Chemical constituents and anti Helicobacter pylori effect of ethyl acetate fraction from Sanchezia nobilis Hook.F

The leaves of Sanchezia Nobilis Hook. F grown in Vietnam are extracted with ethanol then distilled for ethanol retrievement under low pressure. The ethanol concentrate was shaken with n-hexane, then stirred with ethyl acetate and concentrated fractions were obtained. From the high ethyl acetate fraction, four compounds were isolated by normal phase and reversed-phase column chromatography. Their structures were determined by spectral analysis, including 1D and 2D NMR techniques (1H, 13C, DEPT, COZY, HSQC, HMBC and NOESY), High Resolution - Electrochewmical Ionization - Mass Spectroscopy (HR-ESI-MS), and identi(cid:977)ied by comparing with the recorded data. Among the isolated compounds, compound 2 (13-O-acetylfawcettimine) was (cid:977)irst time isolated from nature; and mixture 1 (Fawcettidin), 3 (Apigenin) and 4 (Kaempferol) were (cid:977)irst time isolated from the Sanchezia genus. The high ethyl acetate fraction of Sanchezia Nobilis Hook. F leaves were then evaluated for the anti-Helicobacter pylori (anti-HP) effect by determining the minimum inhibitory concentration method (MIC), which showed a good inhibitory, dose (cid:0) dependent effect on Helicobacter pylori. The ethyl acetate fraction at 1/8 dilution showed a complete inhibitory effect on Helicobacter pylori for as long as 24 hours. At 1/128 dilution, the fraction only showed good results on inhibition after 24 hours.


INTRODUCTION
Sanchezia is a small genus with more than 50 species, mainly distributed in the subtropical and tropical area such as the USA, Africa, the Mediterranean, Oceania, India, and some Southeast Asian countries. Most species are located in tropical rainforests in Central and South America (Ecuador) (Leonard and Smith, 1964). In previous publications, the genus was studied for effects such as antibacterial, antifungal and insecticidal by the agar diffusion method with 15 strains of Gram (+) and Gram (-). The effect on six strains of fungi and Tribolium castaneum was not as good as the comparison antibiotics (Rafshanjani et al., 2014). Evaluation of antioxidant effect by the ORAC method showed a result similar to quercetin. The anticancer effect on a methanolic fraction from Sanchezia speciosa leaves extract on MCF-7, SK-MEL-5, HUVEC cell lines had the best results on CMF-7 cell lines (Paydar et al., 2013). The anticancer effect by the MTT method on Hela cells from S. Nobilis roots had positive results (Shaheen et al., 2017). Researching on anti-in lammatory effect by inhibition of albumin denaturation assay method and antioxidant effect by DPPH method had good results (Loi et al., 2017). The published report on chemical constituents of these species has been showing that they contain several groups of compounds such as lavonoids, saponins, glycosides, alkaloids, steroids, carbohydrates, tannins and phenols. A number of speci ic compounds were isolated, such as ive matsutake alcohol compounds, in which four compounds were irst time isolated from the Acathanceae family and 1 compound was irst time isolated from the nature: 3-O-β-arabinopyranosyl-(1-6)-β-glucopyranosyl-(1-6)-β-glucopyranosyl-1octen-3-ol; 6 other compounds from the methanol extract of S. Nobilis leaves and roots, which 1 compound was irst time isolated from the nature: 9-O-β-xylopyranosyl-(1→6)-O-β-glucopyranosyl-(1→6)-O-β-glucopyranosyltrans-cinnamyl alcohol, 4 compounds were irst time isolated from the Acanthaceae family and 3 compounds were irst time reported from the Sanchezia genus (Mohamed et al., 2013;Ellah et al., 2014;J. C. Omondi and S. Omondi , 2015); 3 lavonoids and the compound 3-Methyl-1H-benz[f]indole-4, 9-dione were also isolated from Sanchezia speciosa leaves grown in Vietnam (Loi et al., 2017);16 fatty acids were also isolated from n-hexane extract of S. Nobilis Hook. F leaves (Nhung, 2018).
In Vietnam, there are only one species has been discovered belongs to the Sanchezia genus, which is Sanchezia Nobilis Hook. F is grown in some provinces such as Tuyen Quang, Nam Dinh, Vinh Phuc, Phu Tho, Thai Nguyen, Quang Nam, and Da Nang (Ho, 2003). In this research, we collected the total ethanol extract of S. Nobilis leaves and then separated the n-hexane, water and ethyl acetate fractions. These fractions would be continued to be isolated and determined the structure of components. We also evaluated the biological effects on peptic ulcer. In this publication, we assessed the inhibitory effect of HP's extract by determining the minimum inhibitory concentration method (MIC).

