Structural Characterization And Assessment Of Anti-Inflammatory And Anti-Tyrosinase Activities Of Polyphenols From Melastoma Normale Part 2

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Compound 3 had the same molecular formula as compound 2, as determined by its HRESIMS data. Comparison between the NMR data of 3 and 2(Table 2) revealed that the methoxyl group at galloyl-C-4 in 2 shifted to galloyl-C-3' in 3. This was confirmed by the HMBC correlations(Figure 2)of the methoxyl protons(8y 3.88) and aromatic proton (bH7.24)with galloyl-C-3'(6c 149.1) in 3, and the chemical shifts of the methoxyl group, galloyl-C-3, galloyl-C-4,and galloyl-C-3' that shifted frombc60.8,151.5,141.3,and 146.5 in 2tobc56.7,146.6,140.7,and149.1 in3,respectively.A positive cotton effect at 237nm and a negative one at 262 nm in the CD spectrum indicated the(S)configuration of the HHDP group for 3 [24]. The β configuration of the sugar moiety was deduced from the coupling constant(=8.5 Hz) of the anomeric proton of the glucosyl moiety. Acid hydrolysis of 3 with 1 M HCl yielded D-glucose, which was confirmed by TLC and GC analyses. Hence, compound 3 was identified as 1-O-galloyl-6-O-(3-methoxygalloyl)-2,3-O-(S)-hexahydroxydiphenoyl-β-D-glucose.

The molecular formula of 4 was deduced from its HRESIMS spectrum (m/z 783.1032, [M-H]-)as C35 H28 O21. Its molecular weight was 16 mass units less than that of 3, which may be attributed to the absence of a hydroxyl and the presence of an aromatic proton (6H6.87)at vanilloyl-C-5' in 4 (Table2). This was confirmed from the presence of a 1，2，4-trisubstituted aromatic moiety （μ7.60 （dd， J=8.3，1.8 Hz），6.87 （d， J=8.3 Hz）， and 7.57 （d， J=1.8 Hz）） and the chemical shift change of galloyl-C-5' at δc 145.8 in 3 to vanilloyl-C-5' at bc 115.9 in 4. The methoxyl protons(6H 3.90)and aromatic proton (H 6.87) correlations with vanilloyl-C-3' in the HMBC spectrum(Figure 2) further confirmed the aromatic proton (H 6.87) at vanilloyl-C-5'. The atropisomerism of the HHDP was shown to be an S configuration by the appearance of positive and negative cotton effects at 239 nm and 263 nm, respectively [24]. The coupling constants of the anomeric proton in 4 were 8.5 Hz reminiscent of β-anomeric configuration. The acid hydrolysis revealed that 4 had the same sugar units as 3. Thus, compound 4 was identified as 1-O-galloyl-6-O-vanilloyl-2,3-O-(S)-hexahydroxydiphenoyl-β-D-glucose.

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2.2. Anti-Inflammatory Actioity Assays

Four new compounds 1-4 were tested for potential anti-inflammatory activity by measuring the inhibition of nitric oxide (NO) production. Unfortunately, none of them displayed significantly anti-inflammatory activity. The IC5o values for the inhibition of NO production by compounds 1-4 are all>50uM. It has been reported that the known flavonols kaempferol(7)[26[, quercetin (10)[27l and myricetin-3-O-α-L-rhamnopyranoside(12)28 have anti-inflammatory activity, but kaempferol-3-rhamnoside (8) [26] and quercetin-3-O-α-L-rhamnoside (11)[29] have no anti-inflammatory activity. From the experimental results and literature data, the flavonoids in the title plant have better anti-inflammatory activity than ellagitannins, it may be the active ingredient corresponding to the anti-inflammatory effect of this plant.

