CHAPTER 5

GOSSYPIN - A BIOACTIVE SECONDARY METABOLITE

FROM HIBISCUS FURCATUS

5.1. INTRODUCTION

Flavanoids represent the most important single group of phenolics in food,

more than 4000 different types have been identified (Harborne, 1964). Biochemistry

of flavanoids (Harborne, 1967) has also been fascinating that ever since 1900

(Ferguson, 2001). Gossypin, a flavanoid compound isolated originally from the

flowers of Hibiscus vitifailius. Here we have isolated the same compound gossypin

from flowers of Hibiscus fLlrcalus and the compound was also detected from the

leaves of this plant in traces.

Hibiscus furcatus is an erect or trailing prickly herb. 2-5 ft hight, found as an

under growth in the forests of Assam, Kerala, Andhra and Mysore (Kirthikar and

Basu, 1935). The plant flzwers in September to November. The flowers are large and

yellow with purple centre. The leaves of H. furcatus are acidic and are edible after

cooking. The leaves are said to improve digestion and are considered as anti-

helmintic. The juice of the leaves mixed with honey is applied in eye diseases. An

infusion of the roots in water is used as a refreshing drink in hot weather. A

decoction of the root bark is given as remedy for poisons and swellings and for

cleansing kidneys (Kirthikar and Basu, 1935). This compound has been shown to

exhibit potent anti-nociceptic response when tested by different animal models

(Ramaswamy and Viswanathan, 1997). Plant tissue culture is found as an alternative

technique for the in vitro production of these low volume high value compounds. In

the present investigation along with isolation and characterization of gossypin from

both ex vitro and in aitro plants of Hibiscus furcatus using state of the art, plant tissue

culture. This chapter also deals with chemosensitivity of the isolated as well as

standard gossypin.

5.2. MATERIALS AND METHODS

5.2.1. Plant Material

Plant parts such as flowers and leaves were collected locally and authentically

identified with the help of herbarium specimens. Intact plants were grown in

shaded net house with prophylactic antifungal treatments for tissue culture studies.

5.2.2. Isolation of gossypin (Fig. 5.la)

In TLC analysis standard gossypin (Sigma Chemical Co. St. Luis, USA) were

chromatographed on silicagel G plates (E. Merck) using appropriate solvent system

and the relative front (Rfl was calculated.

Distance traveled by the spot X f =

J

Distance traveled by the solvent

Fig.5.1. Structure of gossypin

5.2.3. Determination of Melting Point

Melting point of the isolated and standard gossypin was determined- (section

2.2.20).

Fig. 5.la. Schematic representation for the isolation of gossypin from plant parts of Hibiscus @rcatus

I Reflex with 85% aq. Et 'OH Distilled off Et.OH

Aquous concentrate

1 Separated with

Waxy residue (discarded)

I- Diethvl Ether

* (discarded)

Ethyl acetate

Column chromatography

I (Butanol: acetic acid (27%) (19)

Preparative TLC (Butanol : acetetic acid : water)

Crystallized in MeOH

Yellow crystals of gossypin I

MP, MS, IR, NMR and HPLC analysis

5.2.4. HPLC analysis and quantification

The purified samples and standard gossypin were subjected to HPLC analysis

as described in materials and methods (section 2.2.23). The quantity of gossypin

present in leaves was calculated by using standard caliberation curves of authentic

sample (Fig.5.2).

5.2.4.1. Standardization of HPLC for the estimation of Gossypin

HPLC Conditions : Altima C185 Micron (4.6 x 250rnm)

Wavelength : 272 nm and 382 nrn

Flow rate : 0.5 ml/min

Volume of injection : 20 ml

Temperature : 25"C*2OC

Sys tern : Isocratic

Run rate : 10 mins.

Mobile phase: 0.1% w/v sodium lauryl sulphate in water : Acetonitrile (85:15)

Solvent : 0.1 % w/v sodium lauryl sulphate in water : Acetonitrile

(85:15)

Standard : Sigma chemical co. St. Luis (Purity 99%)

Retention time : 3.56 min.

Standard preparation

Weigh accurately about 1.lOmg of standard into a l o r d volumetric flask, dissolve

and make up to volume with solvent.

Sample Preparation

Weigh accurately about l.Omg of sample into a lOml volumetric flask,

dissolve and make up to volume with solvent.

Procedure : Filter both standard and sample solutions through 0.45 m membrane

Fig. 5.2.a; b. HPLC chromatogram of standard gossypin and those from Hibisc~~sfurcntus petals

CLASS-M13A V t r . - 1 . 6 1 3 S Y S = l R E P O n T . I , ! 0 = l 9 DATA=GOSC31.K14 0 1 / 1 0 / 3 ! 1 3 : 4 1 : 4 ? S amp 1 e : GOSSYPXN (G-1 ) ( STAtqlDARD)

, Deteccor : SPD-MlOAvp . Method N a m e : GOS .MET

C * * Chromatogram + * *

CLASS-MLOA Ver.=l.60 SYS=l REPORT,NO=ZO DATA=G05031.K20 01/10/31 13:52:25 Sample : GOSSYPIN (G-2 ) SAMPLE Detector : SPD-MlOAvp Method Name : GOS.MET

mAbs

* * * Chromatogram *+ *

4 00-

2 00-

0

- Chl 272nm m u

in

1: I j i ; j

. .--/-\LL-

mAb s

400 -

2 00-

C h l 272nm

0 rD 51

0---

filter and inject.