Plant material
The leaves of S. Nobilis Hook. F. were collected in Nam Dinh province during January 2018 and authenticated by the School of Medicine and Pharmacy, Vietnam National University, Hanoi,. A voucher specimen has been deposited to that place.

General experimental procedures
The ESI-MS were recorded on a Varian Agilent 1100 LCMSD mass spectrometer. The NMR [ 1 H (500 MHz), 13 C (125 MHz), and DEPT-90 and 135 MHz)] spectra were recorded on an AVANCE spectrometer AV 500 (Brucker, Germany) in the Institute of Chemistry, Vietnam Academy of Science and Technology (VAST). The FT-IR spectra were recorded on an IMPACT-410FT-IR spectrometer (CARL ZEISS JENA). Chemical shifts were reported in ppm downield from TMS with J in Hz. Melting points were measured on Mikroskopheiztisch PHMK-50 (VEB WaegetechnikRapido, Germany). Optical rotation was measured on WXG-4 disc polarimeter. Analytical TLC was performed on Kieselgel 60 F 254 (Merck) plates (silica gel, 0.25 mm layer thickness) and RP-18 F 254 (Merck) plates (0.25 mm layer thickness). Spots were visualized using ultraviolet radiation (at 254 and 365 nm) and by spraying with 10% H 2 SO 4 , followed by heating with a heat gun. Column chromatography was performed on silica gel (70-230 and 230-400 mesh, Merck). Organic solvents were of analytical grade. Optical densities were read on an ELISA plate reader (Bio-rad).

Extraction and isolation
The leaves of S. Nobilis were washed, dried, chopped. 3.0 kg of chopped leaves were submerged in 12 L ethanol 80% (solvent) at room temperature in three days to collect the irst extract. We added more solvent to submerge the herbs in 2-3 cm under the liquid surface (10 L per time) for two times to collect the second and the third extract.
All three extracts were mixed, paper− iltered, distilled for ethanol retrievement under low pressure, about 251.2 g concentrated ethanol extract was collected.
150 g of the concentrated ethanol extract were dispersed in distilled water, partition extracted with n−hexane and ethyl acetate (each solvent three times, 900 mL per time in 30 minutes). The n−hexane and ethyl acetate fractions were distilled for solvent retrievement under low pressure to collect these fractions: H for n−hexane (28.6 g), E for ethyl acetate (56.8 g). The remaining aqueous extract was concentrated to obtain fraction N (45.6 g).
Fraction E (20g) was dispersed in 2% tartaric acid solution (300 mL) and iltered out solid precipitate to collect the iltrate. The iltrate was alkalinized to pH 9 with NaHCO 3 , then extracted with dichloromethane (CH 2 Cl 2 ) (4 times x 600 mL). The obtained extract was concentrated at low pressure to produce E1 extract (5g). Fraction E1 was isolated

Culturing method
Biopsies were Giemsa−stained to determine the presence of H. pylori. Positive samples were crushed, grounded and inoculated into 5% sheep's blood agar media with H. pylori selective supplement. Agar plates were incubated at 37 • C under microaerophilic conditions, created by GasPak TM (BD) bags. The results were read after seven days with the appearance of small, grey, transparent, 1 -2 mm in diameter colonies. Reactions like urease, catalase, oxidase were used to identify the bacteria. Good growing colonies were chosen to activate in liquid media for tests.

