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NutritionResearch,W1.18, No. 5. pp. 841-850.

1998
Copyright0 1998ElsevierScienceInc.
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ELSEVIER PIISO271-5317(98)00069-4

EFFECT OF GARLIC AGGLUTININ AND GABLIC EXTRACTS ON THE RAT


JEJUNDM

Anita Gupta*,PhD and Rajinder S. Sandhu,PhD.

Department of Molecular Biology and BiOChuPiStry,


Guru Nanak Dev University, Amritsar-143 005, INDIA.

In an earlier study it was reported that intragastric


intubation of a mannan specific lectin,
edible garlic (Allium sativum) was bound to ASA&U brush
from
border membranes and epithelial cells of the intestine and
arrested the growth of Swiss albino rats. The aims of
present study were to determine the systemic effects of
on jejunal enzymes and nucleic acids and to
58 the morphological changes in jejunum. Swiss male
albino rats were fed either purified
native/heat denatured crude garlic extZ&O EG
intragastric intubation for 7 days- Although, ;;;Ainic”,;t
heat treated garlic extracts had no
effect on nucleic acids, crude extracts in native form
showed increased levels of nucleic acids in jejunum.
While activities of disaccharidases and acid phosphatase
(ACP) were higher, alkaline phosphatase(ALP), lactate
dehydrogenase (LDH) and adenosine triphosphatase (total and
ouabain sensitive ATPase) were lower in the jejunum of
ASA fed rats. On the other hand,native and heat treated
gar118 extracts significantly enhanced disaccharidases along
with LDH, ACP and ALP, whereas the loss of ouabain sensitive
ATPase was not significantly different from that of
treated rats. However, a significant rise in the p::&!
content was seen in all the rats irrespective of
treatment. Histological examination of jejunum of treated
rats revealed alterations of brush border membrane,
thinning and sloughing off of villus structure and
vacuolisation. It is concluded that garlic lectin
(ASA ) influences biological functions by altering Na/K
ATPa&&' in rat intestine. However, differential effects of
and garlic extracts on nucleic acids and ALP after
;:&! c intubation indicated that other components of garlic
besides ASAaiE9. need to be examined for the assessment of
biological ivity of brush border membrane of intestinal
tract.
0 1998 Eketier Scimcclnc.

Key Words : Garlic effects, Garlic lectin, Jejunal nucleic


acids, Jejunal enzymes, Na/K ATPase
____________________~~~~~~~~-~~~~~~

* Corresponding Address: Department of Biophysics,


Postgraduate Institute of Medical Education and
Research,Chandigarh-160012, INDIA.
841
a42 A. GUPTA and R.S. SANDHU

Foods of plant origin contain specific carbohydrate-


binding proteins called lectins (1) and other antinutritional
factors. These lectins when consumed in raw form, both in food
and feed, may have serious deleterious effects (2). However,
cooking provides safeguard against such lectins. Garlic (Allium
sativum) is by and large eaten in cooked form but its consumption
in raw form under certain conditions is not ruled out
particularly for medicinal purposes. Lectins from Allium sativum,
ASA and ASA are mannan specific agglutinins (3-5) and can
be &&.ly separ@ed on asialofetuin-linked amino activated silica
affinity column. While ASA is a 110 kDa glycoprotein with two
identical subunits of J&t 50 kDa,
heterodimeric lectin comprising two clos~$5si~~la~ ::bunEi
of 12.5 and 13.0 kDa. ASA can induce mitogenesis in human
guinea pig lymphocytes It&!) and binds carbohydrate receptors
localised on various human carcinomas (7).
extracts when fed orally to animals, arrested
treated rats(8). While passing through the alimentary tract,
could withstand enzymatic degradation in functionally and
?~@&hemically intact form a part of which bound to the brush
border membrane as well as 60 the lower half of the villi (8).
These findings could lead to potentially damaging reactions in
the gut with powerful systemic effects. Keeping this in view, the
of the present study was to determine the effects of
iimmucosal enzymes and nucleic acids of the intestine and cola@
them with the effects of native/heat treated garlic extracts &I
y&yg.

