New coumarin from the roots of Prangos pabularia growing wild in Tajikistan

Abstract Dichloromethane and butanol extracts of the roots of Prangos pabularia were analyzed to determine chemical constituents and biological activity. The new coumarin 1, yuganin B ((5-(((2S,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-((2-oxo-2H-chromen-7-yl)oxy)tetrahydro-2H-pyran-3-yl)oxy)-3,4-dihydroxytetrahydrofuran-3-yl)methyl 4-hydroxy-3-methoxybenzoate) along with three phenolic and twenty-four known coumarins were isolated from the roots of Prangos pabularia, and the structures of these isolated compounds were elucidated by UV, HR-ESIMS, and 1 D and 2 D NMR spectroscopy. In addition, the anti-melanogenic effect of several of the isolated individual compounds and their inhibitory effect on B16 cells were evaluated. Isolating and testing compounds may proof to be useful in the treatment of hyperpigmentation and as a skin-whitening agent in the cosmetics industry. Graphical Abstract


Introduction
Investigations of the chemistry of natural products include identification of the biological and pharmacological active compounds from the plants and their structures and biological activities (Numonov et al. 2018).Medicinal plants have been employed in various traditional medicines throughout the world since ancient times.They have been a rich source of chemicals and many bioactive compounds have been isolated in their pure form (Jiang et al. 2015;Numonov et al. 2017).The main objective of natural product chemistry is to identify and comprehensively investigate the active secondary metabolites from the plants and to help develop them into medicinal drugs (Numonov et al. 2020).Coumarinic compounds are a class of lactones structurally constructed by a benzene ring fused to an a-pyrone ring, and essentially possess a conjugated system with electron-rich and good charge-transfer properties (Numonov et al. 2018).Many coumarin compounds are being actively studied as drug candidates with high pharmacological activity that may ultimately be used, to treat various types of diseases (Carneiro et al. 2021).
Prangos, a genus of herbaceous, perennial distributed in the Mediterranean region and Western and Central Asia, belongs to the Apiaceae family (Sharma et al. 2013).Prangos species have been chemically and biologically investigated.P. ferulacea, is a species traditionally used in many countries for its several properties (Bruno et al. 2019), and coumarins in sufficient quantity were isolated and identified from this plant (Bruno et al. 2021).The family Apiaceae is well known for producing a large number of coumarins with isoprenoid units disposed in multiple ways along with a relatively high concentration of secondary metabolites such as furocoumarins (Tada et al. 2002).
Prangos pabularia is one of the most extensively studied species among the 72 species of the genus of Prangos, until now known to be indigenous to India (Sharma et al. 2013).In the Flora of USSR, 19 species are recognized and in the Flora of Tajikistan there are five known species of Prangos: P. fedtschenkoi (Regel & Schmalh.)Korovin, P. sarawschanica Korovin, P. pabularia Lindl., P. bucharica B. Fedtsch., P. ornata Kuzmina.It can grow in the mountainous areas and limestone slopes in altitudes 780 to 3600 m above sea level with large quantities in Tajikistan (Numonov et al. 2018).
In the flora of Turkey there are 13 species of Prangos recorded (Duran et al. 2005) and number of Prangos species are used in folk medicine as tonics and applied externally to stop bleeding and to heal scars (Ulubelen et al. 1995).The roots of P. pabularia have been used to kill aquatic snails in India (Nosrati and Behbahani 2016).Since ancient times, this plant was used in Tajikistan for treatment of wounds and skin diseases, especially for treatment of vitiligo (Numonov et al. 2018).The presence of furanocoumarins in P. pabularia play an important role for the treatment of vitiligo based on the similarity skeleton of the isolated coumarins to the strong activity against vitiligo of 8-methoxypsoralen (Yin et al. 2018).The roots of P. pabularia in combination with Dactylorhiza hatagirea, Physalis alkekengi, Podophyllum hexandrum, Terminalia belerica, Tribulus terrestris, Juniperus macropoda, Pedicularis longiflora and Picrorhiza kurroa are used as against all kinds of kidney and urinary disorders (Ballabh et al. 2008).Four abundant compounds of the roots of P. pabularia, oxypeucedanin, oxypeucedanin methanolate, imperatorin and osthole showed promising antibacterial activity (Sharma et al. 2013).Osthole raised blood pressure and stimulated respiration as well as stimulating cardiac muscles and constricting peripheral blood vessels (Ogawa et al. 2007).
Our previous chemical investigation of the roots of P. pabularia resulted on the isolation of the novel compound yuganin A and another 10 known compounds with anti-vitiligo effects on the proliferation of B16 melanoma cells (Numonov et al. 2018a).
In this context, the aim of our investigation is a comprehensive study of P. pabularia growing in Tajikistan, which led to the isolation and structure elucidation of 28 (1-28) individual compounds from the roots of P. pabularia, including a new coumarin (1) named yuganin B and the anti-vitiligo effect on the proliferation of B16 melanoma cells of several of the isolated in enough amounts pure compounds.