Antibacterial effect evaluation method
The diluting method in liquid medium was proceeded to determine the MIC of the fractions.
Step 3: The 10 8 bacteria/mL suspension was mixed with the dilutions in step 1 in a 1:1 ratio, incubated at 37 • C in 2h, then taken out, inoculated into Pylori agar using a quantitative set.
The concentrations of bacteria in step 2 were also inoculated into Pylori agar to create agar plates of standard bacterial concentrations for comparison with agar plates in step 3. The results were read after 2, 6 and 24h (Cockerill, 2010;Yee and Koo, 2000).

Chemical constituents of Sanchezia Nobilis
The ethanol extract powder was isolated into nhexane, ethyl acetate and water fractions. A part of ethyl acetate fraction was isolated by different chromatography techniques on both stationary phase and reversed-phase silica gel to isolate alkaloids 1, 2 and lavonoids 3, 4. Analyzing the 13 C-NMR showed signals for 1 CH 3 , 8 CH 2 , 4 CH groups and three non-hydrogenated carbon atoms. Furthermore, 13 C-NMR contained signals corresponding to a carbon ketone δC 218.0 (C-5); a non-hydrogenated carbon atom δC 145.0 (C-13); and a methine carbon-nitrogen δC 127.1 (C-14); two carbon nitrogens δC 60.3 (C-1) and δC 56.2 (C-4). When compared to the previously reported researches (Dao et al., 2019;Li et al., 2015) the structure of compound 1 was inferred to be Fawcettidin. This compound was irst isolated in 1963 (Burnell et al., 1963), but the structure was de ined in 1970 (Ishii et al., 1970).

Alcaloid 2: 13-O-acetylfawcettimin
Brown yellow solid.   IR has a maximum adsorption spectrum at 3400(v*max cm −1 ), speci ic to OHgroup; the peak at 2256 (v*max cm −1 )was typical for the covalent oscillation of theC-H bond. Similarly, we had 1651 (v*max cm −1 ) for the C=O and1047 (v*max cm −1 ) for the C-O-C bonds.
Analyzing the NMR showed a secondary acetoxy group (δH 2.11, J = 5.0, δC 21.4, 128.5 and 172.4) in addition to typical resonances for the fawcettimin alcaloid structure frame (for example, carbon carbinolamine resonance at δC 75.7) showed that the two must be an acetoxy derivatize of fawcettimine (Hiroko et al., 2011). Analyzing the NMR (especially HSQC) showed four thin carbon chains [a: - On the HMBC, therewas CH 3 (C-16) group replacing at C-15. On the other hand, the δC changing in C-8 and C-13 compared with reference con irmed the substitute acetoxy group at C-13. Based on the typical signal at δC 128.5 (C-13); 217.5 (C-5); 172.4 (C-17) showed that the acetoxy groupwas located at C-13.
Therefore, compared with the reference (Katakawa et al., 2011), we could con irm that compound 2was 13-O-acetylfawcettimin, and it was irst isolation from natural.   In the 1 H-NMR, the aromatic ring proton signal of 2 doublets connected at δH 6.92 and 6.48 (J = 2.0 Hz) showed the correlation of HSQC with the carbon resonance at δC 102.8 (d), and 98.8 (d), assigned to H-6 and H-8 of ring A. Two doublet directly connected at δH 7.92 and 6.92 (2H, J = 8.5 Hz) showed longdistance links with the13 C-NMR signal at δC 161.4 (C-4'). Therefore, assigned respectively H-2'/6' and H-3'/5' of ring B. Besides, a singlet at δH 6.77 was assigned to H-3. Long-term correlation con irmed the H-3 assignment with C-2 (δC 161.1) and C-1' (δC 121.1). 13 C-NMR at δC 164.1 showed the correlation of HMBC with H-6 and H-8, assigned to C-7. In 13 C-NMR and DEPT, there were signals of 8 carbon grades 4 and 7 CH groups. Based on the data analyzed above and compared with 1 H-NMR, 13 C-NMR in reference (Ersöz et al., 2002), the structure of compound 3 was determined to be Apigenin. 1 H-NMR of 4 showed the proton pairs located in the meta position on the aromatic ring at δH 6.19 (1H, d, J = 2.0 Hz) and 6.43 (1H, d, J = 2.0 Hz), corresponding with H-6 and H-8. 1 H-NMR also showed the two doublet signals at δH 8.04 (2H, d, J = 2.8, 11.5 Hz, H-2' and H-6') and 6.92 (2H,d,J = 2.8,9.7 Hz,, corresponding with four aromatic protons in the B ring, featured for 1', 4' lavones. There were 15 C signals observed in 13 C-NMR, showed by DEPT and HMQC as 14 carbon sp2 atoms and one carbonyl signal at δC 175.9. The unsaturation occupied 8/11 double bond, and the remaining 3 of unsaturation were suitable for the lavonol structure. Comparing NMR data of 4 with Kaempferol (Aisyah et al., 2017), compound 4 was determined to be Kaempferol (Figure 1).

