The Avocado, unflatteringly known in
the past as alligator pear, midshipman's butter, vegetable butter, or sometimes
as butter pear, and called by Spanish-speaking people aguacate, cura,
cupandra, or palta; in Portuguese, abacate; in French, avocatier;
is the only important edible fruit of the laurel family, Lauraceae. It is
botanically classified in three groups: A) Persea americana Mill.
var. americana (P. gratissima Gaertn.), West Indian Avocado;
B) P. americana Mill. var. drymifolia Blake (P. drymifolia
Schlecht. & Cham.), the Mexican Avocado; C) P. nubigena
var. guatemalensis L. Wms., the Guatemalan Avocado. It has
traditionally been used for antibacterial, antifungal, hypotensive, and
anti-inflammatory, anti-immune enhancer effect. Furthermore, Avocado
juice, which is made from the ripe fruit, was very popular of its many health
benefits. The Avocado tree may be erect, usually to 30 ft (9 m)
but sometimes to 60 ft (18 m) or more, with a trunk 12 to 24 in (30-60 cm) in
diameter, (greater in very old trees) or it may be short and spreading with
branches beginning close to the ground. Almost evergreen, being shed briefly in
dry seasons at blooming time, the leaves are alternate, dark-green and glossy
on the upper surface, whitish on the underside; variable in shape (lanceolate,
elliptic, oval, ovate or obovate), 3 to 16 in (7.5-40 cm) long. Because limited
reports on the fruits of Avocado were available in the literature, it
was thought to be desirable to carry out systematic investigations on the
fruits of this plant. Use of natural drugs in gastric gastric lesions is well
documented8,9. Information provided by practitioners of
African traditional medicine suggests that Avocado fruit possesses
anti-gastric lesion remedy. The aim of this present study was to support the
anti-gastric lesion effect by the amelioration of gastric lesions, attributed
to Avocado fruit extract, by evaluating the anti-gastric lesion activity
of an ethanolic as well as indomethacin extract of this fruit.
Chemical composition:
According to Kadam, and Salunkhe, the Avocado
has a high lipid content-from 5 to 25% depending on the cultivar. Among the
saturated fatty acids, myristic level may be, 1%, palmitic, 7.2, 14.1 or 22.1%;
stearic, 0.2, 0.6 or 1.7%. Of the unsaturated fatty acids, palmitoleic may
range from 5.5 to 11.0%; oleic may be 51.9, 70.7 or 80.97%, linoleic, 9.3, 11.2
or 14.3%. Non saponifiable represents 1.6 to 2.4%. Iodine number is 94.4. Amino
acids of the pulp (N = 16 p. 100) are recorded as: arginine, 3.4; cystine, 0.1:
histidine, 1.8; isoleucine, 3.4; leucine, 5.5; lysine, 4.3;methionine, 2.1;
phenylalanine, 3.5; threonine, 2.9; tryptophan, 0.1 ; tyrosine, 2.3; valine,
4.6; aspartic acid, 22.6; glutamic acid, 12.3; alanine, 6.0; glycine, 4.0;
proline, 3.9; serine, 4.1 .
MATERIALS AND METHODS:
Plant material
Fresh fruits of P. americana were
collected from its natural habitat in the Agricultural Research Center, Tepi,
Ethiopia and were authenticated in the department of biology, Mizan-Tapi
University, Tepi. The seed was removed and the edible part was chopped into
small pieces, dried at 50-60°C and ground into powder. Known amount of dry
powder was repeatedly extracted by the process of maceration in an aspirator
using 95% ethanol as menstrum. The extract was concentrated under reduced
pressure by rotary evaporator to obtain thick syrup mass, and stored at 4°C.
The yield was approximately 10% of fresh fruit. Working concentrations of the
extract were made in nonpyrogenic distilled water before use in the
experiment.
Experimental animals
Healthy, male albino rats of Wistar strain
(150-170g) were selected for the present study. The rats were procured from
Tepi Veterinary Center, Tepi, Ethiopia. Before beginning the experiments, the
animals were allowed to acclimatize to animal house condition for a period of
one week.
Pretreatment studies
Six groups of six rats each were pre-treated
with single oral administration of Avocado fruit extract at a
concentration of 300mg/kg body weight 1h before the gastric lesion inducing
procedures. Gastric lesions was induced in 36h fasted rats by the oral
administration of gastric lesion inducing drug, indomethacin (48mg/kg b.w)10
and necrotizing agent, 1ml of absolute ethanol11.The rats
were killed 6h after indomethacin and 1h after the ethanol administration by an
overdose of ether. The gastric gland were removed and opened along the greater
curvature and the ulcer index was evaluated according to severity and gastric
lesion scores.