Results and discussion
Column and flash chromatography, preparative HPLC purification led to the isolation of 20 individual compounds from the dichloromethane fraction and eight individual compounds from the n-butanol fraction of the 95% ethanol extract from roots of P. pabularia.Osthole (12 g), imperatorin (10.7 g) and oxypeucedanin (4 g) were isolated as the major compounds in the roots of the P pabularia.In addition, oxypeucedanin hydrate (2 g) and heraclenol (1.4 g) were also found in significant amounts.Analysis of the 13 C NMR and HSQC spectra of compound 1 showed the presence of 28 carbon signals, represented as nine quaternary carbons, including two carbonyl carbon, 15 methine, including two anomeric carbons, three methylenes, and one methyl carbon atom.Study of the 1 H and 13 C NMR spectra of 1 revealed the existence of coumarin nuclei, one meta-substituted benzene ring, two sugar residues and one methoxy group (d C 56.42).The most downfield signal appeared at d C 167.54 was assigned to an ester carbonyl (C-7 000 ) that was attached to a benzene ring by HMBC correlations of H-2 000 /C-7 000 and H-6 000 /C-7 000 .The 1 H and 13 C NMR chemical shift values of sugar residues indicated that they related to hexose and pentose forms and were identified as glucopyranose and apiofuranose, respectively.

Spectral identification of isolated compounds
The relative configuration of 1 was established on the basis of the vicinal coupling constants of the anomeric protons as well as the NOESY spectrum.From the coupling constant J ¼ 7.6 Hz and NOESY correlations between H-1 0 /H-3 0 , H-5 0 the configuration of glucopyranose was determined as b.The coupling constant value of J ¼ 1.1 Hz and lack of NOE interaction between H-1 00 /H-2 00 as well as the quaternary carbon shift value (d C 79.23, C-3 00 ) indicated the presence of an a-threo-apiofuranose residue in compound 1 (de Santana Julião et al. 2009).The linkage sequences of residues were deduced by analyzing the correlation peaks in the HMBC spectra.The position of b-Dglucopyranoside residue was assigned to the C-7 position of the coumarin on the basis of HMBC correlation of H-1 0 with C-7 (d C 161.54).The HMBC correlations from H-1 00 (d H 5.50) to C-2 0 (d C 77.84) evidenced the linkage of the apiose moiety to carbon C-2 0 position of b-D-glucose.The attachment of benzoic acid at C-5 00 of apiose was confirmed by cross-peak from H-5 00 to C-7 000 in the HMBC spectrum.The position of the methoxy group was established based on the HMBC cross peaks OCH 3 /-3 000 (d C 148.51) at the -3 000 atom of the benzoic acid residue (available in the supplementary material).Based on the above evidences, the structure of 1 was designated as shown in Figure S1 and the detailed 1 H and 13 C NMR spectroscopic data of 1 are given in the Table S1 (available in the supplementary material).

Effects of pure isolated compounds on the proliferation of B16 melanoma cells
Several of the isolated compounds obtained in enough quantities were assessed for their effects on the growth of B16 melanoma and 8-MOP-treated B16 melanoma cells.The results show that the cell viability after treatment with isolated pure compounds at 10 and 50 lM and the value of intracellular melanin content (%) were significantly increased.Isooxypeucedanin, at 1, 10 and 50 lM concentrations, resulted in intracellular melanin content (%) of 197, 214, and 225, respectively, and at 50 lM showed a significant effect on the proliferation of B16 melanoma cells.The all tested compounds except PPE2 and PPE14 showed moderate effect on the proliferation of B16 melanoma cells at the 50 lM concentrations (Table S2 available in the supplementary material).The cell viability with 8-MOP at 50 lM was 194% and with DMSO at 2 lM was 116%.