XATERIALS AND HEFHODS

Preparation of Lectin

Purification of lectin from bulbs of Allium sativum was achieved


on asialofetuin-linked amino activated silica affinity column
using 0.02 M glycine-HCl buffer, pH 3.5 and 2.5 (5) - Active
fractions were pooled separately, dialysed against 0.01 M
phosphate buffered saline, pH 7.2 (PBS) and concentrated Using
Amicon concentrators (Amicon Inc. MA, USA). Haemagglutinating
activity was determined by the method of Burger (9) and protein
was assayed by the method of Lowry et al (10). Purity of lectins
was checked on SDS-polyacrylamide gel electrophoresis (11).
Lectin showing single band on SDS-PAGE corresponding to 47 kDa
used for the present study whereas lectin with
12.5 and 13.0 kDa (ASA ) was frozen for further use.
showed 11% specig? c activity with 58% yield.

mal feedinaexneriments

Male Swiss albino rats weighing 90-100 gm were fed standard


pellet diet from Hindustan lever Ltd, Mumbai and water ad
libitum. After acclimatisation, rats were divided into four
GARLIC AND THE JEJUNUM 843

groups of six animals each and were given treatments for 7 days
bY intragastric intubation route in two shifts of doses as
follows:

Control (Group I)-animals were given PBS as vehicle.


Lectin treated (Group II)-animals were fed 16 mg ASAllO per
100 gm body weight per day.
Native extract treated (Group III)-animals were fed native
garlic extract containing 50 mg protein per 1OOgm body weight
and lectin activity nearly similar to Group II per day.
Heat Denatured extract treated (group IV)-animals were
fed heat treated (boiled at 100 C for 10 min) crude garlic
extract containing equivalent amount of protein as in
Group III but having no Pectin activity.

On 8th day, two hours before sacrifice, animals were given an


additional dose of treatment, their intestines were excised and
divided into 3 equal segments (S to s ). A 1 cm piece from the
proximal end of each S segment b as ta a en, cut into 0.2-0.4 cm
pieces and fixed in bufgered formalin for histologic evaluation.
The remainder of each S segment representing the jejunal segment
(12) was rinsed with2 ice cold 0.9% normal saline, everted
carefully and mucosa homogenised in ice cold buffer. Homogenates
were assayed for sucrase, maltase, lactate dehydrogenase (LDH),
alkaline phosphatase (ALP), acid phosphatase(ACP) and adenosine
triphosphatase (ATPase) activities.

Nucleic acids were extracted by the method of Schneider (13).


DNA was assayed by measuring the blue colour of diphenylamine at
600 nm and RNA was estimated by reading green colour of orcinol
at 660 nm. Protein was assayed by the method of Lowry et al
(IO). The activities of sucrase (E.C.3.2.1.10) and maltase
(E.C.3.2.1.48) were measured in intestinal homogenates by the
method of Dahlquist (14) using glucose-oxidase peroxidase.
Alkaline phosphatase (E.C.3.2.3.2) was estimated by hydrolysing
p-nitrophenyl phosphate at pH 10.5 and acid phosphatase
(E.C.3.1.3.22) was estimated by hydrolysing p-nitrophenyl
phosphate at pH 4.8 . Na/K ATPase estimation was carried out in
presence (ouabain insensitiys ATPgse) an6 in absence of ouabain
(total ATPase) along with Mg Na and K ions (15) and released
inorganic phosphate was estimaied. The Na/K stimulated ATPase was
computed by subtracting ouabain insensitive ATPase from total
ATPase. LDH (E.C. 1.1.1.27) was estimated by oxidation of
pyruvate in presence of NADH and observing the change in optical
density per min at 340 nm (16).

Statistical Analvsis

Statistical analysis was done by the least significant difference


evaluated by student's \t' test.

