Two new glycosides from the fruits of Morinda citrifolia L Текст научной статьи по специальности «Фундаментальная медицина»

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References:

[1]Delectis Florae Reipublicae Popularis Sinicae, Agendae Academiae Sinicae Edita.Flora Reipublicae Popularis Sinicae, Tomus 24. Beijing: Science Publishing House,1986, 147-148.

[2]Wu J R. Research on the Original Plants of Crude Drug"Sang Ji Sheng"(Ramulus Taxilli).Chinese Traditional Herbal Drugs, —1981—12(5):32

[3]Peng H Y, Zhang Y H, Han Y et al. Studies on the anticancer effects of total alkaloid fromViscum coloratum. China Journal of Chinese Materia Medica. —2005—30(5):381~387.

Two NewGlycosides from the Fruits ofMorinda eitrifolia L.

Hong-CaiZhang1^, Yu Wang1, Shu-Min Liu 1*

1Academy of traditional Chinese medicine,Heilongjiang University of Chinese Medicine, Harbin 150040, China; E-Mails: [email protected] (H.-C.Z.); [email protected] (Y.W.)

*Authors to whom correspondence should be addressed; E-Mails: [email protected] (S.-M.L.);[email protected] (L.L.); Tel.: +86-139-451-33028 (S.-M.L.).

Abstract: To study the chemical constituents of the fruits of noni(Morinda citrifoliaL.), and find novelcompounds, an n-butanol extract of the ethanol soluble fraction was subjected to repeated silica gel and ODS column chromatography and HPLC. Two new glycosides were isolated and their structures elucidated by NMR and HRFAB-MS spectrometry as(2£',4£',7Z)-deca-2,4,7-trienoate-2-O-ß-D-glucopyranosyl-ß-D-glucopyra-noside (1) andamyl-1-O-ß-D-apio-furanosyl-1,6-O-ß-D-glucopyranoside (2), respectively.

Keywords:Morinda citrifoliaL. ;glycosides

1. Introduction

Morinda citrifolia L. (family Rubiaceae), also known as Tropical Radix Morindae Officinalis (RMO) and Medicinal Morinda Root Seasonal Fruit, is usually a small tree or bush occurring in the South Pacific tropical islands and widely distributed in the Hainan Province and Paracel Islands of China and in Taiwan. The fruits of Morinda citrifolia L. are oval and juicy with a strong odor, and have been used for a long time as a medicinal plant in Southeast Asia and the Pacific Islands. All parts of the plant can been used, including fruit, leaf, root, bark, flower, stem and seed[1]. Reported traditional uses include as a treatment of boils, abscesses, and inflammations of various origins, fungal infections, and constipation as well as diarrhea[2,3]. Pharmacological research has revealed a number of biological activities in recent years, such as anticancer[4], anti-inflammation[5], antioxidant[6], liver protection[7], and anti-AIDSproperties [8]. In this work silica gel column chromatography was employed to separate the glucoside constituents of an n-butanol extract of the ethanol soluble fraction of Morinda citrifolia L. fruits. 2D-NMR techniques, HR-ESI-MS and hydrolytic reactions were used to elucidate the structures of the extracted compounds.

2. Results and Discussion

Compound 1 (Figure 1) was obtained as a white powder (15.6 mg). The molecular formula was determined to be C22H34Onby the HR-FAB-MS [M+H]+ peak at m/z507.2071. Acid hydrolysis

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of 1 only gave D-glucose. The H and C-NMR spectra of 1 indicated an alkenoic acid ester moiety and two glucose groups. The alkenoic acid ester moiety was confirmed by *H-NMR (Table 1) signals at 5h6.00 (1H,d, J = 15.2Hz), 7.74(1H,ddd, J = 15.2,11.6,0.8Hz), 6.21 (1H, t, J = 11.3 Hz),

