Indian Journal of Experimental Biology Vol. 38, February 2000, pp. 155-159

Non-cellulosic mixed linkage ~-D-glucan in sorghum, Sorghum bicolor (L.) Moench-Localization and biological activity studies

H P Ramesh & R N Tharanathan*

Department of Biochemistry and Nutrition, Central Food Technological Research Institute. Mysore 570 013. India

Received 29 December 1998; revised 4 October /999

Fluorescence of Calcofluor and Congo Red was observed in stained sections of sorghum grai n (SorX/1I(1II hic%r (L.)

Moench), especially in the sub-aleurone cells indicating the presence of mixed linkage P-D-glucan. Relatively less Iluores­cence intensity was observed in the single layered (-20 f.lm thick) aleurone. Alkali extracted P-D-glucan (fraction 2) of sor­ghum showed 30% activation of rat peritoneal macro phages (ill vitro) at 100 f.lg mr' concentration in 10 min . Thi s activa­tion was found mediated mainly through PLA2 pathway. A phagocytic index k of 0.1 02±O.008 was observed ill vivo carhon clearance test in mice in the group treated with fraction 2. Accumulation of colloidal carbon particles in sp leen and li ver of mice was moderate in this group, compared to control.

(l ~311 ~4)-~-D-glucans, hereafter called MLG (mixed linkage ~-D-glucan) are one of the minor components of cell wall polysaccharides, especially in the seeds of monocotyledons'. These ~-D-glucans influence various functions, viz. water holding ca­pacity, porosity and plasticity of the plant tissues2

.

Through fluorescent microscopy, which is an impor­tant technique both for morphological and/or histo­chemial analysis of cereals/grains, and using Congo Red and Calcofluor, MLG was found to be localized in aleurone, sub-aleurone and endosperm cell walls of oats and barley" .

A majority of the (I ~3)- linked ~-D-glucans found as constituents of fungi, algae and higher plants4 are excellent biological response modifiers with signifi­cant immunomodulatory activities5

.6

. They exhibit antibacterial, antiviral and also antitumour activi­ties7

-1O

• MLGs of cereals, on the other hand have also been found to possess beneficial implications in nu­trition and health of the consumer, for example, cho­lesterol lowering properties3., 1.12. lowering of blood glucose (in hyperglycemic conditions), modulation of hormonal responses and colon cancer, and also in the bio-availability of micronutrients' 3. Some induction of lysosomal hydrolase synthesis and secretion by barley glucan have also been observed in mouse peritoneal macrophages'4. The present communica­tion deals with the (a) localization of MLG in sor­ghum through their fluorescence intensity with re-

*Correspondent author.

spect to Congo Red and Calcofluor dyes , and (b) stud~ of its biological activity.

Materials and Methods Sorghum {Sorghum hicolor (L.) Moench}. variety

SB 905) was procured from Agricultural station, Dharwad. Barley glucan, Sephacry l S-400, DEAE­cellulose, amyloglucosidase (EC. No.~.2.1 . ~, 10 units mg" solid), phorbolmyristate acetate (PMA) , capsai­cin, Calcofluor, Congo Red , nitroblue tetrazolium (NBT) and zymosan were obtained from Sigma, USA, dexamethasone was from Cadila Lab, India and adrenaline was from Harson Labs, India.

Preparation offraction 2 (M LC (i sorghul1I )-Sor­ghum flour (100 mesh, 100g) was gelatinized and subjected to amylolysis using amyloglucosidase and the residue obtained after centrifugation was ex­tracted with boiling water and centrifuged' ). The residue was further extracted with IN NaOH and centrifuged. The supernatant was adjusted to pH 4.5 and centrifuged. Hemicellulose B was obtained from the supernatant by adding 3 vol. of ethanol and it was further fractionated on DEAE-cellulose (CO/ ) col­umn. The fraction obtained by water elution was further purified on Sephacryl S-400 to get a minor fraction I and a major glucan (MLG) fraction 2'6.

Fluorescent histochemical localizatioll (~r MLC­Grains were kept in ice-cold water (-2 hr) and thin sections (8-10 ~m) were taken in a freezing micro­tome (American Opticals, USA) . They were stained with Fluorescent Brightner 28 (Clacofluor, 0 .0 I % in

. 156 INDIAN J EXP BIOL, FEBRUARY 2000

water or 50% ethanol) or with Congo Red (50% etha­nol). After mounting in fluorescent-free immersion oil, observations were made in a Leitz-Laborlux S fluorescent microscope using different filter combi­nations (excitation BP-340-380 and 546114, absorp­tion LP-430 and LP-580 for Calcofluor and Congo Red, respectively). Thickness of aleurone was meas­ured using an ocular micrometer (Erma, Japan).

