INTRODUCTION:

Hibiscus cannabinus Linn. of Malvaceae family is widely present in the Deccan parts of India which comprises Andhrapradesh, parts of Karnataka, Madhyapradesh, and Bihar¹.

The gynaceum part of the flowers of Hibiscus cannabinus Linn has been chosen for phytochemical investigation.

MATERIALS AND METHODS:

Extraction and Fractionation:

Fresh gynaceum part of Hibiscus cannabinus collected from in and around the region of Kumbakonam, Tamil nadu were extracted with 90% MeOH under reflux. The alcoholic extract was concentrated with benzene, peroxide free Et₂O and EtOAc. The benzene and Et₂O fractions did not yield any isolable material.

EtOAc Fraction:

The residue from EtOAc fraction was taken up in a small quantity of Me₂CO and kept under chilled conditions for one week. A yellow solid on crystallization from aq. MeOH (MP 219-221^oC) was obtained. With dil. alkali it gave a yellow solution that changed to green colour. It answered Wilson’s Boric acid, Gibb’s, Shinoda and Molisch’s tests. It did not respond to Horammer-Hansel test. Its R_f values are tabulated in table I.

It had λ_max MeOH nm 260, 315 sh, 362; + NaOMe 271,406; +AlCl₃ 269,301, 329, 426; + (AlCl₃+HCl) 269, 360, 390 sh; + NaOAc 270, 398 and (NaOAc + H₃BO₃) 259, 374. The compound has been identified as Myricetin -3-O-glucoside by a direct comparison with authentic samples of Primulus sinensis².

Hydrolysis of Glycoside:

Approximately 0.05 g of the glycoside was dissolved in 5 ml of hot MeOH and subjected to hydrolysis with an equal volume of 10% H₂SO₄ in a boiling water bath for 2 hr and the hydrolytic products were identified as described below.

Identification of Aglycone:

The residue from Et₂O fraction was taken up in Me₂CO and in a ice chest for a day. Shining yellow crystals with M.P 357-360^oC was obtained. It appeared yellow under UV with or without NH₃. It had λ _max MeOH nm 254, 272sh, 301sh 374; + NaOMe 262sh, 285sh, 322, 423; + AlCl₃ 271,316sh, 450; + (AlCl₃+HCl) 266, 275sh, 308sh, 360sh,428; + NaOAc 269, 335; + NaoAc+H₃BO₃) 258, 304sh, 392. It had R_f values depicted in table I

RESULTS AND DISCUSSION:

The gynaceum part of the fresh flowers of Hibiscus cannabinus have been found to contain Myricetin-3-O-glucoside.

In the UV spectrum of the glycoside the band I absorption maxima was located at 362 nm which suggest a flavonol skeleton. A comparison of band I absorption of the glycoside and that of the aglycone reveals that there may be 3-O-glycosylation in the flavonol³.

Table – 1: Rf ( X 100 ) values of the constituents of the flowers of B.Ceiba ( Whatman No. 1, Ascending 30 ± 2⁰ c )

Compound	a	b	c	d	e	f	g	h	i
Myricetin-3-O-glucoside( authentic )	05	10	25	40	80	47	32	40	25
Glycoside isolated from EtOAC fraction	05	10	25	40	80	47	32	40	25

Solvent Key :-

a) H₂O b) 5% aq.HOAc, c) 15% aq.HOAc, d) 30% aq. HOAc,

e) 60%aq.HOAc, f) BAW( n-BuOH:HOAc:H₂O=4:1:5 ) g) water saturated phenol

h) t-BuOH:HOAc:H₂O=3:1:1 i) Forestal( HOAc:con.HCl:H2O=30:3:10)

Table – 2 ¹³C-NMR spectral data and their assignment for the glycoside from the flowers of Hibiscus Cannabinus

Carbon Number	Myricetin-3-O-glucoside ( from literature δ ppm )	Glycoside isolated from EtOAc fraction (δ ppm)
C2	147.1	146.8
C3	136.1	133.0
C4	176.0	175.8
C5	161.0	160.6
C6	98.5	98.1
C7	164.2	163.9
C8	93.5	93.5
C9	156.4	156.0
C10	103.3	102.9
C1’	121.2	121.0
C2’	107.5	107.7
C3’	146.0	145.5
C4’	136.1	136.0
C5’	146.0	145.5
C6’	107.7	107.7
C1’’	101.8	102.9
C2’’	73.4	73.3
C3’’	77.1	77.2
C4’’	69.8	69.6
C5’’	76.5	75.9
C6’’	61.0	60.6

It was also supported by the fact that the glycoside did not answer Horhammer-Hansel test whereas the aglycone did. The bathochromic shift of 44 nm observed in the NaOMe spectrum (band I) of the glycoside indicated the presence of a free –OH at C-4’. An additive bathochromic shift of 36 nm observed in the AlCl₃ spectrum with respect to AlCl₃/HCl spectrum indicated the presence of a catechol type of ortho dihydroxyl grouping in the B ring⁴. This was also confirmed by the bathochromic shift of 12 nm in its NaOAc+H₃BO₃ spectrum^5,6. The presence of a free –OH at C -7 is evident from the bathochromic shift of +10 nm (bandI) on the addition of NaOAc. The appearance of four absorption maxima of AlCl₃ spectra a bathochromic shift of +28 nm (band I). In the AlCl₃/HCl spectrum with respect to the methanolic spectrum⁵ and the positive Wilson’s Boric acid test indicated the presence of a free 5-OH in the A ring.

In the 1H NMR spectrum (400 MHz, DMSO-d₆, TMS) of the glycoside the signals of the protons at C-2’ and C-6 merge at δ 6.27 ppm. The proton at C-8 resonates at δ 6.38 ppm. The H 1” of the glucose moiety resonates at δ 5.46 ppm.The remaining sugar protons appear in the region of δ 3.18-3.37 ppm. The 5-OH proton resonates at δ 12.45 ppm. The 7-OH proton is found at δ 10.75 ppm.

The various signals noticed in the ¹³C NMR spectrum of the glycoside (100 MHz, DMSO-d₆, TMS) has assigned to the different carbons are presented in table 2.

From the above chemical, spectral evidences the glycoside from the EtOAc fraction has been characterized as Myricetin 3-O-glucoside by comparing it with an authentic sample.

CONCLUSION:

The gynaceum part of the flowers of Hibiscus Cannabinus was taken for analysis. on phytochemical investigation of the EtOAc soluble fraction yielded Myricetin 3-O-glucoside.

ACKNOWLEDGEMENTS:

The authors wish to acknowledge Indian Institute of Science, Bangalore and National Institute of Technology, Trichirappalli for technical assistance.

REFERENCES:

1. Wlth. India, Raw materials, CSIR, New Delhi, 1959,5, 77

2. J.B.Horborne and H.S.A. Sherratt, Biochem.,J. 1961, 78,298

3. K.R.Markham and T.J.Mabry The flavonoids, J.B.Harborne, T.J.Mabry and H.Mabry, Eds., Chapman and Hall, London, 1975, 48

4. K.R.Markham and T.J.Mabry, Phytochem., 1968, 7, 1197

5. L.Jurd, Arch.Biochem.Biophys.,1956, 63, 376

6. K.R.Markham and T.J.Mabry Phytochem., 1968 57,45

Received on 11.09.2009 Modified on 05.11.2009

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 81-82