J. Saudi Chem. Soc., Vol. 10, No. 3; pp.

501-508 (2006) 501

ISOLATION AND CHARACTERIZATION OF KAPPA CARRAGEENAN FROM HYPNEA

MUSCIFORMIS (RED ALGAE) OF KARACHI COAST

Fatima Bi, Muhammad Arman, Mahmood-ul-Hussan and Seema Iqbal

PCSIR Laboratories Complex Karachi, Pakistan
(14th Nov. 2005; Accepted 20 th July 2006)

‫ ﺍﻟﱵ ﲨﻌﺖ ﻣﻦ ﺳﺎﺣﻞ ﻛﺮﺍﺗﺸﻲ ﰲ ﺑﺎﻛﺴﺘﺎﻥ ﻟﻔﺼﻞ ﻣﺮﻛﺐ ﻛﺎﺭﺍﺟﻴﻨﺎﻥ ﺍﻟﺬﻱ ﻳﺴﺘﺨﺪﻡ‬Hypnea musciformis ‫ﺍﺳﺘﺨﺪﻣﺖ‬
‫ ﻭﻗﺪ ﺍﺳﺘﺨﺪﻣﺖ ﺧﻄﻮﺍﺕ ﻓﺼﻞ ﻣﺘﻌﺪﺩﺓ ﻭﻛﺎﻧﺖ ﻧﺴﺒﺔ ﺍﻟﻜﺎﺭﺍﺟﻴﻨﺎﻥ ﺍﻟﱵ ﰎ ﺍﳊﺼﻮﻝ ﻋﻠﻴﻬﺎ ﺗﺘﺮﺍﻭﺡ‬.‫ﻛﻌﺎﻣﻞ ﺗﻐﻠﻴﻂ ﻭﺍﺳﺘﺤﻼﺏ‬
-١٩,٩ ‫ﺎﻳﺪﺭﻭﺟﺎﻻﻛﺘﻮﺯ ﰲ ﺍﻟﺴﻜﺮ ﺍﻟﻜﻠﻲ ﰲ ﻣﺪﻯ‬‫ﺃ‬-٦،٣ ‫ ﻭﻧﺴﺒﺔ‬،%٥٥-٣١,٨ ‫ ﻭﻧﺴﺒﺔ ﺍﻟﺴﻜﺮ ﺍﻟﻜﻠﻴﺔ‬.%٤٤-٣٤ ‫ﺑﲔ‬
‫ ﻭﻳﺪﻝ ﺍﻟﺪﻭﺭﺍﻥ‬.‫ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ‬%٥٣-١٥,٤ ‫ ﻭ‬%٤١-١٤,٨ ‫ ﺃﻣﺎ ﻧﺴﺒﺔ ﺍﻟﻜﱪﻳﺘﺎﺕ ﻭﺍﻟﺮﻣﺎﺩ ﻓﻘﺪ ﻛﺎﻧﺖ ﻣﺮﺗﻔﻌﺔ‬.%٢٧,٦
‫ ﻭﺃﻇﻬﺮﺕ ﺩﺭﺍﺳﺎﺕ ﺃﻃﻴﺎﻑ ﺍﻷﺷﻌﺔ ﲢﺖ‬.‫ ﰲ ﺗﺮﻛﻴﺒﻬﺎ‬α-D glycosidic ‫ﺍﳌﻮﺟﺐ ﻟﻌﺪﻳﺪﺍﺕ ﺍﻟﺘﺴﻜﺮ ﻫﺬﻩ ﻋﻠﻰ ﺳﻴﻄﺮﺓ ﺍﺭﺗﺒﺎﻁ‬
‫ ﻭﱂ ﻳﺘﻢ ﺍﻟﻌﺜﻮﺭ‬،‫ﺍﳊﻤﺮﺍﺀ ﻭﺟﻮﺩ ﻛﺎﺑﺎ ﻛﺎﺭﺍﺟﻴﻨﺎﻥ ﻛﻤﺮﻛﺐ ﺃﺳﺎﺱ ﻣﻊ ﻭﺟﻮﺩ ﻧﺴﺒﺔ ﺷﻮﺍﺋﺐ ﺻﻐﲑﺓ ﺟﺪﺍﹰ ﻣﻦ ﻧﻮﻉ ﺃﻳﻮﺗﺎ ﻛﺎﺭﺍﺟﻴﻨﺎﻥ‬
‫ ﻭﺃﻇﻬﺮﺕ ﻋﺪﻱـﺩﺍﺕ ﺍﻟﺘﺴﻜﺮ ﺍﻟﱵ ﰎ ﺍﳊﺼﻮﻝ ﻋﻠﻴﻬﺎ ﻓﻌﺎﱄـﺓ ﺍﺳﺘﺨﻼﺹ ﻣﻮﺟﺐـﺓ ﰲ ﺑﺎﺯﻻﺀ ﺍﳊﺪﺍﺋﻖ‬.‫ﻋﻠﻰ ﻧﻮﻉ ﻻﻡـﺩﺍ‬
.‫ ﺣﺰﻣﺔ ﺭﺋﻴﺴﺔ ﻭﺍﺣﺪﺓ‬HPLC ‫ ﻭﺃﻋﻄﺖ ﲢﺎﻟﻴﻞ‬.(Pisum sativum)
Hypnea musciformis collected from Karachi coast of Pakistan was used for isolation of a thickening and
emulsifying agent “Carrageenan”. Various extraction procedures (extraction 1-6) were employed and the
yield of carrageenan obtained was in the range 34-44%. Total sugar was found 31.8-55.4%, 3,6-
anhydrogalactose of total sugar was in the range of 19.9-27.6%. Sulphate and ash content were high 14.8-
41% and 15.4-53% respectively. The positive rotation of these polysaccharides indicated a predominance
of α-D glycosidic linkages in their structure. IR spectral studies showed kappa carrageenan as major
phycolloid with a very small contaminant of iota type carrageenan, λ type was not detected.
Polysaccharide obtained showed a positive elicitor activity in Garden Peas (Pisum sativum). HPLC
analysis provided a single major component.

