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Novel biologically active flavonoidal constituent from Smithia conferta Sm.

R.N. Yadava

Natural Products Laboratory,Department of Chemistry, Dr. H. S. Gour University, SAGAR 470 003 (M.P.) INDIA
E-mail: rnyadava@rediffmail.com, Telephone No. 07582-265465

Abstract

A novel bioactive flavone glycoside was isolated from the acetone soluble fraction of the flowers of Smithia conferta Sm. Its structure was characterized as 5,6-dihydroxy 7,8,3’-trimethoxy flavone–4’-O-β-D-glucopyranosyl -(1→4)-O-β-D-xylopyranoside by various spectral analysis and chemical degradations. This novel natural product shows antimicrobial activity against various plant pathogenic bacteria and fungi.

Keywords

Smithia conferta, Leguminosae, Novel flavonoidal constituent, Microbial activity.

Introduction

Smithia conferta1,2 (N.O. Leguminosae) is commonly known as Lakshmana Booti in Sanskrit and Naichi Bhaji in Marathi. It is found almost throughout in India. The plant is laxative and used as tonic. It is used in the treatment of biliousness, ulcers and rheumatism. Its powdered leaves are used to cure sterility in woman. It is also used to remove the effects of old age and wrinkles. Earlier workers3-6 have reported the presence of various bioactive constituents from this plant. The present paper deals with the isolation and characterization of novel biologically active flavone glycoside from the flowers of this plant.

Results and discussion

The acetone soluble fraction of the powdered flowers of this plant afforded a yellow crystalline compound A which responded Molisch test and all positive tests for flavonoidal glycoside7,8. UV λmax (MeOH) at 278 and 345 nm and its changes in the presence of diagnostic shift reagents9, indicated the presence of free OH group at C-5 and C-6 positions and blocked C-7, C-8, C-3’-positions.

Compound A on acid hydrolysis with 7% H2SO4 gave an aglycone B and sugars which were identified as D-xylose (Rf 0.29) and D- glucose (Rf .17) (co-pc and co-tlc). The aglycone B, was identified as 5,6,4’-trihydroxy 7,8,3’-trimethoxy flavone by comparison of (m.p., mmp, UV, IR, 1H-NMR, 13C-NMR ) with reported literature10. Alkaline degradation of the compound B yielded two compounds m.f. C10H12O6, [M+] 228 identified as 3,4 dimethoxy, 2,5,6tri hydroxy acetophenone and m.f. C8H8O4, [M]+ 168 identified as ρ-hydroxy 3-methoxy benzoic acid, confirmed the position of hydroxyl and methoxyl groups in compound A. Formation of 3,4-dimethoxy 2,5,6-tri-hydroxy acetophenone confirmed the position of hydroxyl group at C-5 and C-6 position in ring A. UV spectrum of the compound B showed bathochromic shift of 37 nm in band I with AlCl3 and 45 nm in band I with AlCl3 + HCl relative to methanol confirmed the presence of hydroxyl groups at C-5 and C-6 position in ring A11-12 A bathochromic shift at 32 nm with NaOAc + H3BO3 in band I relative to methanol confirmed the presence of –OH groups at C-4’13.

Compound A

Compound B

Compound A on acetylation with Ac2O/pyridine gave an octacetate derivative, suggested eight acetylisable -OH groups. Its 1H-NMR spectrum showed eight singlets at δ 2.48, 2.36, 2.02, 2.08, 3.09, 2.05, 2.10, and 2.15 indicating the presence of eight hydroxyl groups in the compound A. The three sharp singlets at δ 3.75, 4.01 and 3.82 showed the presence of three methoxyl groups in B. The 1H-NMR spectrum also exhibited A2B2 pattern of B ring as it showed two double doublets at δ 7.22 (2H, d, J 8.5 Hz, H-2’, 6’) and δ 6.85 (2H, d, J = 8.6 Hz, H-3’, 5’). The aromatic protons of sugars showed doublets at δ 5.51 and 5.63 which were assigned for H-1’’ and H-1’’’ of D-glucose and D-xylose.

The remaining sugar protons were appeared at δ 3.95-4.42 as a multiplate for eleven protons. 13C-NMR spectrum of the A revealed the presence of 29 carbon atoms, confirmed its structure [see Table –1]. The FABMS of [A] showed fragments at m/z 521 and 522 due to removal of disaccharide. The RDA fragmentation patterns were appeared at m/z 213, and 164 due to [A1 + H+]+ and [B1]+ fragment. The formation of [A1 + H+]+ and [B1]+ fragments confirmed the presence of two methoxyl and two hydroxyl groups in A ring and one methoxyl group in B ring.