General experimental procedures
NMR spectra were recorded on a Varian MR-400 (400 MHz for 1 H and 100 MHz for 13 C) spectrometer in DMSO-d 6 and CD 3 OD.TMS (d 0.00) signal was used as an internal standard for 1 H NMR shifts, and DMSO-d 6 (39.52 ppm vs. TMS) signal was used as a reference for 13 C NMR shifts.B16 melanoma cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China).Column chromatographic separation was performed on Sephadex LH-20 gel (Amersham Pharmacia Biotech, Sweden) and silica gel (100-200 mesh, Qingdao Haiyang Chemical Factory, China).The spots on the TLC plate were identified by spraying vanillin solution and also observed under UV-wavelength at 245 nm.HPLC analysis was performed using a DIONEX Ultimate 3000 HPLC system (Thermo-Fisher, USA) equipped with autosampler and coupled to variable UV wavelength detector.The chromatographic separation was performed using X-Bridge TM C18 column (10 Â 150 mm, particle size 5 lm, Waters), operated at 35 C. The HR-ESIMS data were collected with a QStar Elite mass spectrometer (AB SCIEX, Framingham, MA, USA).

Plant material
The roots of P. pabularia were collected from Takob valley of the Republic of Tajikistan in August 2018.The roots were authenticated by Dr. Sodik Numonov and voucher sample (No.TAS 23659) has been deposited in the herbarium of the Xinjiang Technical Institute of Physics and Chemistry Urumqi, Chinese Academy of Science.

Extraction and isolation procedures
The 3.400 kg of dried, pulverized roots of P. pabularia were extracted by using 95% ethanol three times (each time 24 h) at 45 C. The extracts were reduced to dryness under reduced pressure.Yield of extract was 898 g (23.5%).Obtained crude extract was suspended in water and successively partitioned in petroleum ether (35.40 g), dichloromethane (295 g), n-butanol (171.5 g) and water-soluble part (393 g) respectively.
The dichloromethane fraction (250 g) was mixed thoroughly with 1 kg of dry silica gel (200-300 mesh), placed onto a silica gel column and eluted with system of increasing polarityhexane: ethylacetate (from 50:0 to 0:1).Similar fractions were combined after developing their TLC profile and as a result obtained twelve (1-12) working fractions.

Melanin content determination
The B16 melanoma cells were cultured in HG-DMEM, and then supplemented with 10% FBS, 100 mg/mL streptomycin and 100 U/mL penicillin.The cells were maintained in a humidified incubator with 5% CO 2 at 37 C, and they were sub-cultured every 2 days to maintain logarithmic growth.Cells were seeded in a 96-well plate at a density of 5 Â 10 3 cells/well.After 24 h of incubation, different concentrations of the test compounds were added to each well of the plate.After the plate was incubated for an additional 48 h, the attached cells were incubated with MTT (0.5 mg/mL, 1 h) and subsequently solubilized in DMSO.The absorbance at 550 nm was then measured using a micro-plate reader (SpectraMax M2Multi-Mode Microplate Reader, Molecular Devices, Sunnyvale, CA, USA) to calculate the percentage cell viability.

Conclusions
Prangos pabularia is one of Tajikistan's important medicinal plants and the roots of this plant have been used in Tajik traditional medicine, which can provide a natural source of anti-vitiligo drugs.Phytochemical investigation of the dichloromethane and n-butanol extracts of the roots of P. pabularia led to the isolation of twenty-four known coumarins and three phenolic compounds.The presence of furanocoumarins in P. pabularia may play an important role in the treatment of vitiligo based on the structural similarity s of the isolated coumarins to the high bioactivity of 8-methoxypsoralen against vitiligo.Yuganin B (1) is a new coumarin and this is the first report that all compounds from the n-butanol fraction have been isolated from P. pabularia.
Our study showed that this plant is a rich source of coumarin derivatives that deserves further investigation in the future.
methoxybenzoate (named as yuganin B) was obtained as white powder.HR-ESI-MS analysis of compound 1 in negative ionization mode produced molecular ion at m/z 605.1516 [M-H] -(calcd.for C 28 H 29 O 15 -) and in positive ionization mode produced molecular ion at m/z 607.1654 [M-H] þ (calcd.for C 28 H 31 O 15 þ ), respectively (Figure S7, S8 in the supplementary material).The chemical structure of the compound 1 was established based on the results of the 1 D NMR and with COSY, HSQC and HMBC experiments.The 1 H NMR spectrum of 1 indicated the presence of simple coumarin nuclei signals at d H 6.19 and 7.69 (each 1H, d, J ¼ 9.5 Hz, H-3, 4), an ABX coupling system at d H 7.26 (1H, d, J ¼ 8.6 Hz, H-5), 6.88 (1H, dd, J ¼ 8.6, 2.4 Hz, H-6) and 6.85 (1H, d, J ¼ 2.4 Hz, H-8) (Table S1 available in the supplementary material).In addition, the proton signals of another ABX coupling system at d H 6.66 (1H, d, J ¼ 8.