INTRODUCTION spectroscopic techniques (Greer et al 1984) [3].

Commercially important polysaccharides
from red seaweeds (Rhodophyta) belong to a
family of polydisperse, long chain, water-soluble
galactans. They are built up of alternating 3-linked
β-galactopyranose and 4-linked α-galactopyranose
which can be variably modified and/or substituted.
In carrageenans, the 4-linked units are in the D-
configuration, whereas in agars they are in the L-
configuration (Usov 1992) [1]. The gelling and
thickening properties and protein reactivity of
these phycolloids have led to their wide spread
commercial use in industry i.e. food and
beverages, pharmaceuticals and cosmetics
(Nishizawa 2002) [2]. These are also used as
biofertilizer in agriculture sector. Because of the
wide commercial applications of carrageenans,
various extraction procedures are given in
literature to obtain this product from red algal
plants and their structures are determined by using
Karachi has a large coastal area and produces a
huge amount of marine algae, unfortunately the
seaweeds are not utilized in Pakistan either as
seavegetables or for extracting commercial
compounds (Husain et al 2001) [4]. A lot of
money is spent to import seaweed products. The
aim of the present study is the exploitation of
seaweeds and to develop an effective extraction
procedure that gives good quality and quantity of
carrageenans from Hypnea musciformis. Another
objective is to explore these polysaccharides as
inducers of hypersensitive response characteristic
to resistance mechanism of plants against diseases
especially in terms of induced browning and
production of phytoalexins (Nicklson and Wood
2001) [5]. Garden peas (Pisum sativum L.) were
used as the test plant in the elicitor activity
experiments.
502 Fatima Bi, Muhammad Arman, Mahmood-ul-Hussan and Seema Iqbal
Material and Method:
Hot water extraction:
Hypnea musciformis (red algae) was
collected from Karachi coast in February and
November 2003. The plant was cleaned from
epiphytes, washed, dried and ground to a fine
powder. 25 g plant material was pretreated with
HCl (0.1N)/formaldehyde (20%) and extracted
with water (tap/distilled) at 70-80 oC with constant
stirring for 6 hours. Supernatant was collected,
residue was re- extracted twice under similar
conditions. Experimental conditions varied for
optimizing the extraction procedures and carra-
geenan were obtained as follows by ethanol
precipitation or by direct drying on water bath.
Extraction 1:
Dry plant treated with HCl (0.1N), stirred for
half hour in an icebath, washed extensively with
water to remove traces of acid. Then extracted
with distil water. The supernatant were direct
dried on water bath.
Extraction 2:
Dry plant treated with HCl (0.1N) , washed
and extracted with distil water. Supernatant
precipitated with 95% ethanol (three volumes of
extract) and precipitate were dried.
Extraction 3:
Dry plant material pretreated with HCl
(0.1N), washed and extracted with tap water and
direct dried.
Extraction 4:
Dry plant pretreated with HCl (0.1N),
washed and extracted with tap water, precipitated
with alcohol and precipitates were dried.
Extraction 5:
Dry plant material pretreated with HCl
(0.1N), washed and extracted with distil water.
Supernatant was precipitated with 0.3 M KCL
(solid) and precipitates were dried.
J. Saudi Chem. Soc., Vol. 10, No. 3
Extraction 6:
Dry plant mixed with formaldehyde (20%),
left overnight, washed and extracted with distil
water. Supernatant was direct dried.
Commercial Carrageenan:
The commercial carrageenan was purchased
