ISOLATION AND CHARACTERISATION OF A NEW ALLELOCHEMICAL FROM PARTHENIUM HYSTEROPHORUS L.

ISOLATION AND CHARACTERISATION OF A NEW ALLELOCHEMICAL FROM PARTHENIUM HYSTEROPHORUS L.

R. N. Yadava* and Shirin Khan

Natural Products Laboratory, Department of Chemistry, Dr. H. S. Gour Central University, Sagar, Madhya Pradesh, India

ABSTRACT

Parthenium hysterophorus L.  is commonly known as “Gajar ghas" in Hindi and belongs to family Composite. It is an annual herb erect up to 1.5 m in hight. Its stems is branched and covered with trichomes. Its leaves are pale green, branched and covered with soft fine hairs. In Homoeopathy system, allergies caused by Parthenium can be treated by a drug prepared from Parthenium. Root decoction is useful in dysentery. In the present  paper, we report the isolation and structurel elucidation of  a new allelochemical identified (I) as 3 , 5, 7, 4′ tetrahydroxy-3'-methoxyflavone-3-O-b-L-galactopyranosyl-(1→3)-O-b-D-arabinopyranosyl-7-O-a-L-rhamnopyranoside alongwith two known compounds Lutexin (II) and Cirsilineol (III) from methanolic extract of the stems of this plant by several colour reactions, chemical degradations and spectral analysis.

Parthenium hysterophorus L., Composite, Stem, flavone glycoside, Antiviral Activity

INTRODUCTION: Parthenium hysterophorus L.^1-3  is commonly known as “Gajar ghas" in Hindi which belongs to family Composite. It is an annual herb erect up to 1.5 m in hight. Its stems is branched and covered with trichomes. Its leaves are pale green, branched and covered with soft fine hairs. The small white flowers have five distinct corners and grow on the stem tips. It is considered a highly invasive weed. It releases chemicals that inhibit the germination and growth of pasture grasses and other plants. This weed is common throughout the World. In Homoeopathy system, allergies caused by Parthenium can be treated by a drug prepared from Parthenium. Root decoction is useful in dysentery.

Earlier workers ^4-8 have reported the presence of various constituents from this plant. In the present  paper, we report the isolation and structure elucidation of  a new allelochemical (I) identified as 3 , 5, 7, 4′ tetrahydroxy-3'-methoxyflavone-3-O-b-L-galactopyranosyl-(1→3)-O-b-D-arabinopyranosyl-7-O-a-L-rhamnopyranoside by several colour reactions, chemical degradations and spectral analysis.

EXPERIMENTAL SECTION:

General experimental procedure: All the m.p. were determined on a thermoelectrically melting point apparatus and are uncorrected. The IR spectra were recorded in KBr disc; ¹H-NMR spectra at 300 MHz in CDCl₃ using TMS as internal standard; ¹³C-NMR spectra were recorded at 300 MHz using CDCl₃ as solvent; UV spectra were determined in MeOH and Mass spectra on a Jeol D-300 mass spectrometer.

Plant material: The leaves of the plant were collected locally around Sagar region and were taxonomically authenticated by the Department of Botany, Dr.H.S.Gour University, Sagar (M.P.) India. A voucher specimen has been deposited in the Natural Products Laboratory, Department Of Chemistry, Dr. H. S. Gour Central University, Sagar (M.P.) India.

 Isolation of the Compound:            Air-dried and powdered leaves (5 kg) of the plant were extracted with 95% ethanol in a Soxhlet apparatus for five days. The ethanolic extract of the leaves of this plant was concentrated under reduced pressure to give light brown viscous mass which was successively partitioned with pet-ether (40-60^oC), chloroform, ethyl acetate, acetone and methanol. The methanol soluble fraction was concentrated under reduced pressure to yield light brown mass (1.24 gm), which was subjected to TLC examination showed three spots indicating it to be mixture of three compounds. These compounds were separated and purified by column chromatography over silica gel G and studied separately.