Table 1. 13C-NMR 1 (90 MHz, DMSO-d6) of Compound A

Atom

δ value

Atom

δ value

C-2

161.72

-OMe

57.91

C-3

101.81

-OMe

62.92

C-4

178.5

-OMe

64.89

C-5

155.64

C-1’’

105.71

C-6

131.88

C-2’’

75.69

C-7

140.45

C-3’’

76.81

C-8

96.45

C-4’’

78.61

C-9

154.36

C-5’’

61.99

C-10

104.56

C-1’’’

105.63

C-11

120.60

C-2’’’

75.47

C-1’

121.8

C-3’’’

76.59

C-2’

128.6

C-4’’’

78.97

C-3’

144.54

C-5’’’

64.39

C-4’

153.43

C-6’’’

50.56

C-5’

116.81

   

C-6’

128.30

   

Permethylation14 of A followed by acid hydrolysis yielded permethylated aglycone identified as 4’ hydroxy 5,6,7,8,3’-penta (methoxy) flavone, showed that sugar units are attached with C-4’ of the aglycone. The methylated sugars were identified as 2,3,4,6, tetra-O-methyl-D-glucose and 2,3,di-O-methyl-D-xylose, revealed that C-4’’ of D-xylose was attached C-1’’’ of D-glucose and C-1’’ of D-xylose was linked with C-4’ of the aglycone and linkage (1→4) between sugars were in pyranose form15,16.

Enzymatic hydrolysis of compound A with almond emulsin liberated D-glucose first then D-xylose, indicating β-linkage between D-xylose and D- glucose, as well as between D-xylose and aglycone.

On the basis of above evidences, the structure of a novel compound A was characterized as 5,6-dihydroxy 7,8,3’-trimethoxy flavone–4’-O-β-D-glucopyranosyl (1→4)-O-β-D-xylopyranoside.

Compound A was tested against various bacteria and fungi. The results recorded in Table-2 (see in experimental) shows that the antibacterial activity of the compound A, was found to be fairly good against gram negative bacteria viz. Escherichia coli and gram positive bacteria Bacillus coagulas. Antifungal activity of the compound A was found to be more active against Penicillium digitatum and Aspergillus niger even on very dilute concentrations. Thus compound A may be potentially used as therapeutic agent diseases caused by these microorganisms.

Experimental

General Experimental Methods

M.PS are uncorrected; UV spectra were determined in MeOH and IR recorded in KBr discs; 1H-NMR spectra were run at 300 MHz using TMS as internal standard and CDCl3 as solvent. 13C-NMR spectra were measured at 90 MHz using DMSO-d6 as solvent.

Plant Material

The plant material was procured by M/s United Chemicals and Allied Products, Calcutta, and authenticated by Botany Department of this University, A voucher specimen has been deposited in Chemistry Department Dr. H.S. Gour University, Sagar (M.P.) INDIA.

Extraction and Isolation

Air dried and powdered flowers (3 Kg) of the plant were extracted with 95% EtOH in a Soxhlet extractor. The total ethanolic extract was concentrated and partitioned with petroleum-ether (40-60C), benzene, chloroform, ethyl acetate, acetone, and methanol. The acetone soluble part was concentrated and examined by TLC, afforded two spots showing it to be mixture of two compounds A and A’ which were separated by TLC and purified by column chromatography. The quantity of compound A’ was found in very small quantity therefore it was not possible to examine it further, hence rejected. Compound A was further purified by column chromatography which was found to be homogeneous on TLC.