Post treatment studies
Ethanol-induced Gastric lesion Model
Male Wistar rats weighing about 150-170g
were divided into different groups each comprising of a minimum of 6 rats as
detailed below.
Group I- Control rats (received 1ml of
water)
Group II- Ethanol-Induced gastric lesion
rats (1mL-1 rat)
Group III- Ethanol-Induced gastric lesion
rats orally treated with Avocado fruit extract at a dose of 300mg/kg
b.w., for a period of 15days.
Group- IV Ethanol induced gastric lesion
rats orally treated with Ranitidine (100mg/kg b.w.,) for a period of 15days.
Indomethacin-Induced Gastric lesion Model
The experimental set up adopted for
indomethacin–induced gastric lesion model was as follows:
Group I -Control rats.
Group II- Indomethacin-Induced gastric
lesion rats (100mg/kg. p.o).
Group III- Indomethacin- Induced gastric
lesion rats orally treated with Avocado fruit extract at a dose of
300mg/kg b.w., for a period of 15days
Group IV Indomethacin-Induced Gastric lesion
rats orally treated with Ranitidine (100mg/kg b.w.) for a period of 15 days.
The treatment schedule was once a day and
the rats were weighed periodically. At the end of the experimental period, all
the groups of rats were subjected to pylorus ligation according to the
procedure of12 as modified by13. Feed was
withheld 12h prior to the operative procedure. The rats were anaesthetized with
ether and the stomach was open through a mid-line incision. The pylorus was
secured and lighted with silk sutures; proper care was taken not to ligate the
blood vessel. The gastric walls were closed and the animals were allowed to
recover from anesthesia. After pyloric ligation, drinking water was withheld
and the gastric juice was allowed to collect for a period of 4h. The rats were
then killed by an overdose of ether and the abdomen was removed after clamping
the esophagus.
The gastric mucosa was washed with 3ml of
lukewarm distilled water and collected in graduated centrifuge tubes. The
gastric juice and washing were homogenized and centrifuged at 500rpm for 5min.
The biochemical parameters assayed in gastric juice include gastric volume,
total acidity, pH and glycoproteins. Dissolved mucosa substances were estimated
in 90% alcoholic precipitate of gastric juice14. The
precipitate thus obtained was either dissolved in 1ml of 0.1N sodium hydroxide
or 1ml of 0.1N sulphuric acid. The former was used for the estimation of
protein, total hexose, hexosamine and fucose, while the latter was used for the
estimation of sialic acid. Hexose was estimated by the method of15.
Hexosamine was estimated by its color reaction with Ehrlich’s reagent16.
Sialic acid was estimated by the method of17.
Fucose was estimated by the method of18. Total
protein was estimated by the method of19. Ratio of total
carbohydrate (TC= sum of total hexose, hexosamine, fucose and sialic acid) to
protein (P) was used as the index of mucin activity.
Determination of Degree of Gastric lesions
The surface area (A) mm2 covered
by each lesion was measured20 and the sum of erosion areas
per rat abdomen was calculated. Percentage Gastric lesion surface was
calculated through Ulcer Index (US).
%US = 
Ulcer index was calculated from percentage
gastric lesion surface as described by21.The following
scores, was used in order to calculate ulcer index; 0. No gastric lesion;
1.US<0.5; 2. 0.5=2.5; 3.2.5=5; 4.5=10; 5.11=15; 6.15=20; 7.20=25; 8.25=30;
9.30=35; 10.US>35.
Statistical Analysis
The values are expressed as mean+ SD
for six rats in each group. All the data were analyzed with SPSS/7.5 student
software. Hypotheses testing method included one way analysis of variance
(ANOVA) followed by post hoc performed with Least Significant Difference (LSD)
test. The p value of less than 0.05 was considered to indicate statistical
significance.
RESULTS:
Pretreatment studies
Table 1 indicates the ulcer index and
gastric lesion surface in control and experimental groups of both gastric
lesion models. A marked increase was observed in gastric lesion surface
and ulcer index of both the gastric lesion-induced groups of rats. In
pretreated groups of rats, a significant decrease in gastric lesion severity
was evident by decreased ulcer index.