Study of Compound (I): It  has  m.p. 246-248^oC, [M]⁺ 756 (FABMS); Found (%): C 52.98, H 5.12, calcd. for m.f. C₃₃H₄₀O₂₀ C 53.01, H 5.14; UV (MeOH) λ_max nm: 254, 366; IR (KBr) ν_max (cm-¹); 3346, 2923, 1653, 1625, 1245, 1063, 868, and  835; ¹H-NMR: (300 MHz, CDCl₃) d (ppm); 12.14 (1H, s, 5- OH), 10.16 (1H, s, 4′-OH), 3.81 (3H, s, 3′-OMe), 6.19 (1H, d, J  1.83Hz, H-6), 6.64 (1H, d, J 1.84 Hz, H-8),  7.18 (1H, d, J  8.7 Hz, H-2′), 6.91 (1H, d, J 8.3 Hz, H-5′ ), 7.12( 1H, d, J 8.1 Hz, H-6′), 5.46 (1H,  d,  J  7.1 Hz, H-1''), 4.41 (1H, d, J 3.8 Hz H-2"), 4.12 (1H,dd, J 3.7, 10.3 Hz H-3"), 4.15 (1H, m,  H- 4'' ), 4.39 (2H, d, J 8.4 Hz H-5'' ), 5.12 (1H, d J 7.5 Hz H-1''' ), 3.80 (1H, dd J 3.6, 9.97 Hz H-2''' ), 3.78 (1H, dd, J 3.7,9.98 Hz H-3''' ), 3.54 (1H, dd, J 3.7, 9.98 Hz H-4''' ), 4.01 (1H, m H-5''' ), 5.36 (1H, d, J 1.5 Hz, H-1''''),  4.80 (1H, dd J 3.7, 9.99 Hz H-2"′′), 4.76 (1H, dd, J 3.6, 9.97 Hz H-3"′′), 4.58 (1H, dd, J 3.6, 10. Hz H-4"′′), 4.31 (1H, m, H-5"′′), 1.19 (3H, m, 6''''-Me); ¹³C-NMR:(300 MHz, CDCl₃) d (ppm); 158.3 (C-2), 135.7 (C-3), 177.4 (C-4), 163.4 (C-5), 99.9 (C-6), 164.8 (C-7), 95.01 (C-8), 157.89 (C-9), 105.0 (C-10), 123.4 (C-1′), 132.1 (C-2′), 115.4 (C-3′), 160.5 (C-4′), 116.4 (C-5′), 130.8(C-6′), 100.21 (C-1''), 74.2 (C-2''), 73.1 (C-3''), 70.5 (C-4''), 69.4 (C-5''), 104.3 (C-1'''), 72.08 (C-2'''), 72.0 (C-3'''), 69.5 (C-4'''), 62.8 (C-5'''), 65.23 (C-6'''). 107.2 (C-1''''), 80.3 (C-2''''), 72.0(C-3''''), 74.40(C-4''''), 67.2(C-5''''), 18.2(C-6''''); [M]⁺ 756 (FABMS).

FIG. 1: COMPOUND (Ι)

Acid hydrolysis of Compound (I): The compound A (650 mg) was dissolved in ethanol (30ml) and refluxed with 20 ml of 10% H₂SO₄  on  water  bath  for 72  hrs. The reaction mixture was concentrated and allowed to cool and residue was extracted with diethyl ether. The ether  layer was washed with water and the residue was chromatographed over silica gel using CHCl₃: MeOH (2 : 4) to give compound A₁ which was identified as 3, 5, 7, 4'-tetrahydroxy 3'-methoxy flavones by comparison of its known spectral data. The aqueous hydrolysate was neutralized with BaCO₃ and BaSO₄ filtered off. The filtrate was concentrated and subjected to paper chromatography examination using  n-BAW (4 :1: 5) as solvent  and  aniline  hydrogen  phthalate  as detecting  agent, confirmed  the  presence of D-galactose (R_f  0.19), L-arabinose (R_f 0.22) and L-rhamnose (R_f 0.36) by (Co-Pc )

Permethylation of Compound (I): Compound A (25 mg) was refluxed with MeI (5 ml) and Ag₂O (5 mg) in DMF (15 mg) for one day and then filtered. The  filtrate  was hydrolyzed with 10% ethanolic  H₂SO₄  for 72 hrs to yield  methylated  aglycone  identified as  3, 7-dihydroxy-5, 3', 4'-trimethoxy flavone and methylated sugars which were identified as 2,3,4-tri-O-methyl-L-rhamnose (R_G 1.07), 2,3,4,6-tetra-O-methyl-D-glactoes (R_G 1.02)  and  2,4-di-O- methyl- D-arabinoes (R_G 0.67).