from local market.
Analytical methods:
Moisture, ash and total carbohydrate
contents were determined. In these experiments
acid hydrolysis was done with time i.e. 0.5, 1.5
and 3 hours and monosaccharide components
were identified by paper chromatography. Details
are given as before (Fatima and Seema 1999) [6].
3,6-anhydro galactose was determined colorimet-
rically using the modified resorcinol method
(Yaphe and Arsenault 1965) [7]. Sulphate content
was determined by modified method (Dodgson
and Price 1962) [8]. Optical rotations of
carrageenan solutions of known concentration
(1% of extracts 1,2,3,4, commercial carrageenan
and 0.1% of extract-5 and 6) were determined
with Digital polarimeter (Jas.Co. Dip. 360) using
50 mm tubes and sodium D line at 589 cm
wavelength. FTIR analysis were performed on a
Nicolet Avatar 370 DTGS Infra red fourier
transform spectrometer.
Elicitor activity and extraction of Induced
Secondary Metabolites (ISM) from Garden
Peas:
A general method of elicitor application was
employed (Whitehead et al 1982) [9]. 100 gram
fresh garden peas (Pisum sativum) purchased from
local market, pealed and cotyledons were sterlized
with 1% sodium hypochlorite solution, washed
extensively with distil water and finally by sterile
water. 20 μL elicitor preparation of extract-5 at a
concentration of 100 µg glu eq/mL and sterile
water were applied to the cut surfaces of
cotyledons for treated and control samples.
Browning induced in samples was recorded after
24 hour incubation and samples were dipped into
 Isolation and Characterization of Kappa Carrageenan from Hypnea Musciformis (Red Algae) of …. 503

distilled 95% alcohol for extraction of induced RESULT AND DISCUSSION

secondary metabolites.
Separation of ISM by HPLC:
Hplc separation was accomplished on 4.6
mm x 25 cm reverse phase C18 column, using
branded instrument of Perkin Elmer with variable
uv detector (254 nm) and isocratic solvent system.
A guard column of pellicular C18 hydrocarbon
chemically bonded to glass beads was placed
before analytical column. Initially 70:30 aceto-
nitrile: water having 0.5% acetic acid was run for
10 minutes and then acetonitrile (100%) was run
for further 25 minutes.
Sample preparation for HPLC:
Dry alcoholic extract of treated and control
sample were dissolved in 1 mL of initial solvent
(70:30 acetonitrile: H 2O). 100 µL of this solution
was further diluted with 2 mL of the same solvent
and filtered with 0.45 µm filters and clear solution
of 20 µL was applied on column.
In this study various extraction procedures
were applied to isolate and characterize
carrageenan from H. musciformis (red algae). The
average total recovery of galactans from hot
water extractions was about 39% of the dry algae
(Table 1) and similar in range as described earlier
(Knutsen et al 1995) [10]. Generally yields were
high in the extracts pretreated with mild acid (38-
44%). Moisture and ash contents of the products
were in the range of 3.1-7.7% and 15.4-18.4%
respectively whereas extract-5 had very low
moisture (0.32%) and highest ash content i.e.
53%. Total sugar contents were in the range of
31.8-55.4%. High sugar content in aqueous hot
extract of H.musciformis was also reported in our
previous communication (Fatima and Seema
1999) [6]. It is documented that plants belong to
Rhodophyceae (red algae) are commonly
sulphated galactan (Miller and Blunt 2002) [11].
High sulphate contents (23.6-41.0%, except
extract-5) and 3,6-anhydro galactose (20-27%),
which were derived from total sugar confirm the
findings for these contents by Chiovitti et al [12].