RESULTS

In order to assess the biochemical effects of ASA lectin,


membraneous and mucosal enzymes, nucleic acids aA&' protein
contents were measured in intestinal tissues in absence and in
presence of lectin and native and heated garlic extracts.
844 A. GUPTA and R.S. SANDHU

B ffec on
The normal range of jejunal DNA and RNA was 5.2850.5 and 1.54kO.2
mg/gm tissue respectively. and heat treated garlic
extracts did not effect nucleic
ASAW! ds content in general (Table
1) * However, native crude garlic extracts increased DNA to
7.53k1.4 and RNA to 2.14+0.4 mg/gm tissue (~~0.05). In contrast,
the protein contents in Sejunum was always higher irrespective of
the treatments. Maximum increase upto 40-60% in protein content
was obtained with native and heat treated garlic extracts
(PCO.01).

Table 1.

EFFECT OF ASA AND GARLIC EXTRACTS FEEDING ON NUCLEIC


ACIDS AND%OTEIN CONTENT IN HAT INTESTINE.

Treatment Protein DNA ~1000


(mg/gm tissue) (mg/gm tissue) (mg/rtissue)
:Protein
(yg/mg)
____________________~_~~~~~~~-~~~~-~-~~~~~~~~~~~~~~-~~~~---~~~-~~
Control 72.6 + 8.4 5.3 +0.6 1.5OkO.2 72.9

Lectin Treated 99.5 +10.8** 5.4 50.6 1.46kO.3 54.4


*
??
Native extract 104.8 212.6 7.5 51.4* 2.14+0.5* 71.9

Heated extract 107.5 & 8.3** 5.5 +0.8 1.18+0.1* 50.9

mean+S.D. (n-61,
Significantly different from controls +p<O.O5, ??
*p<O.Ol.

Enzyme activities were measured to check the functional


disturbances due to presence in the brush border
membranes. The enzyme activities Of ASA&@ d in terms of micromoles
expre
of substrates reacted in unit time by either 1 mg protein or 1 gm
of fresh weight of intestine are given in Table 2. Sucrase and
maltase activities in jejunum of rats studied by glucose-oxidase
peroxidase method(14) were significantly elevated following
intubation of pure lectin or crude garlic extract given in
native/heated forms. The results were more significant when
enzyme activity was expressed on fresh weight basis. ASA fed
animals, though showed higher activity of sucrase and &&Ptase,
results failed to reach to statistically significant different
level (Table. 2).

Unlike disaccharidases, ALP activity was considerably lost


followingenhanc~~O
ASA treatment. However, feeding,of ga;;;Es e;i;;z;;
showed activity of ALP on fresh weight
having any effect on the specific activity of the enzyme. In
contrast to ALP, activity of ACP per gm tissue was significantly
higher in jejunum of ASAll and native garlic extract fed rats
GARLIC AND THE JEJUNUM 045

(p<O.Ol). Heat treatment of garlic extracts tended to abolish the


rise of ACP which was shown by native extracts or purified
ASA110.
Lactate dehydrogenase on protein basis appeared to be
sensitive for loss of activity after ASA treatment (Table. 2).
In contrast, garlic in native or denat&h&d form enhanced the
activity of LDH on tissue weight basis.

Table 2.

EFFECT OF ASA AND GARLIC EXTRACTS FEEDING ON


INTE&NAL ENZYMES IN RATS.
_--_____---_________~---~~~~-------~~~~~~~_----~--~~~~~~-~-~~~~~~~~-~~
Enzyme Control Lectin Native Heated
treated extract extract
_-_______--_________~~~~~~~~~~~~~~~~~~~____~~~--~~~~~~----~~~~~~~~~---

Sucrase
Units/gm tissue 122.7+45 179.4+62 227.8226: 216.8 +61*
Units/mg protein 1.52kO.5 l-78+0.7 2.20+0.2 2.05TO.5

Maltase ??
Units/gm tissue 300.7291 398.1+82 466.0237: 484.3+28,
Units/mg protein 3.36kO.5 4.0320.8 4.5420.6 4.51kO.2