Peak area of sample x Con, of standard x Purity of std Calculation = = %

Peak area of std. x Con. of sample

Stabilize the column with mobile phase for I h before analysing the samples.

5.2.5. IR Spectra

The functional groups present in CFT were determined by IR spectra. The

spectra were measured using Perkin Elmer PF 2 800 in KBr (Fig.5.3).

5.2.6. Electron Spray Mass spectrometry (ESMS)

The mass of the isolated gossypin was recorded on micro mass quattrol triple

quadrupole mass spectrophotometer. The samples were dissolved in suitable

solvents such as CHCb, MeOH, acetonitrile, H20 were introduced into the ESI

source through a syringe pump at 0.4ml/ h. The ESI source capillary was 3.5 KV and

the con voltage 25-30V. The spectra were collected in 4 Sec (Fig. 5.4).

5.2.7. IH-nuclear magnetic resonance

The number of protons present in the purified samples was recorded by

Bruker DRX-300 MHz FT NMR in CDCb solutions (Fig. 5.5).

5.2.8. Tissue culture studies of Hibiscusfurcahts

The explants used were stem and leaves. After surface sterilization these

explants were incubated in MS and 8 5 medium with different hormonal

combinations (Table. 5.1).

5.2.8.1. Culture environment

Primary explants as well as pieces of calli were cultured in media and

incubated in a culture room maintained at 25 *C. The callus induction started under

Fig. 5.3.a. IR spectrum of standard gossypin

Fig. 5.3.b. IR spectrum of isoIated gossypin

Fig. 5.4.a. Mass spectrum of standard gossypin

Fig. 5.4.b. Mass spectrum of isolated gossypin

Fig.5.5.a. 'HNMR spectrum of standard gossypin

Fig.5.5.b. 'H NMR spectrum of isolated gossypin

- 1.

EEEE E rm ,/.---.- r

Table.5.1. Growth hormones incorporated for inducing synthesis of Gossypin from

Hibiscus furcatus

S .No.

1

2

3

4

Growth regulator

NAA + BA

NAA + KN

2,4-D+BA

2,4-D+KN

5 + + +

Concentration

0.25 + 1 .O 0.5 + 1.0 0.75 + 1 .O 1.0 + 1.0

0.25 + 1 .O 0.5 + 1 .O 0.75 + 1.0 1.0 + 1.0 0.25 + 1.0 0.5 + 1.0 0.75 + 1 .O 1.0 + 1.0' 0.25 + 1.0 0.5 +1 ,o 0.75 + 1 .O 1.0 + 1.0

6

7

Response

- + + - - - - - - - - - - - - +

IAA + KN

I A A NAA 2,4-I)

%gossypin

- ND ND

0.25 + 1 .O 0.5 + 1.0 0.75 + 1 .O 1.0 + 1.0

1.0 + 2.0 1.0 + 2.0 1.0 + 2.0

+ - - + + + -

dark and calli were sub cultured every 4" week into the same medium or other

combinations of media. All cultures were alternatively exposed to 16h photoperiod

at a photon flux density 30 - 50 mE rn -2 S light fluorescent lamp (Philips India

Ltd.)

5.2.8.2. Culture initiation and callus culture establishment

For callus initiation different based media liquid/solid supplemented with

different combinations and concentrations of growth regulators were tested for

culture initiation (Plate. No. 5).

5.2.8.3. Callus growth measurement

Piece of calli (200 mg, fw) were estimated to fresh nutrient media and

incubated for various periods of time. To determine the growth kinetics of the callus

cells, calli grown on different media were collected and fresh weight (fw) and dry

weight (dw) of calli were determined.

5.3. RESULTS AND DISCUSSION

Column chromatography of ethyl acetate fraction yield gossypin yieding

fraction in TLC (BuOH; Acetic acid 1:l v/v). These fractions were pooled and

purified by preparative TLC. TLC analysis of the purified fraction should give

yellow spots with an Rf of 0.60. The solvent system (BuOH: HOAC: H20; 4:1:5) was

used for the isolation of gossypin using preparative TLC. The crystalized gossypin

showed a melting point at 230-232O C. The ethyl acetate fraction contains a gossypin

content of only traces were detected in HPLC. But the intact flowers contain 2%

gossypin on a dry weight basis when compared with standard gossypin (99%

purity). The callus induced in tissue culture has only trace amount of gossypin.

These data strongly proved once again that morphological differentiation is

necessary for secondary metabolite production.

Plate No. 5. A & B- Habitat view of Hibiscusficrcatus

C & D- In vitro callus from Hibiscus furcatus

E & F- Callus regeneration in MS medium (IAA 0.5 mgl +BA lmgl)

Plate No. 5 --

CHAPTER 6