5.89 (1H, dt, J = 15.8,7.8Hz), 3.08(2H, brt, J = 7.5Hz),5.33(1H,m),5.46(1H,m),2.12 (2H,dt, J = 7.5,1.1Hz), and 0.99 (3H, t, J = 7.5 Hz) and by 13C-NMR signals at 5c167.0 (s), 121.9 (d), 142.0 (d), 127.3 (d), 141.5 (d), 27.4 (t), 126.5 (d), 134.2 (d), 21.5 (t), and 14.6 (q). A combination of the COSY, HSQC, and HMBC data allowed assignment of the 13C-NMR (Table 1) signals from the disaccharide. The characteristic features of the two glucose moieties appeared in the 13C-NMR spectra, which exhibited signals at 5c94.5 (d), 83.0 (d), 77.6 (d),71.2 (d), 77.8 (d), and 62.6 (t) for the first glucose and at 5c105.7 (d), 75.9 (d), 77.9 (d), 71.9 (d), 77.7 (d),and 62.2 (t) for the second glucose[9]. The XH-NMR signals at 5h5.69 (1H, d, J = 7.8 Hz) and 4.53(1H, d, J = 7.8 Hz), and the 3C-NMR signals at SC 105.7 (d) and 94.5(d) indicated the presence of anomeric protons and carbons in the disaccharide moiety that had a P configuration according to the coupling constant. The HMBC correlation between the anomeric proton SH4.53 (H-1'') and SC83.0(C-2') connected the terminal glucose to the inner glucose. The linkage between the fatty acid ester moiety and the disaccharide was also established by the HMBC correlation between anomeric proton SH5.69 (H-1') and Sc167.0 (C-1). Important HMBC interactions of compound 1 are shown in Figure 2. Thecoupling constant of 15.2 Hz between H-2 and H-3 indicated A2'3 to be Econfiguration.Thecoupling constant of 15.8 Hz between H-4 and H-5 indicated A4'5 to be ¿configuration. On the basis of the above data, the structure of 1 was deduced to be (2E,4E,7Z)-deca-2,4,7-trienoate-2-0-y#-D-glucopyranosyl-y#-D-glucopyranoside. Figure 1. Structures of compounds 1 and 2.

Compound 2 (Figure 1) was obtained as a white powder (17.9 mg). The molecular formula was deduced from the HR-FAB-MS 383.1911 [M+H]+ and the 13C-NMR data to be C16H30O10.

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Acid hydrolysis of 2 only gave D-glucoseand apiose. The H- and C-NMR spectra of 2 indicated a heptanol moiety, a glucose group and an apiose group. The heptanol moiety was supported by !H-NMR signals at 5r3.56 (2H,m), 1.53(2H,m), 1.24(4H,m), and 0.82 (3H,t, J = 7.0Hz) and by 13C-NMR signals atSc71.4 (t), 32.4 (t), 24.5 (t), 23.4 (t), and 14.3 (q). A combination of the COSY, HSQC, and HMBC data allowed assignment of the 13C-NMR signals of the disaccharide. The

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characteristic features of the glucose moiety and the apiose moiety appeared in the C-NMR spectra, which exhibited signals at 5c105.9 (d), 75.1 (d), 78.1 (d),71.6 (d), 77.7 (d), and 68.4 (t) for the glucose and signals at Sc111.0 (d), 78.0 (d), 80.6 (s),75.4 (t), 65.7 (t) for the apiose[10]. The HSQC correlation between anomeric proton SH4.45 (1H, d, J =7.8 Hz) and anomeric carbon SC 105.9 (d) indicated that the glucose moiety had a P configuration. The HSQC correlation between anomeric proton SH4.86 (1H, d, J =2.3 Hz) and anomeric carbon SC 111.0 (d) indicated that the apiose moiety had a P configuration. The HMBC correlation between anomeric proton SH4.86 (H-1'') and SC68.4(C-6') connected the terminal glucose to the inner glucose linkage. The linkage between the fatty acid ester moiety and the disaccharide was also established by the HMBC correlation between anomeric proton SH4.45 (H-1') and SC71.4 (C-1). Important HMBC interactions of compound 2 are shown in Figure 2. On the basis of the above data, the structure of 2 was deduced to beamyl- 1-O-y9-D-apiofuranosyl- 1,6-O-y9-D-glucopyranoside.