Alkaline Phosphatase
Units/gm tissue 428 +16 313 +9* 532 +20+ 613 +12*
Units/mg protein 4.5520.5 3.33+0.4* 5.4Ok1.5 5.11+0.4

Acid Phosphatase
Units/gm tissue 82.2+20 134.5221: 156.7+24= 125.6+23
Units/mg protein 1.08kO.l 1.3520.2 1.45kO.l 1.03+0.04

LDH
Units/gm tissue 63.8214 70.0+17 87.2+18* 87.2+14*
Units/mg protein 0.8420.1 0.66+0.1* 0.8520.1 0.8320.1
_____---____---_-___~~~~~~~~~~~~~~~~~~~~~~~~~--~~~-~~~~-~-~~~~~~~-~~~~
Mean+S.D., (n=6)
Significantly different from controls tpx0.05.

Total ATPase and ouabain sensitive ATPase activities seemed


to be inhibited following garlic and ASA intubation. Ouabain
sensitive ATPase which is responsible foh'ffa/K transport across
the intestinal microvilli was inhibited upto 65-72% on feeding
with ASA crude native garlic extracts and their heat
denatured'@epzEations (p<O.Ol). The effect could be demonstrated
on protein as well as on fresh weight basis. Feeding of heat
treated garlic extract could not protect the loss of intestinal
Na/K ATPase. However, ouabain insensitive ATPase remained
unaffected since marginal increase in this fraction of ATPase was
nonsignificant (Table. 3). The total ATPase also declined after
various treatments but consistent decline was seen in rats fed
with crude or heated garlic extracts.
846 A. GUPTA and R.S. SANDHU

Histological examination in jejunum of rats fed with ASA


crude garlic extracts revealed following alterations: a)
and sloughing off of the villous structure; b) lengthening of the
villi and c) increase in number of goblet cells. Vacuolisation of
intestinal mucosa was common feature in jejunum of treated
animals. A great deal of cell debris could be observed in the
lumen of intestine probably due to collapse of villi (Plate 1).

Table 3.

EFFECT OF ASA AND GARLIC EXTRACTS FEEDING ON INTESTINAL


ATPase IN HATS.
_____________________~~~__~~~_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
E,nzymes Control Lectin Crude Heated
treated extract extract
____________________~~~~-~~~~~--~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Total ATPase ??
Units/gm tissue 132.2244.5 98.9228.5, 83.5k13.1, 90.6+15.1;
Units/mg protein l-84+0.61 0.99kO.28 0.80+0.12 0.84kO.77

Ouabain Sensitive ATPase


Units/gm tissue 84.4k18.9 34.7+21.7;* 24.5+15.0;* 25.2+7.1*;
Units/mg protein 1.16kO.27 O-35+0.22 0.2320.28 0.2320.07

Ouabain Insensitive ATPase


Units/gm tissue 48.4k28.2 64.2k46.0 59.0218.8 64.6k29.3
Units/mg protein 0.6720.39 0.65kO.46 0.5650.18 0.6020.28
________--_______--_~~~-~~~~~~~~~~~~~~~~~-~~~~~~~~~~~~~-~~~~~~~~~~
Mean+S.D., (n=6)
Significantly different from controls ?? p<O.O5, ??*p<O.Ol

DISCUSSION

In our previous report, it was shown that garlic lectins


arrest the growth of the treated rats, which was associated with
decrease in food intake (8). As lectin passed through the gut, it
could withstand all enzymatic reactions. However, its binding to
the mucosal epithelium of gastrointestinal tract was indicative
of deleterious effects. In view of previous report, the present
study was mainly designed to check the functional disturbances
in intestinal tissuee~e~s~lapea~~e~~~~~ct~t~~~ts. ;f;,,stizx
suggests that some
modified by the unknown factors present in garlic."Qhe increase
in protein, DNA and disaccharidases along with a decrease in
other brush border membrane enzymes like ATPase, ALP and LDH
indicated selective disturbances in brush border cells caused by
Although, feeding of ASA
4Z&&%. se of disaccharidases, ,rud~l%a~:~en~k~~~e~~~~~~~ iihoEz
statistically significant stimulation of these and other enzymes
(ALP, ACP and LDH) which were associated with increase in DNA,
RNA and protein.
a47

