 ml of 5% sulfuric acid was heated at 100 °C for 3 h, and the precipitate that had formed was separated off. The hydrolysate was neutralized with barium carbonate, concentrated in vacuum, and subjected to TLC in the butanol-pyridine-water (6:4:3) system, which revealed the presence of D-glucose.
Conclusion. In this study, we investigated the iridoid constituent of Phlomis severtzovii growing in Uzbekistan and for first time we have isolated new iridoid identified on basis of UV, IR, 1H and 13C NMR spectra as Diasetogin.
Disclosure Statement
No potential conflict of interest was reported by the authors.
Acknowledgements
Authors would like to thank the ICPS (Uzbek Academy of Sciences) for providing all necessary equipment, chemicals and animals for the study. High resolution mass spectra and NMR spectra were recorded in the Department of Structural
Studies of Zelinsky Institute of Organic Chemistry, Moscow. Funding
The project was funded through a grant from the Republic of Uzbekistan State Foundation [grant number PZ-20170927342].
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