2

Table 1. NMR data of 1 and 2 in CD3OD in ppm, J in Hz, recorded at 400 MHz and 100 MHz, respectively)._

No. 1 2

5C (DEPT) Sh (J, Hz) SC(DEPT) Sh (J, Hz)

1 167.0 (C) 71.4 (CH2) 3.56 m

2 121.9 (CH) 6.00 d (15.2) 32.4 (CH2) 1.53 m

3 142.0 (CH) 7.74 ddd (15.2,11.6,0.8) 24.5(CH2) 1.24 m

4 127.3 (CH) 6.21 t (11.3) 23.4(CH2) 1.24 m

5 141.5 (CH) 5.89 dt(15.8,7.8) 14.3(CHb) 0.82 t (7.0)

6 27.4 (CH2) 3.08 br t (7.5)

7 126.5 (CH) 5.33 m

8 134.2 (CH) 5.46 m

9 21.5(CH2) 2.12 dt (7.5,1.1)

10 14.6 (CH3) 0.99 t(7.5)

1' 94.5 (CH) 5.69 d (7.8) 105.9 (CH) 4.45 d (7.8)

2' 83.0 (CH) 75.1 (CH)

3' 77.6 (CH) 78.1 (CH)

4' 71.2 (CH) 71.6 (CH)

5' 77.8 (CH) 77.7 (CH)

6' 62.6 (CH2) 68.4 (CH2)

1'' 105.7 (CH) 4.53 d (7.8) 111.0(CH) 4.86 d (2.3)

2'' 75.9 (CH) 78.0 (CH) 3.79 d (2.3)

3'' 77.9 (CH) 80.6 (C)

4'' 71.9 (CH) 75.4 (CH2) 3.86 d (9.8) 3.64 d (9.8)

5'' 77.7 (CH2) 65.7 (CH2) 3.47 s

6'' 62.2 (CH2)

Figure 2. Key HMBC correlations of 1 and 2.

1 2

3. Experimental

3.1. General

IR and NMR spectra were recorded on Shimadzu FTIR-8400S and Bruker DPX 400 (400

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MHz for 'H-NMR and 100 MHz for C-NMR) instruments, respectively. Chemical shifts are given as 5 values with reference to tetramethylsilane (TMS) as an internal standard, and coupling constants are given in Hz. The HR-ESI-MS analyses were conducted on a Waters LCTPremier XE TOF-MS instrument. A Hypersil ODS II (5 pm, 4.6 x 250 mm, Dikma, Lake Forest, CA, USA) column was employed for analytical HPLC (Waters, 2695-2998 instrument). Preparative HPLC

(Agilent1100 system, Santa Clara, CA, USA) was performed on a Pegasil ODS II (5 pm, 9.4 x 250 mm, Agilent) column. Silica gel (200-300 mesh, Haiyang, Qingdao, China) was employed for column chromatography and ODS-A (120 A, 50 pm) was obtained from YMC Co. (Kyoto, Japan).

3.2. Plant

The fresh fruits of Morinda citrifolia L. were collected from Hainan Province of China, in July 2010.The voucher specimen (20100720) was deposited in Heilongjiang University of Chinese Medicine, Harbin, China.