Biological activity studies-Macrophage activating property of MLG (fraction 2) was studied by in vivo

. carbon clearance test)7 in male mice (Mus musculus, OUT B, Swiss Albino, INO-cft). Animals weighing 27-30 g were grouped statistically into 3 numb~rs, each containing 8 animals. One group was injecte (IP) with fraction 2 (50 /l-g g-) body weight) in saline once a day for 5 days. Second group was injected with zymosan (same dosage, as above), and the third group was kept as control which received only saline. All the animals were fed with standard pellet diet and water ad libitum. At 24 hr, after day 51h

, the animals were injected through tail vein diluted colloidal car­bon containing 1 % gelatin in phosphate buffer saline.

Blood samples were obtained from the orbital plexus at 0, 3, 6 and 9 min, and its absorbance in 0.1 % sodium carbonate was recorded at 660 nm. Phagocytic index k was calculated using the equation.

k = In 00) -In 002

t2 - t)

where 00) and 002 are the optical densities at times t1 and t2 respectively. Results were expressed as the arithmetic mean ± SO of eight mice. Histopathologi­cal studies of liver and spleen of control and experi­mental animals were also carried oue s. In vitro stud­ies on isolated peritoneal macrophages from male

pc

a[

en

albino rats (Rattus norvef?icLls, OUT B. Wistar Al­bino, INO-cft) were carried out by the NBT reduction assay)9. Macrophages (106

) in 0 .5 ml of Hank ' s bal­ance salt solution (HBSS) containing 0.4% BSA were pre-incubated at 37°C for 5 min . The reacti on was initiated by adding fraction 2 (I 00-300 ~lg ml -I

) or PMA (100 nM) . After 10 min at 37°C, 0 .04 ml of NBT was added. After 15 min , the reaction was stopped by adding 2.6 ml of ice-cold HBSS and cen­trifuged. To the pellet 3.0 ml dioxan was added and kept in boiling water bath (10 min, to extract reduced NBT) . After cooling it was centrifuged and aD of the clear supernatant was read at 540 nm. Various inhibitors, viz. capsaicin, adrenaline and dexameth­asone, of activation pathways were included to under­stand the mechanism of action 20

.

Results and Discussion Figure I shows a diagrammatic representation of

various tissues identified along the transverse section of sorghum grain. It may be seen that these morpho­logical features are similar to those reported before"). The peri carp is 2-3 layer thick and consists of rectan­gular cells containing pigmentary materials , whereas the endocarp is composed of cross and tube cells . Tube cells conduct water during grain germination while cross cells hinder moisture loss by forming a seal in tube cells21

. Aleurone is the outer cover over endosperm and consists of a single I ,-~yer of rectangu­lar cells. Fig. 2a represents sorghum histology.

Figures 2b and 2c show white fluorescence of Cal­cofluor and Fig. 2d shows Congo Red fluorescence . These indicate the appearance of MLG in aleurone, sub-aleurone and inner endospermic cell walls of sor­ghum grains . Calcofluor fluorescence is quite sig-

ep

me tc cc

at Sell

e n

st

Fig. I-Diagrammatic illustration of various tissues in the transvers section of sorghum grain. pc: pericarp: al: alcuronc: cnd: endosperm: ep: epicarp, me: mesocarp; tc: tube cells, cc: cross cells; sal : subaIeurone and sl: starch .

RAMESH & THARANATHAN: p-D-GLUCAN IN SORGHUM 157

nificant over that of Congo Red. It is clear that fluo­rescence is not uniform through different regions of the grain indicating variations in the concentration of MLG (Table 1). Sub-aleurone (outer endospermic) cell walls showed intense fluorescence of Calcofluor dye (Fig. 2b), whereas it was less in aleurone and in­ner endospermic cell walls. Similar observations have also been made in oat and barley grains23

,24 .

Overall, the fluorescence intensity observed was high in barely (results not shown) as compared to that in sorghum, mainly because of wide variations in the content of MLG. Barley contains -3.0-10.6% of MLG, whereas in sorghum it is 0.12%. Significant histochemical difference between the two grains with respect to aleurone lies in its thickness . In sorghum it is single layered with one cell thickness of - 20 flm as compared to that of barley where it is 2-3 layers with a total thickness of -50 flm.