Evaluation of anti-Helicobacter pylori effect
Agar plates with test samples were ethyl acetate fraction mixed with H. pylori and compared with standard agar plates of H. pylori (10 8 bacteria/mL). The H. pylori inhibition effect levels are shown in Table 1.
The results of the research in Table 1 showed that ethyl acetate fraction at 1/8 dilution compared to the original solution, the fraction effectively inhibited H. pylori after 2 hours, after 6 hours and after 24 hours of exposure. When at 1/16 concentration after 2 hours of exposure to the fraction, the intensity of bacteria decreased to 10 2 , and after 6 hours, after 24 hours of exposure, H. pylori is completely inhibited. At 1/32 concentration and 1/128 concentration after 2 hours, 6 hours of exposure, the bacterial concentration also decreased, and after 24 hours, H. pylori is completely inhibited. Thus, the MIC of the extract fraction is 1/16.
H. pylori and gastric juice are considered to be the two main factors causing stomach ulcers in humans. H. pylori can cause in lammation and necrosis of gastric parietal cells. This bacterium also secretes the urease enzyme that breaks down urea in the stomach into ammonia, which damages the mucus layer of the stomach and produces other toxins that make the epithelial cells oedema, necrosis facilitates acid and pepsin attack causing ulcers (Malfertheiner et al., 2009;Sokic-Milutinovic, 2015). Therefore, eliminating H. pylori was an important target in the treatment of gastritis with its infection. Some researches have shown that extracts are resistant to bacteria may be due to antibiotic extracts, which may also contain extracts that make the environment unsuitable for bacterial growth, or synergistically with other extracts to improve the antimicrobial effect. With the results of our research above, ethyl acetate extract from S. Nobilis leaves had ability to anti-HP. (Konstantinopoulou et al., 2003;Wu et al., 2008) It was possible to explain the above results because the chemical constituents of the extract fraction had the effect of inhibiting bacteria including H. pylori, such as Apigenin and kaemferol (Wang et al., 2019;Escandón et al., 2016).

CONCLUSIONS
Four compounds were isolated from the ethyl acetate fraction of Sanchezia Nobilis Hook. F leaves extract. They were 1: Fawcettidine, 2: 13-Oacetylfawcettimine, 3: Apigenin and 4: Kaemferol. Compound 1, 3, 4 were irst isolated from the Sanchezia genus, and compound 2 was irst isolated from natural. The ethyl acetate fraction has also been shown to affect H elicobacter pylori.