3.3. Extraction and Isolation

The fruits of Morinda citrifolia L. (26 kg) were ground and sieved through standard mesh

sieve

No. 10 and extracted with 95% EtOH (3 x 10 L) for 2 h. Concentration under reduced pressure gave the EtOH extract (210 g) which was dissolved in water (10 L), and successively extracted with petroleum ether (60-90°C), EtOAc, and n-butanol, (3 x 10 L) respectively. Solvents were removed to give the petroleum ether (20.2g), CHCl3 (5.7 g), EtOAc (6.9 g) and n-butanol (162.4 g) extracts. The n-butanol fraction was repeatedly column chromatographed on silica gel with a gradient of CHCl3/MeOH (1:0—>0:1) as eluent to afford five fractions: Frx (6 g), Fr2 (13 g), Fr3 (16 g), Fr4 (8 g),Fr5 (21 g),Fr6 (10 g),Fr7 (4 g),and Fr8 (24 g).

Fr3 (2.6 g) was subjected to ODS column chromatography with MeOH/H2O (1:9—1:0) and finally purified by preparative HPLC on a Pegasil-ODS H column with MeOH/H2O (2:8) to afford 1 (15.6 mg, tR=43 min). Fr5 (21g) was subjected to repeated silica gel chromatography with CHCl3/MeOH (30:1—0:1) elution to afford a number of subfractions B1-B4. B2 (2.7 g) was subjected to ODS column chromatography with MeOH/H2O (1:9—1:0) and finally purified by preparative HPLC on a Pegasil-ODS H column with MeOH/H2O (3:7) to afford 2 (17.9 mg, tR=28 min).

(2E,4E,7Z)-deca-2,4,7-trienoate-2-O-fi-D-glucopyranosyl-fi-D-glucopyranoside(1). White amorphous powder. mp. 132-134 °C. IR (KBr): 3396.3, 1740.6, 1472.1, 1071.6, 988.4, 905.3 cm-1. HR-ESI-MS m/z507.2071 [M+H]+ (calc. C22H34O13, 507.2078); 1H and 13C-NMR (CD3OD) data see Table 1.

Amyl-1-O-^-D-apiofuranosyl-1,6-O-^-D-glucopyranoside (2). White amorphous powder. mp. 77-79 °C. IR (KBr): 3403.1, 1469.3, 1375.8, 1071.4 cm-1. HR-ESI-MS m/z383.1911 [M+H]+(calc. C16H30O10,383.1917); XH and 13C-NMR (CD3OD) data see Table 1. 4. Conclusions

Two novel glucosides were isolated from the n-butanol fraction of Morinda citrifolia L.fruits. Compound 1 is (2E,4E,7Z)-deca-2,4,7-trienoate-2-O-y#-D-glucopyranosyl-y#-D-glucopyranoside and compound 2 is amyl-1-O-y#-D-apiofuranosyl-1, 6-O-y5-D-glucopyranoside. Acknowledgments

Nature Science of Heilongjiang Province (No. 2009062501). References

1. Potterat, O.; Hamburger, M.Morinda citrifolia (Noni) fruit-phytochemistry, pharmacology, safety.Planta Med.2007,73, 191-199.

2. McClatchey, W. From Polynesian healers to health food stores: Changing erspectives of Morinda citrifolia (Rubiaceae). Integr. CancerTher.2002,1, 110-120.

3. Dixon, A.R.; McMillan, H.; Etkin, N.L. Ferment this: The transformation ofNoni, a traditional Polynesian medicine (Morinda citrifolia, Rubiaceae). Econ. Bot. 1999,53, 51-68.

4. Hirazumi, A.; Furusawa, E.; Chou, S.C.; Hokama, Y.Immuno-modulation contributes to the anticancer activity ofMorinda citrifolia(Noni) fruit juice. Proc. West. Pharmacol. Soc. 1996, 39, 79.

5. Nitteranona, V.; Zhanga, G.D.; Darienb, B.J.; Parkin, K. Isolation and synergism of in vitro anti-inflammatory and quinone reductase (QR)inducing agents from the fruits of Morinda citrifolia (noni).Food Res. Int.2011, 44, 2271-2277.

6. Liu, C.H.; Xue, Y.R.; Ye, Y.H.; Yuan, F.F.; Liu, J.Y.; Shuang, J.L. Extraction and characterization of antioxidant compositions from fermented fruit juice of Morinda citrifolia (Noni). Agric. Sci. Chin.2007, 6, 1494-1501.