Table 1: Yield & Chemical Composition of Algal Extracts of H. musciformis (% w/w)

Sugar Content
Extracts Yield Moisture Ash 3,6-anhydro Sulphate
Total
Sugar galactosea

1 40.0 7.7 15.7 45.3 20.9 31.1

2 38.0 4.6 18.4 50.8 21.8 25.4
3 44.2 6.9 16.3 41.1 23.1 35.4
4 39.1 5.1 18.1 46.5 27.6 30.1
5 40.0 0.32 53.0 31.8 25.9 14.8
6 34.2 5.4 15.4 55.4 22.3 23.6
commercial ndb 3.1 15.7 40.1 19.9 41.0
carrageenan

a: 3,6-anhydro galactose is derived from total sugar

b: not determined

J. Saudi Chem. Soc., Vol. 10, No. 3

504 Fatima Bi, Muhammad Arman, Mahmood-ul-Hussan and Seema Iqbal
Results for the various characteristic
properties of these phycolloids are summarized in
Table 2. After extraction and drying of samples,
no odour was found in any extract. Most of the
extracts were brown in colour. The alkali
treatment of extract-5 gave sample a creamish
white colour, close to the colour of commercial
carrageenan. Except extract 4 and commercial
carrageenan all other samples were soluble in
water (2%) at 60-70 oC with in half hour, a very
small amount of insoluble material was left in
some samples. Precipitates formed with
methylene blue and positive test with milk are the
characteristic tests of carrageenans. pH of aqueous
solution (1%) of the extracts 1-6 showed acidic
nature (pH = 4-5) whereas commercial
carrageenan had slightly alkaline nature (pH =
7.7). The gelling strengths of aqueous solution
(2%) were recorded at room temperature and also
at 4oC. Some of the extracts i.e. 1,3,4 were non-
gelling but extract 2 and 6 formed thick and
viscous gels at 4oC. Literature survey revealed
that the presence of 3,6- anhydro sugar causes
gelling but if this is replaced by the 6-sulphate the
gelling power is considerably lessened and the 2,6
disulphate in place of 3,6 anhydro sugar results in
the complete loss of gelling power (Percival and
Mcdowell 1990) [13]. This suggest that gelling
property not only depend on anhydro sugar but
also dependent on sulphate content of the
phycolloids. It is also possible that acid treatment
has hydrolyzed the linkages at 3,6-anhydro-
galactose and made the samples totally non-
gelling whereas extract-5 with low sulphate
(14.8%) showed highest gelling strength and
formed thick and stable gel at room temperature.
Some non-gelling polysaccharides obtained from
marine algal plants were also reported by Parekh
et al 1989 [14]. It was surprising that commercial
carrageenan only formed viscous solution at low
temperature i.e 4oC, may be due to its high
sulphate content (41%). In market the carrageenan
are found as blended material for different
purposes rather a pure product (Thomas 1997)
[15]. The positive rotation of these poly-
saccharides indicates a predominance of α-D-
glycosidic linkages in their structure.
J. Saudi Chem. Soc., Vol. 10, No. 3
Monosaccharide composition determined by
acid hydrolysis and paper chromatography
showed galactose as the major sugar component
of each extract and released as early as 0.5 hour of
hydrolysis, regular increases were observed and
maximized at 3 hour of hydrolysis. Xylose/
arabinose and glucose along with minor amount of
fucoses were also visible in the chromatogram.
The intensity of spots were high in the extracts
pretreated with mild acid, this may be due to some
addition of glucose from starch as contaminant.
Infra red technique has been commonly used
in the characterization of carrageenans (Falshaw
et al 1996 [16]; Manuel et al 2002 [17]). The IR
spectra of the crude extracts 1-6 along with
commercial carrageenan were recorded (Fig. 1).
All the spectra displayed a broad absorption band
at 1210-1220 cm-1 corresponding to sulphate ester
and is common to all sulphated polysaccharides
and increases in size with the sulphate content. In
commercial carrageenan spectra this band is fairly
sharp and strong and is in accordance with the
high sulphate content of the sample (Table 1). The
diagnostic region (940 cm-1-800 cm-1) of the IR
spectra of polysaccharides resembled that of
kappa carrageenans elaborated by red algal plant
i.e. H.musciformis (Knutsen et al 1995) [10]. The
characteristic bands at 930 cm-1 and 840 cm-1
represent the anhydrogalactose and galactose- 4-
sulphate respectively. The lack of absorbencies at
820 and 830 cm-1 in all spectra suggests that these
samples are not galactans of only one common
type. However extracts-4, 5 and commercial
carrageenan had a small but prominent band at
805 cm-1 showing iota carrageenan type structure
present in these phycolloids (Chiovitti et al 1996)
[12] while other spectra displayed complete
elimination of this absorption band. Greer et al
1984 [3] found that polysaccharides obtained from
H.musciformis comprised of 73% of kappa
carrageenan and 17% of iota carrageenan.
Analysis of carrageenan from different algal
sources has revealed the hybrid nature of these
polymers (Bellion et al 1982) [18]. A careful
examination of all the spectra lead to a conclusion
that carrageenans derived from H.musciformis had
features of both kappa and very small portion of
iota carrageenan.
Table 2: Characteristics of Phycolloids of H. musciformis