The in vitro macrophage activation by fraction 2 is shown in Fig. 3. The percent activation, expressed with respect to PMA (100%) by fraction 2 was 30% at a concentration of 100 flgmrl and in control it was only - 9%. PMA is a known activator and its stimu­lation is calcium independent and is blocked by ki­nase inhibitors25

. Figure 4 shows the dose dependent activation of macrophages by fraction 2. Up to 100 flg

Table I-Fluorescence intensity of calcotluor/Congo Red in dif­ferent regions of sorghum grain

Stain Aleurone Sub-aleurone Inner cndosper-mic cell walls

Calcotluor + ++++ + Congo Red + ++ +

mrl concentration a steep increase was noticed in the activation, which gradually decreased as concentra­tion increased. In Fig. 5 'is shown a time dependent (-10 min) increase in NADPH oxidase activity . After 10 min, a decline in the activation was evident. In order to understand the mechanism involved in this activation, specific pathway mediating the respiratory burst was established by using specific inhibitors ~()~27. From Fig. 6, it is evident that capsaicin had a veI:Y little effect on fraction 2 induced respiratory burst , the inhibition was only 8%, which ruled out the in­volvement of Ca2

+ calmodulin pathway in this actiYa­tion. 65% inhibition by adrenaline indicated partial involvement of protein kinase A. However. dexa­methasone was very effective in inhibiting the macrophage activation to the extent of 80%. It is clear that fraction 2 triggered respiratory burst is mainly mediated by PLA2 pathway. It is based on the fact that dexamethasone inhibits both m-RNA synthesis and post-translational expression of group II PLA}. Further PLA2 involvement in the respiratory burst is weB known in several other systems~x .

100 .-

";J!. 80 c

60 .!:! -0 > 1,0 -v « 20

0 n A B c

Activators

Fig. 3-111 vitro macrophage acti vation: A. PM A: B. fraction 2 and C, control.

Fig. 2-Histology (a) and Iluorescence histochemical localization of MLG in sorghum; al and a2-Cnlcotluor staining: aJ-Congo Red staining, x 300

158 INDIAN J EXP BIOL, FEBRUARY 2000

In the carbon clearance test (CCT), phagocytic index k (Fig. 7) observed in the group treated with fraction 2 was fairly increased (k=0. 102± 0.008) as compared to that of control group (k=0.069 ± 0.006). Animals treated with zymosan, a known activator form yeast showed as expected, a high phagocytic index k 0.192 ± 0.001. Zymosan is a potential acti­vator of component29

.

These observations complimented the histological data on liver and spleen of treated animals, which

100

~ 80

c: 60 .9 0 1.0 >

.oJ U <{ 20

0 0 10 100 200 300 400 500

Concenlra lion '( tJ giml).

Fig. 4-A dose dependent response of fraction 2 on macrophage activation.

~ c 0

.oJ

0 > -u <{

20

a 1 5 10 15 20 25 30

Time(min}

Fig. 5-Time dependency of macrophage activation by fraction 2

showed selective and differential accumulation of carbon particles in the kupffer cells and in the macro­phages of red pulp, respectively. As shown in Figs 8b and 9b, severe accumulation of carbon particles was observed in liver and spleen of zymosan treated ani­mals indicating maximum clearance of carbon parti­cles injected into the blood. In the group treated with fraction 2, only a moderate accumulation of carbon particles was observed in li ver (Fig. 8c ) and (Fig.

100 .--r--

~ o . 80 c 0 60 0 > 40 U

0-

<{ 20

0 n A 8 c o

Inhibit ors

Fig. 6-Effect of inhibitors on macrophage acti valion. A. fraclion 2; B, fraction 2 + adrenaline; C. Fraction 2 + capsaicin and d, fraction 2 + dexamethasone.

J::

x 0.20 QJ

'0 C +

0 .15 u

'" 0 .10 u 0 01 rt 0

J::. 0. 05 0..

A 8 c Fig. 7-Phagocytic index k in different groups. A, control: B. zymosan and C, fraction 2.

Fig. 8-HistoIogy of liver showing varied response of phagocytosis in CCT in mice. a, without activation ; b. zYlllosan activation and

e, fraction 2 activation, x 250.

RAMESH & THARANATHAN: ~-D-GLUCAN IN SORGHUM 159

Fig. 9-Histology of spleen showing varied response of phagocytosis in CCT in mice. a. without activation; h. zymosan activation and Co

fraction 2 activation, x 250.

9c). On the other hand, in animals subjected to CCT without any prior treatment (Figs 8a and 9a), a mini­mum (base line) activation was still observed. Similar observations were also made in the in vitro studies (Figs 4 and 5). From all these studies, it was noted that fraction 2 of sorghum has a mild dose and time dependent macrophage activating property.

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