Study of the compound A

It was crystallized from methanol as yellowish crystal (1.80gm) m.p. 286-288C [M]+ 654 (FABMS), m.f. C29H34O17, (found C, 53.19%, H 5.20%, calcd C, 53.21%, H 5.17%). UV λmax (MeOH) 278, 345; (+NaOMe) 272, 391; (+AlCl3) 260, 380; (+AlCl3 + HCl), 260, 388; (+NaOAc) 274, 394; (+NaOAc / H3 BO3) 277, 376. IR (KBr) νmax 3445, 2942, 2872, 1665, 1620, 1585, 1120 and 825 cm-1. Compound A (75 mg) was mixed with Ac2O/py (10 ml). It formed octacetate derivative, C45H50O25, m.p. 186-188C, [M]+ 990. The 1H-NMR (300 MHz, CDCl3) δ3.75 (3H, s, 7-OMe), 4.01 (3H, s, 8-OMe); 3.82 (3H, s, 3’-OMe); 7.22 (2H, d, J 8.5 Hz, H-2’, 6’); 6.85 (2H, d, J 8.6 Hz, H-3’, H-5’); 2.42 (3H, s, 4’- OAc), 2.48 (3H, s, 5-OAc), 2.36 (3H, s, 6-OAc), 5.45 (1H, d, J 8.8 Hz, 1’’-anomeric proton), 2.02 (3H, s, 2’’-OAc), 2.08 (3H, s, -3’’-OAc), 3.09 (3H, s, 6’’-OAc), 5.69 (1H, d, J 9.0Hz, 1’’’-anomeric proton), 2.05 (3H, s, 2’’’-OAc), 2.10 (3H, s, -3’’’-OAc), 2.15 (3H, s, 5’’’-OAc), 3.95-4.42 (11H, m, protons of sugar residue). [M]+ absent, m/z 522, 521, 360, 332, 331, 213, 212, 184, 164 (FABMS).

Acid Hydrolysis

Compound A (500mg) was hydrolysed with 7% H2SO4 by refluxing for about 8 hrs yielded aglycone B. The hydrolysate was neutralized with BaCO3 and BaSO4 filtered off. The filtrate was concentrated to yield a yellow viscous mass which was subjected PC examination and found to contain D-xylose (Rf 0.29) and D-glucose (Rf 0.17) (co-pc and co-tlc).

Identification of the Aglycone B

It crystallised from methanol to yield (0.98 gm), m.p. 230-232C, [M]+ 360 [FABMS], m.f. C18H16O8, (Found C 60.0%, H 4.38%, calcd C 60.02%, H 4.30%), UV λmax (MeOH) nm 262, 344; (+NaOMe) 278, 390, (+AlCl3) 268, 381; (+AlCl3-HCl) 266,389; (NaOAc), 258, 393; (+NaOAc/H3BO3) 245, 376 IR (KBr) νmax cm-1 3445, 2942, 2872, 1665, 1620, 1585, 825; 1H-NMR (300MHz, CDCl3) of the acetylated derivative of B, δ 3.78 (3H, s, 7-OMe), 4.05 (3H, s, 8-OMe), 3.75 (3H, s, 3’-OMe), 7.23 (2H, d, J 8.5, H-2’, 6’), 6.86 (2H, d, J 8.6, H-3’, 5’), 2.42 (3H, s, 4’-OAc), 2.47 (3H, s, 5-OAc), 2.32 (3H, s, 6-OAc) [M]+ : 360 m/z 332, 331, 213, 212, 184, 164, 163 (FABMS).

Permethylation and Hydrolysis of the A

Compound A (20 mg) was refluxed with CH3I (3ml) and Ag2O (15mg) in DMF (5ml) at room temperature for 48 hrs. The reaction mixture was filtered and the residue was treated with EtOH (5 ml). The syrupy residue was hydrolysed with 10% H2SO4 for 7-8 hrs. After usual workup, it gave methylated aglycone identified as 4’-hydroxy 5,6,7,8,3’-pentamethoxy flavone and methylated sugars were identified as 2,3, di-O-methyl-D-xylose and 2,3,4,6-tetra-O-methyl-D-glucose.

Alkaline degradation of compound B

Compound B (100 mg) was refluxed with 50 ml of 35% KOH in 20 ml of MeOH for two days. The reaction mixture was cooled, acidified with HCl and extracted Et2O. The ethereal layer was washed with water and dried over Na2CO3. The Et2O distilled in vacua gave (a) m.f. C10H12O6, m.p. 174-176C, [M]+ 228, (Found C 52.68%; H 5.29%, calcd C 52.63%, H 5.26%), identified as 3,4-dimethoxy-2,5,6-trihydroxy acetophenone. The aqueous phase was acidified with HCl and extracted with Et2O and washed with water to yield (b) m.f. C8H8O4, m.p. 152-154oC, [M]+ 168, (Found C 57.10%, H 4.81%, calcd C 57.14%, H 4.76%), identified as p-hydroxy-3-methoxy benzoic acid.

Enzymatic Hydrolysis of the Compound A

Compound A (35 mg) was dissolved in ethanol (20 ml) and treated with almond emulsin (30 ml) in a (100 ml) conical flask fitted with stopper. The reaction mixture was allowed to stand for 3 days at room temperature and filtered. The aglycone and hydrolysate were studied separately. The hydrolysate was concentrated and subjected to P.C. on Whatman filter paper No.1 using (n-BuOH:Ac2OH:H2O 4:1:5) as solvent system confirming the presence of D-xylose (Rf 29) and D-glucose(Rf 17) and also revealed the presence of β-linkage between both sugars as well as between sugar and aglycone.