Enzymatic hydrolysis of Compound (I): The compound A (25 mg) was dissolved in MeOH (30 ml) and hydrolysed with an equal volume of takadiastase at room temperature in a 150 ml round bottomed flask fitted with air condenser. The contents were left for two days and filtered to yield L-rhamnose (R_f 0.36) and proaglycone, confirming the presence of α-linkage between L-rhamnose and   proaglycone.

Proaglycone   on further  hydrolysis with almond  emulsin  liberated D-glactoes (R_f 0.17) first, then  D-arabinoes (R_f 0.27)  and  aglycone, which confirmed the presence of the β-linkage between D-glactoes and D-arabinoes as well as between D-arabinoes and aglycone.

Study of Compound A-1: It was crystalysed to acetone to yield 950 mg. It has m.p. 309-310^oC,  m.f. C₁₆H₁₂O₇ , M⁺ 316, (EIMS) Found (%) C, 63.57; H, 3.97, Calcd for m.f. C₁₆ H₁₂O₇  C, 63.57; H, 3.97. UV: (MeOH) λ_max (nm) 255, 274, 362, 372; IR: n (cm^-1); 3405, 2890, 1731, 1650, 1602, 1567, 1447, 1383, 1365, 1301, 1204, 1072, 1025 cm^-1. ¹H-NMR: (300 MHz, CDCl₃) d (ppm); 10.40 (1H, s, 3-OH), 12.01 (1H, s, 5-OH), 9.79 (1H, s, 7-OH), 9.42 (1H, s, 3′-OH), 9.29 (1H, s, 4′-OH), 6.20 (1H, d, 2.2 Hz, H-6), 6.60 (1H, d, J 2.4 Hz, H-8), 7.62 (1H, d, J  2.1 Hz, H-2′), 6.82 (1H, d, J 8.4 Hz, H-5′), 7 (1H, d, J 8.0 Hz H-6′), ¹³C-NMR:(300 MHz, CDCl₃) d (ppm); 162.9 (C-2), 104.2 (C-3), 180.4 (C-4), 160.4 (C-5), 101.2 (C-6), 163.0 (C-7), 95.8 (C-8), 157.89 (C-9), 105.0 (C-10), 130.0 (C-1′), 117.2 (C-2′), 128.87 (C-3′), 132.5 (C-4′), 116.0 (C-5′), 124.0 (C-6′).

FIG. 2: COMPOUND (A-1)

Compound (II): It has m.p. 261-262^oC, [M]⁺ 448 (FABMS); found (%): C 54.95, H 4.47, calcd.for m. f. C₂₁H₂₀O₁₁ C 54.82, H 4.55; UV (MeOH) λ_max nm:, 256, 268, 351, (+AlCl₃) 277, 335, 418; (+AlCl₃–HCl) 265, 274, 311, 355; (+NaOMe) 267, 276, 403; (+NaOAc) 268, 275, 326; (+NaOAc–H₃BO₃) 265, 302, 374; IR (KBr) ν_max (cm-¹); 3420, 1658, 1610; ¹H-NMR: (300 MHz, DMSO-d₆) d (ppm); 13.14 (1H, s, 5- OH), 9.14-10.79 (3H, s, 3′, 4′, 7′-OH), 7.50 (1H, d, J 2.2 Hz, H-2′,6′), 6.89 (1H, d, J 8.3 Hz, H-5′), 6.63 (1H, s, H-3), 6.54 (1H, s, H-6), 5.02 (1H, d, J 7.0 Hz)3.32-3.91 (sugar protons, m); ¹³C-NMR:(300 MHz, DMSO-d₆) d (ppm); 164.14 (C-2), 102.45 (C-3), 181.01 (C-4), 161.45 (C-5), 98.34 (C-6), 162.83 (C-7), 104.68 (C-8), 156.10 (C-9), 104.20 (C-10), 121.95 (C-1′),114.05 (C-2′), 145.93 (C-3′), 149.87 (C-4′), 115.75 (C-5′), 119.43 (C-6′), 73.49 (C-1"), 70.89 (C-2"), 78.86 (C-3"), 70. 82 (C-4"), 82.01 (C-5"), 61.72 (C-6"); (ESIMS): 448 [M] ⁺, 430 [M-H₂O] ⁺, 314 [M-C₈H₆O₂] ⁺, 299 [M-aglycone-CH₂] ⁺, 286 [M-162] ⁺, 134 [M-314]⁺, 69 [MM-379] ⁺: Thus, it was identified as Lutexin by comparison of its spectral data with reported literature value.