2.3. Tyrosinase Inhibitory Actioity Assays

All compounds were investigated for potential tyrosinase inhibitory activity. As shown in Table 3, new compounds 2-4 displayed moderate tyrosinase inhibitory activities. New compound 1exhibited weak tyrosinase inhibitory activity. Quercetin (10)[30] has a significant tyrosinase inhibitory activity, and its anti-tyrosinase activity is better than quercetin-3-O-w-L-rhamnoside(11)[26] and kaempferol (7) [26], which is consistent with the literature data. This indicated that 3'-and 4/-hydroxy groups on the B ring and 3-hydroxy groups on the C ring in flavonols were crucial to their activities. The anti-tyrosinase activity of kaempferol (7)[26] is better than kaempferol-3-rhamnoside (8)[26]and compounds 8 and 12 [31] has no significant anti-tyrosinase activity, which further supports the above structure-activity relationships.

3. Experimental 3.1.Materials

The roots of M.normale were collected from Yanshan Town, Guilin City, Guangxi Province, in September 2018, and authenticated by Professor Yusong Huang (Guangxi Institute of Botany). A voucher specimen (20180912) was deposited in the Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization, Guangxi Institute of Botany, China.

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3.2. General Experimental Procedures

Optical rotations were measured with an ADP440+ polarimeter （λ 589 nm， path length 1.0 cm). The NMR spectra were acquired on a Bruker Advance 500 spectrometer （Bruker Biospin AG， Fällanden， Switzerland）， and the residual solvent peaks were used as references. Coupling constants and chemical shifts were given in Hz and on a δ（ppm）scale, respectively. The ESIMS and HIRESIMS data were recorded on a BRUKER HCT mass spectrometer and LCMS-IT-TOF spectrometer (Shimadzu, Kyoto, Japan), respectively. GC was performed on an Agilent 7890ON(Agilent Technologies, Inc. Chandler, AZ, USA)system with a 0.32 mm i.d.×25 mL-Chirasil-Val column. Analytical HPLC was run on a Shimadzu LC-2030C HPLC(Shimadzu, Kyoto, Japan)system using a 4.6i.d.×250 mm Agilent Eclipse XDB-C18(5 um) column. semi-preparative HPLC was conducted on a Shimadzu LC-20AT HPLC system using a 20.0i.d.×250 mm Dr. Maisch reprosil 100 C18 (5 um) column at a flow rate of 4 mL/min. Column chromatography(CC)was performed using Sephadex LH-20 （25-100 μm; GE Healthcare Bio-Science AB， Uppsala， Sweden）， silica gel column (200-300 mesh, Qingdao Marine Chemical Co. Ltd., Qingdao, China), MCI gel CHP 20P(75-150 um; Mitsubishi Chemical Co., Tokyo, Japan), and Chromatorex ODS (50 um, Merck, Darmstadt, Germany) columns.

3.3. Extraction and Separation

Air-dried, powdered roots (10 kg) of M. normale were extracted with 80% aqueous acetone(3×7 days) at room temperature and each extract filtered. The filtrates were dried under reduced pressure to afford a crude extract (0.6 kg). The extract was suspended in H2O(1 L)and successivelypartitioned withpetroleum ether(3×2L),EtOAc (3×2 L).The EtOAcextract (80g)was divided into ten fractions(Fr.1-10)by silica gel CC (8 i.d.×20 cm)eluting with a gradient of CH2Cl-MeOH(100:0,95:5,90:10,80:20, 70:30,50:50,0:100,v/v). Fr.4(32 g) was loaded onto an MCI gel column(6i.d.×20 cm) and eluted with a gradient of MeOH-H，O（0∶100-100∶0，v/ø）to afford eighteen subfractions（Fr4-1-4-18）.Separation of subfraction Fr4-14(3.0g)was done by another silica gel column (3i.d.×20 cm) eluting with a gradient of CH2Cl2-MeOH (97:3-80:20, o/v) and Sephadex LH-20 CCeluting with CH2Cl2-MeOH(1:1,v/o)to afford 7(11.3 mg),8(10.6 mg),9(9.3 mg),10 (20.6 mg),11 (7.6 mg), and 12 (6.2 mg). Fr 6(4.2 g) was applied to ODS C18 column and eluted with MeOH-H2O (20:80-80:20) to obtain 9 subfractions(Fr 6-1-6-9). Further separation of subfraction Fr 6-4(1.2 g)using Sephadex LH-20 CC(eluted with MeOH-H2O,10:90-100:0)yielded compounds 1(9.6 mg),5(7.2 mg), and 6(7.3 mg),respectively. Compounds 2(tR 130.5 min,5.2mg),3(tR 146.8min,9.6 mg),and 4(tR 177.2min,15.3mg)were obtained from Fr7(2.5g)via semipreparative HPLCeluting with a gradient of MeOH-H2O(20:80-40:60, / u, 0-250 min).