Tests Ext.1 Ext.2 Ext.3 Ext.4 Ext.5 Ext.6 Commercial carrageenan

Colour Reddish Light Dark Brown Light Creamish Brown Pinkish White powder
Brown Brown Brown White

Solubility in water Dissolved at Dissolved at Dissolved at Dissolved at Dissolved at Dissolved at Dissolved at room
(2% w/v) 60-70oC 60-70oC 60-70oC room 60-70oC 60-70oC temperature
temperature

Methylene blue test ppt formed ppt formed ppt formed ppt formed ppt formed ppt formed ppt formed

Milk reactivity Positive Positive Positive Positive Positive Positive Positive

pH of aqueous 4.4 4.2 4.7 4.8 5.2 4.8 7.7

solution (1% w/v)

Aqueous gel strength Non gelling Gel formed Non gelling Non gelling Gel formed Gel formed Viscous solution
(2% w/v) at 4oC at room at 4oC at 4oC
temperature
+ 53.8 o
Optical rotation [α]D25 + 58.2o + 48.1 o
+ 58.0 o
+ 52.0 o
+ 30.0 o
+ 64.0 o
506 Fatima Bi, Muhammad Arman, Mahmood-ul-Hussan and Seema Iqbal

Fig. 1: FTIR Spectra of polysaccharide preparations from H.

musciformis (Ext. 1-6) and commercial carrageenan of
market (7).

Papers have been published showing
seaweed polysaccharides as an elicitor of plant
defence mechanism in treated tissues of chickpea
in terms of induced browning and phytoalexin
production (Fatima and Seema 2003) [19]. On the
basis of chemical composition and IR spectral
studies extract-5 was found the close
representative of kappa carrageenan and was
investigated for its elicitor activity in treated
tissues of garden peas (Pisum sativum). In this
study peas tissues were inoculated with 100 µg
glu eq/mL preparation of elicitor (ext.5). After 24
hours of incubation high intensity of browning
was produced in the treated sample as compared
to the control. Typical chromatogram in Fig. 2
showed the resolution of alcoholic extract of
elicited tissues of peas by HPLC analysis. The
prominent and sharp peak – A represents induced
secondary metabolites eluted in the organic phase
(100% Acetonitrile). It is reported that the
phytoalexin 6a-hydroxy pterocarpan and phenolic
contents as well as enzyme activity in peas was
increased on fungal infection and elicitor
treatment (Katoch et al 2002 [20]; Banks and
Dewick 1982 [21]). Quantitation and charac-
terization of phytoalexins induced in this system is
going on.
As a result of these studies it is
recommended that seaweed of Karachi coast can
be utilised for the production of commercially
viable product “Carrageenan” which has immense
use in various industrial sectors.

J. Saudi Chem. Soc., Vol. 10, No. 3

 Isolation and Characterization of Kappa Carrageenan from Hypnea Musciformis (Red Algae) of …. 507
Fig. 2: HPLC separation of induced secondary metabolites (Peak-A) in pea cotyledons treated with elicitor
preparations (Polysaccharide) of H. musciformis
Acknowledgement:
The authors are thankful to Mr. Muhammad
Sadiq Ali, Junior Technical Officer and Mr.
Muhammad Javaid, Senior Technician for their
assistance in Laboratory work.
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