Plate 1 : Photomicrographs showing morphological changes in


jejunum of rat intestine after feeding with ASA and garlic
extracts. (a) control; (b) treated with ASA 10;anhl?c) with crude
garlic extract. Five micron thick section & were stained with
haematoxylin/eosin and photographed [120 x]. Note the changes in
brush border membranes. Thinning and sloughing off the villi,
increase in number of goblet cells and vacuolisation in
and garlic extract treated sections is very clear. ASAllO
848 A. GUPTA and R.S. SANDHU

It shows that garlic extracts can stimulate a selective


population of intestinal cells, which could not be stimulated for
DNA and
E;,l'ly g;;f;;z ;;;&&$,~o$;;Jld;~ ai?dswt;; ;&;ii
adequate
contents in the garlic fed rats, and increased glycolysis as
evident by high LDH and low DNA/protein ratio, it appeared that
the observed enhancement could be due to both cellular
hypertrophy and hyperplasia as shown by lengthening of
intestinal villi (16) and proliferation of human and guinea
pig lymphocytes by garlic lectins (6). It is therefore,
possible that some garlic constituents by direct interaction with
selective population of intestinal cells or by indirect effect
via gut endocrine cells are able to stimulate gut cellular
proliferation in a manner similar to that found with soybean
lectin (17) and kidney bean lectin (18).

Among the loss of enzyme activities by ASA 1O, ouabain


sensitive ATPase(Na/K ATPase) was the most sensitiv a showing a
diminution of about 70% activity after lectin treatment.
Nonetheless, changes in alkaline and acid phosphatases after
lectin feeding may be attributed to the manifestations of
adaptive responses on account of dietary manipulations as has
been reported for ALP and leucine aminopeptidase following the
feeding of Concanavalin A (Con A) and Winged bean lectin (WBL)
(12,19). Heat denatured garlic extracts did not restore the
activity of ouabain sensitive ATPase suggesting that the
secondary conformation The;d o;E;hbP is not essential in the
inhibition of ATPase. tions along with morphological
changes in brush border membranes support the possibility of
direct interactions of garlic components including lectins with
intestinal mucosa (8). This in part, may alter : (a) the dietary
intake of nutrients by affecting the transport across the
intestinal microvillous membranes and (b) some antinutritional
effects and stunned growth after garlic feeding. Similar
observations have been reported with many lectins like Con A
(28), PHA (Phaseolus vulgaris) (18), Wheat germ agglutinin (28)
and WBL (12,21). Effects of ASA like other lectins appear to
be initiated by its binding to th&.Oepithelial cells that results
in the impairment of the ability of these cells for digestion
and/or absorption of nutrients (Gupta and Sandhu, unpublished)
and hindering the normal cell maturation (21).

Thus, although the discrete mode of action of ASA ’ not


clearly understood at present, but it is concluded"~h~~ the
lectin present in garlic induces toxic effects on membrane ATPase
after its gastric intubation. Since lectin and heated extracts
had no effect on nucleic acids whereas native extracts affected
differently, it indicates that other components in garlic need to
be examined for biological effects on intestinal tract. How
changes in garlic
extracts re~~~~~a~lu~~~~s~et~~~~~smf~~~l~~o~~~~ora~~d is the
subject of future investigation.

The authors acknowledge Prof.R.Nath, Department of Biochemistry,


PGIMER for his assistance in providing the laboratory facilities
and CSIR, New Delhi for providing financial assistance.
GARLIC AND THE JEJUNUM 849

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Acceoted for oublication Feb. 9- 1998.

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