3.4.Spectroscopic Data

Whiskey tannin C (1):Brown amorphous powder; [α]-12.6°(c= 0.16, MeOH);UV （MeOH）λmaxnm （log e）∶203（2.02），276（1.19）; CD （MeOH）λmax（△e）263（-7.8），240（10.9），225(2.9).1Hand 13C NMR data,see Table 1;HRESIMSm/z:[M-H]-1005.1240(calcd for C4sH33O27-, 1005.1215). 1-O-(4-methoxygalloyl)-6-O-galloyl-2,3-O-(S)-hexahydroxydiphenoyl-β-D-glucose(2): Brown amorphous powder amorphous powder; 【α】5-5.5°（c=0.17， MeOH）; UV（MeOH） 入max nm （log e）∶203 （2.52），275（2.01）; CD （MeOH）λmax（△8）264（-8.0），238（30.6），218（1.3）.'H and 13C-NMR data,see Table 2; HRESIMS m/z:799.0983[M-H]-(calcd for C3sH2O22-, 799.099).

1-O-galloyl-6-O-(3-methoxygalloyl)-2,3-O-(S)-hexahydroxydiphenoul-6-D-glucose(3); Brown amorphous powder;【α】7-33.5°（c=0.12， MeOH）; UV（MeOH）入max nm （log e）∶203 （2.40），273（1.68）; CD （MeOH）λmax（△e）262（-3.1），237（11.1），210（-2.7）.'Hand13CNMR data，see Table 2;HRESIMS m/z:799.1006 [M-H]-(calcd for C35H2O22-,799.0999). 1-O-galloyl-6-O-vanilloyl-2,3-O-(S)-hexahydroxydiphenoyl-β-D-glucose (4): Brown amorphous powder; 【α】7-20.6°（c=0.15， MeOH）; UV（MeOH）λmax nm （log e）∶203 （2.42），278（1.86）; CD（MeOH）λmax（△e）263（-12.1），239 （42.5）， 219（0.9）.'H and 13CNMR data，see Table 2;HRESIMS m/z: 783.1032 [M-H]-(calcd for C35H27O21-,783.1050).

3.5. Acid Hydrolysis of 2-4

Compound 2 (2 mg) was treated with 1 M HCl (5 mL) at 80 °C for 4 h, and then extracted with ethylacetate(3×5 mL). The aqueous phase was dried under a stream of N2 to generate a neutral residue that was analysed using TLC(SiO2)with EtOAc-pyridine-EtOH-H, O(7:1:1:2)as solvent system. The R-value of the neutral residue was the same as that of authentic D-glucose indicating the sugar component of 2 was glucose. The neutral residue and 5 mg L-cysteine methyl ester hydrochloride were dissolved successively in 3 mL of anhydrous pyridine and warmed at 80°C for 1 h. After removal of the solvent by evaporation under reduced pressure, the reaction mixture was subsequently reacted with 0.6 mL of N-trimethylsilylimidazole at 80°C for 1 h. The reaction mixture was partitioned with n-hexane and H, O, and then the n-hexane layer was analyzed by a GC instrument. The injector and detector temperatures were set at 250 °C and 280°C, respectively. The initial column temperature was held at 160 °C for 1 min, then increased to 280C at 5°C/min, and held for 10 min. The authentic D-glucose and authentic L-glucose were silylated and analyzed in the same way, and their retention times were 19.09 and 19.25 min, respectively. The results of the GC analysis indicated that the sugar component of 2 was D-glucose(t 19.1 min). The sugar components of 3 and 4 were determined using the same procedure.