FIG. 3: COMPOUND (ΙΙ)

Compound  (III): It has m.p. 194-195^oC, [M]⁺ 344 (EIMS); Found (%): C 62.79, H 4.65, calcd. for m.f. C₁₈H₁₆O₇, C 62.56, H 4. 60; UV (MeOH) λ_max nm: 282, 325; (AlCl₃); 342, 376 IR (KBr) ν_max (cm-¹);1690, 1595, 1351, 1203, 1115, 1026. ¹H-NMR: (300 MHz, CDCl₃) d (ppm); 3.98 (3H, s, 6-OCH₃), 3.82 (3H, s, 7-OCH₃),  3.80 (3H, s, 3′-OCH₃), 7 (1H, s, 3-H), 6.86 (1H, s, 8-H), 7.62 (1H, s, 2′-H), 7.30 (1H, d, J 8.81 Hz, 5′-H), 7.65 (1H, d, J 8.84 Hz, 6′-H); ¹³C-NMR:(300 MHz, CDCl₃) d (ppm); 55.80 (3′-OCH₃), 56.20 (7-OCH₃), 60.39 (6-OCH₃), 164.95 (C-2), 103.91 (C-3), 183.31( C-4), 153.74 (C-5), 133.16 (C-6), 159.43 (C-7), 91.57 (C-8), 153.63 (C-9), 106.32 (C-10), 122.47 (C-1′),  112 (C-2′), 149.10 (C-3′), 152.65 (C-4′), 117.05 (C-5′), 121.41 (C-6′). Thus it was identified as Cirsilineol by comparison of its spectral data with reported literature value.

FIG. 4: COMPOUND (ΙΙΙ)

Antiviral activity of Compound (I): Compound (I) was tested for antiviral activity against Japanese  Encephalitis Virus  in vitro (Vero cells) and  result is given in following Table 1. Result shown in Table 1 showed that no antiviral activity was found.

TABLE 1:ANTIVIRAL ACTIVITY OF COMPOUND (I)

RESULTS AND DISCUSSION: The methanol soluble fraction of the leaves of the  plant afforded a new compound (I) (Fig. 1), m.p. 246-248^oC, m.f. C₃₃H₄₀O₂₀ [M]⁺ 756 (FABMS). It responded Molisch and Shinoda tests ⁹ showing its flavonoidal glycosidic nature. Its IR  spectrum showed absorption bands at 3346, 2923, 1653, 1625, 1245, 1063, 868, and  835 cm-¹. In ¹H NMR spectrum, a singlet at δ 12.24 confirmed the presence of -OH group at C-5 position. A singlet at δ 3.81 integrating three proton intently confirmed the presence of  methoxy group at H-3' position. A doublet at δ 7.98 was assigned for H-2' and H-6'. A doublet at δ 6.95 was assigned for H-5'. A singlet at δ 10.16 was assigned for –OH group at C-4' position. The ortho coupled doublets at 6.18 (d, 1H, J 1.85 Hz ) and 6.44 (d, 1H, J 1.85 Hz ) were  assigned at C-6 and C-8 positions respectively. The anomeric proton signals at δ 5.10 (1H, d,  J  7.2 Hz ), δ 5.12 (1H, d, J 7.5 Hz) and δ 5.36 (1H, d, J 1.5 Hz) were assigned to H-1'', H-1''' and H-1'''' of D-arabinose (R_f 0.19), D-galactose (R_f 0.25)  and L-rhamnose ((R_f  0_.37) respectively ¹⁰. In ¹³C-NMR spectrum, a singlet at δ 58.5 showed the presence of methoxy group at C-3' position.   