7. Wang, M.Y.; Nowicki, D.; Anderson, G.; Jensen, J.; West, B. Liver protective effects of Morinda citrifolia(Noni). Plant Foods Hum. Nutr. 2008, 63, 59-63.

8. Selvam,P.; Maddali,K; Marchand,C. Studies of HIV integrase inhibitory activity of Morinda citrifoliaL. Fruit Extracts.Antivir. Res. 2010, 86, 45-46.

9. Akihisa,T.; Seino,K.; Kaneko, E.;Watanabe, K.; Tochizawa, S.; Fukatsu, M.; Banno, N.; Metori, K.; Kimura, Y. Melanogenesis inhibitory activities of iridoid, hemiterpene-, and fatty acid-glycosides from the fruits of Morinda citrifolia(Noni). J. Oleo Sci. 2010, 59, 49-57.

10. Miyase,T.; Ueno,A.; Takizawa,N.; Kobayashi, H.; Oguchi, H. Studies on glycosides of epimendium grandiflorum Morr.var.thunbergianum(MIQ.)Nakai.III.Chem. Pharm. Bull.1988,36, 2475-2484.

Sample Availability:Samples of (2E,4E,7Z)-deca-2,4,7-trienoate-2-O-y#-D-glucopyranosyl-y#-D-glucopyranoside (1) and amyl-1-O-y5-D-apiofuranosyl-1,6-O-y5-D-glucopyranoside (2) are available from the authors.

About efficiency of complex therapy of refractive amblyopia

Sorokina E.V.

Amur State Medical Academy, Blagoveshchensk, Russia

Abstracts: The results of complex treatment refractive ambliopia in patients with high myopia, includes eximer laser refractive surgery (Epi-LASIK) in combined with using the drug Ceraxon and electrical stimulation of retina and optic nerve, was studied. Complex treatment refractive amblyopia in patient with high myopia leads to increasing vision acuity after treatment in average by 25%, in fact improve hemodynamics of ophthalmic artery, central retinal artery, and short posterior ciliary arteries, decrease period of revitalizing of visual function and made postoperative period of rehabilitation faster.

Key words: high myopia, refractive amblyopia, hemodynamics, Ceraxon (citikolin), electrical stimulation of retina and optic nerve, Epi -LASIK.

Refractive anomalies are the most widespread pathology of the visual analyzer at present. Actual direction is the development of correction methods in patients with amblyopia, which developed on the base of refractive anomaly. Recently, a number of foreign and domestic scholars have noted improvement in visual acuity in patients with refractive and anisometropic amblyopia after the excimer laser refractive operation. In recent years, there are reports in the literature about the using pharmacological drugs: piracetam, nootropil, instenon, vizobalans, mildronate, pikamilon, fezam, in complex treatment of amblyopia in children. Results of complex treatment was evaluated positively, suggesting that the use of pharmacological drugs that contribute to successful treatment of amblyopia - one of today's promising. Citikolin (Ceraxon by firm «Nycomed») is of special interest, whose effectiveness is proved by numerous clinical studies and publications. Citikolin registered in over 40 countries and over 18 years, is widely used in neuroscience as a drug neurotransmitter, neuroreceptor, neurotrophic actions in patients with acute cerebral insufficiency of various origins. Pharmacotherapeutic group: nootropic drugs. Citikolin (cytidine-5-difosfoholin) - is an organic substance, which belongs to the group of nucleotides - of biomolecules, which play an important role in cellular metabolism. Citikolin (cytidine-5-difosfoholin) is an essential precursor of phosphatidylcholine (lecithin), a major phospholipid of all cell membranes, including neuronal membranes. Choline is also involved in the synthesis of acetylcholine, and citikolin is a donor of choline in the synthesis of acetylcholine. All these effects contribute to the activation energy

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