Determination of antimicrobial activity of the compound a

The antimicrobial activity of the compound A (400 mg) was screened at its various dilutions using acetone as solvent. The compound A was prepared in acetone and water in different concentrations. The antibacterial activity of the compound A was determined by filter paper disc diffusion method17. The sterile filter paper discs (6 mm) were soaked with the standard antibacterial agent and various test samples and were dried at 50C. The discs were then placed on the soft nutrient agar (2%) petri plates previously seeded with suspension of each bacterial species. The diameters of zone of inhibition were measured at 371C after 24 hrs.

For antifungal activity Saboraud’s broth18 media with 4% agar was used for the preparation of plates and inoculated with the spore and mycelium suspension of fungi obtained from 10 days old culture. The plates after inoculation were incubated at 271C after 48 hrs. The diameters of zone of inhibition were measured and various results are reported in Table 2.

Table2. Antimicrobial activity of the compound A


Bacterial species

Diameters of zone of inhibition (mm)*

Compound A at concentration (%)

Std**

100

80

60

40

20

100

(-) Escherichia coli

14.2

11.8

8.4

6.9

5.4

17.3

(-) Psedomonas aerugenosa

11.4

9.4

6.2

3.4

-

22.2

(+) Bacillus coagulas

16.4

12.8

11.2

9.6

8.4

21.2

(+)Staphylococcus aureus

11.4

8.5

6.1

3.2

-

19.9

             

Fungal Species

         

Std***

Aspergillus niger

12.5

10.4

8.7

6.5

5.2

14.6

Tricoderma viride

11.8

9.3

4.1

2.2

-

23.1

Fusarium oxysporum

12.4

9.5

7.3

3.2

-

21.5

Penicillium digitatum

14.5

12.4

10.6

8.2

6.4

16.8

* The diameters of zone of inhibition (mm) taken in different directions.
** Streptomycin used as standard antibacterial agent.
*** Griseofulvin used as standard antifungal agent.

Acknowledgements

Thanks are due to the Head, R.S.I.C., Central Drug Research Institute, Lucknow (U.P.) India for recording the various spectra and elemental analysis, Head, Department of Chemistry, Dr. H.S. Gour University, Sagar for providing laboratory facilities and Dr. Archana Mehta, Department of Botany, Dr. H.S. Gour University, Sagar for providing necessary facilities to complete antimicrobial activity.

References

Chopra RN, Nayar SL and Chopra IC (1996). Glossary of Indian Medicinal Plant. C.S.I.R. Publication, New Delhi, 228-229.

Wealth of India (1981). A Dictionary of Indian Raw Material and Industrial Products. C.S.I.R. Publication, New Delhi, Vol. IX, 368-369.

Lal SD and Lata K (1980). Eco. Bot., 34(3), 273-275.

Choudhary RR and Haq M (1980). Bull. Medico. Ethno. Bot. Res., 1(4), 546-553.

Pant PC and Joshi MC (1993). J. Res. Edu. Indian Med., 12(2), 19-29.

Yadava RN and Agrawal PK (1999). J. Inst. Chemists, 71(6), 227-230.

Jurd L (1962). Chemistry of Flavonoid Compounds. T.A. Geismann Ed., Pergamon Press, London, 107-155.

Brigga LH and Locker RH (1951). J. Chem. Soc., 3136.

Mabry TJ, Markhaam KR and Thomas MB (1970). The Systematic Identification of Flavonoids. Springer, New York.

Broucke Chris O Van Den, Dommisse Roger A, Esmans EL and Lemli Jozef A (1982). Phytochemistry, 21, 2581-2583.

Jurd L and Geismann TA (1956). J. Org. Chem., 21, 1395.

Harborne JB and Mabry TJ (1982). The Flavonoids Advances in Research, London, 241.

Ulubeler A, Miski M, Neuman P and Mabry TJ (1979). J. Nat. Prod., Vol. 42(3), 262.

Hakomoni S (1964). J. Biochem., 55, 205-208.

Petek F (1965). Bull. Soc. Chem. Fr., 263-267.

Hirst EL, Jones JKN (1949). J. Chem. Soc., 1659-1661.

Maruzzella JC and Henry PA (1958). J. Am. Pharm. Assoc., 47, 471-476.

Vincent JC and Vincent HW (1944). Proc. Soc. Exp. Biol. Med., 55.

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