Acid hydrolysis of compound (I) with 10% ethanolic H₂SO₄  yielded aglycone 1-A, m. p. 268-270^oC, m.f. C₁₆H₁₂O₇, [M]⁺ 316 (FABMS), identified as 3,5,7,4'-tetrahydroxy 3'-methoxy flavone (see ¹HNMR and ¹³CNMR data in experimental section) ^11-12. The aqueous hydrolysate obtained after hydrolysis of the glycoside was neutralized with BaCO­₃ and the BaSO₄ was filtered off. The filtrate was concentrated under reduced pressure and subjected to paper chromatography examination showed the presence of L-rhamnose (R_f  0_.37), D-galactose (R_f 0.25) and L-arabinose (R_f 0.19). Quantitative estimation of sugars carried out by the procedure of Mishra and Rao, showed that all three sugars were present in equimolar ratio (1:1:1). Periodate oxidation of compound (I),confirmed that all the sugars were present in the pyranose form ¹³.

The position of sugar moiety (ies) in compound (I)was determined by permethylation ¹⁴followed by acid hydrolysis  yielded methylated aglycone identified as 3,7-dihydroxy-5, 3', 4'-trimethoxy flavone which confirmed that glycosidation was involved at C-3 –(OH) and C-7 –(OH) position of the alycone. The methylated sugars were identified as  2, 3, 4-tri-O-methyl-L-rhamnose (R_G 1.07),  2, 3, 4, 6- tetra-O-methyl-D-galactose (R_G 1.02)  and  2, 4-di-O-methyl-D-arabinose (R_G 0.67) ¹⁵.

Therefore it was concluded that C-1"'-OH of D-galactose was linked with C-3"′-OH of D-arabinose and C-1"-OH of D-arabinose attached with –OH group at C-3 position of aglycone. The interglycosidic linkage (1→3) was found between D-galactose and L-arabinose ¹⁶.

Enzymatic hydrolysis of  compound (I) with  takadiastase liberated  L-rhamnose and  proaglycone confirming the presence of α-linkage between L-rhamnose and 3, 5, 7, 4'-tetra hydroxy 3'-methoxy flavone-3-O-β-D-glactopyranosyl (13)-O-β-D-arabino pyranoside  as proaglycone. Proaglycone on further hydrolysis with almond emulsin liberated D-galactose first followed by D-arabinose and aglycone, confirming the presence of β-linkage between D-galactose and D-arabinose as well as between D-arabinose and aglycone. On the basis of above evidence, the compound A was characterized as 3, 5, 7, 4′ tetrahydroxy-3'-methoxyflavone-3-O-b-D-galacto pyranosyl-(1→3)-O-b-D-arabinopyranosyl-7-O-a-L-rhamnopyranoside.

Compound (I) was  tested  for antiviral activity  against  Japanese  Encephalitis Virus  in vitro (Vero cells)  but  no  antiviral  activity was shown by compound  (I).

Compound (II) (Fig. 3), was analyzed for m. f. C₂₁H₂₀O₁₁,   m. p. 261-262^oC, [M]⁺ 448 (EIMS) It was identified as Lutexin by comparison of its spectral data with reported literature values ¹⁷.

Compound (III) (Fig. 4), was analyzed for m. f. C₁₈H₁₆O₇,  m. p. 194-195^oC, [M]⁺ 344 (EIMS). It was identified as Cirsilineol by comparison of its spectral data with reported literature values ¹⁸.

CONCLUSION: Parthenium hysterophorus L. belongs to composite family we have isolated a new allelochemical along with two known compound Lutexin, Cirsilineol from the stem of

Parthenium hysterophorus L . A new allechemical have show no antiviral activity. The secondary plant metabolites are called an allechemical, These chemicals include flavonoids, alkaloids, tannins etc.

ACKNOWLEDGEMENT: Authors are thankful to the Director CDRI Lucknow for recording various spectral data and elemental analysis and Head, Department of Chemistry, Dr. H. S. Gour Central University, Sagar (M.P.) India, for providing necessary laboratory facilities.

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    How to cite this article:

    Yadava RN and Khan S: Isolation and Characterization of a New Allelochemical from Parthenium hysterophorus L. Int J Pharm Sci Res. 2013; 4(1); 311-315.

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