3.6. Anti-Inflammatory Activity

3.6.1.NO Production by LPS-Stimulated RAW 264.7 Cells

The RAW 264.7 cells were cultivated in DMEM supplemented with 10% FBS at 37 ℃℃in a humidified atmosphere of 5% CO2 for 24 h. Cells in a 24-well plate (5×104 cells/well)were treated with 200 ng/mL LPS and the test compounds. After 22 h, the media were collected, and the level of nitrite was measured using the Griess Reagent System (Promega, Madison, WI, USA).

3.6.2. Cell Viability

MTT assay was carried out to measure the cytotoxicity of test samples on the RAW264.7 cell line. Cells that were in the logarithmic growth phase were seeded in a 96-well plate at the density of 5× 10+-6×10* cells/well and incubated at 37°C with 5% CO, for 24 h. Next， the cells were treated with 100 μL of culture medium at various concentrations of test samples for 24 h. The medium was discarded and 100 uL of FBS free medium containing MTT(1 mg/mL) was added to each well. After incubation in the incubator for 4 h, the supernatant was discarded and 100 μL of DMSO was added to each well to dissolve the formazan. The absorbance was measured at 570 nm using a microplate reader.

3.7. Anti-Ty/rosinase Actioity

The anti-tyrosinase activities of the isolates were investigated according to the procedure described by Aoki et al. with slight modifications [21]. L-DOPA and kojic acid were used as substrate and positive control, respectively. Solution of L-DOPA at 5.0 mM, mushroom tyrosinase at 100 U/mL, as well as samples at different concentrations were prepared in phosphate buffer （pH6.8）.20 μL of the sample solution and 10 μL of mush-room tyrosinase solution were mixed and pre-incubated at 37°C for 10 min, then 40 uL of L-DOPA solution was added and incubated at 37°C for 5 min. The reaction system of the anti-tyrosinase activity experiment is shown in Table 4. The absorbance was measured at 475 nm using the Spark 10M multimode microplate reader(Tecan Trading AG, Zurich, Switzerland). All assays were repeated three times and each time in triplicate. The percent inhibition of tyrosinase activity was calculated using the following formula:

where T represents the absorbance with sample and tyrosinase; To represents the absorbance with a sample but no tyrosinase; C represents the absorbance with tyrosinase but no sample; Co represents the absorbance without tyrosinase and sample.

4. Conclusions

The study is intended to explore more anti-inflammatory and enzyme inhibitory activities of polyphenols from the roots of M.normale on the basis of our previous works [20]. As expected, twelve polyphenols were obtained from the tile plant for the first time and compound 1-4 were new ellagitannin. The successful isolation and structure identification of ellagitannin provide materials for the screening of anti-inflammatory drugs and enzyme inhibitors and also contribute to the development and utilization of M.normale. Ellagitannins 1-4 have no significant tyrosinase inhibitory activities and anti-inflammatory activities, which makes it less likely to be a potential anti-inflammatory drug or enzyme inhibitor. Fortunately, they possess new effects, such as anti-obesity [6,7]. Flavonoids have better anti-inflammatory and inhibitory enzyme activities than ellagitannins in the roots of M. normale, and they are more likely to be anti-inflammatory and anti-enzyme inhibitors. The study of the structure-activity relationship is helpful to find new anti-inflammatory drugs and enzyme inhibitors.

This article is extracted from Molecules 2021, 26, 3913. https://doi.org/10.3390/molecules26133913 https://www.mdpi.com/journal/molecules