Table 1: Effect of
pretreatment with Avocado fruit
extract on indomethacin-induced and ethanol induced gastric lesion rats
|
Groups
|
Gastric Lesion surface (%)
|
Ulcer index
|
|
Control
|
0.0
|
0
|
|
Indomethacin-induced gastric
lesion
|
41.3+ 3.1a*
|
9
|
|
Avocado + Indomethacin
|
2.4 +0.15b*
|
2
|
|
Ethanol induced gastric
lesion
|
20. 5+
0.12b*
|
6
|
|
Avocado + Ethanol
|
23+ 0.14b*
|
1
|
Values are expressed as Mean + SEM of
six animals in each group. One way ANOVA followed by post hoc test (LSD). *
P<0.05. Comparisons are made between a control, b
indomethacin, Ulcer index: No gastric lesion; 1. US<0.5; 2. 05< 2.5;
3. 2.5< 5; 4.5< 10; 5.11< 15; 6.15< 20;
7.20< 25; 8.25< 30; 9. 30< 35; 10.US>35
Post Treatment Studies
Table 2 and 3 show the percentage-gastric
lesion surface, ulcer index, total gastric volume, gastric acidity and pH of
control and experimental groups of rats in both gastric lesion models. A high
degree of ulcer index and gastric lesion surface were obtained in both the
gastric lesion -induced groups of rats when compared to control animals. Ulcer
index and gastric lesion severity were found to be comparably higher in
indomethacin-induced gastric lesion rats. Treatment with Avocado fruit
extract exhibited decrease in both ulcer index and severity of gastric lesion.
A significant increase was observed in the
volume, acidity and a concomitant decrease in pH of gastric juice in both
indomethacin and ethanol induced groups of rats. Administration of Avocado
fruit extract was observed to significantly decrease the acidity, volume and
increase the pH in the gastric juice of both the gastric lesion -induced
rats. The beneficial effect of ranitidine was more pronounced in
indomethacin-induced gastric lesion when compared ethanol-induced gastric
lesion group.
Levels of
Glycoprotein
The levels of
glycoprotein contents and TC: P ratio in the gastric juice of control and
experimental groups of rats on indomethacin induced and ethanol induced are
presented in table 4 and 5, respectively. From this result it is evident that
the level of hexose, hexosamine, sialic acid, fucose and TC: P ratios in both
the gastric lesion model were
considerably lowered when compared to
control groups of rats. Oral administration of Avocado fruit
extract significantly elevated (p<0.05) the levels of glycoprotein contents
after 15 days of treatment in both the gastric lesion-induced groups.
DISCUSSION:
In most cases, the stable incident of
gastric lesion in rat models provides a powerful and convenient tool for the
investigation of therapeutic modalities for the disease and for its
complications. Non-Steroidal Anti-inflammatory Drugs (NSDAIDs) like
indomethacin and aspirin are known to induce numerous punctiform and filiform
gastric lesions during the course of anti-inflammatory therapy and hence
indomethacin induced gastric lesion model was used in the present study.
Although the Mechanisms underlying the ulcerogenicity of indomethacin are not
completely understood, inhibition of prostaglandin synthesis may be an
important22. This view is supported by the fact that
prostaglandins normally have a protective function in the stomach by
maintaining gastric microcirculation23 and caused gastric
secretion of bicarbonate and mucus24. Consumption of alcohol
produce sever hemorrhagic lesions in the gastric mucosa and hence ethanol
induced gastric lesion model was included in the present study. Ethanol-induced
gastric lesion formation may be multifactor. The factors involved in the
formation of gastric lesion using ethanol have been described25.
Further26, has suggested that the gastric wall mucus
depletion induced by ethanol is one of the pathogenic mechanisms responsible
for gastric lesion. The numbers of lesion present on the gastric mucosa are
indicative of the severity of gastric lesion disease27. The
diameters of the lesion are used for the determination of ulcer index, a
measure of gastric lesion in the gastric mucosa.
The observed decrease in the ulcer index in
P. Avocado fruit extract pretreated groups of rats may be due to its
anti-secretory or cytoprotective proteins or both. Through, the mechanism of
gastric lesion formation by indomethacin as well as ethanol is quite different,
the efficacy of the drug was found to be the same in controlling the gastric
lesion.
Although there is considerably controversy
about the role of mucus in the prevention of gastric mucosal injury28,
the gastric mucus coat is considered to be important both in preventing damage
to the gastric epithelium as well as in facilitating its repair. The incidence
of ethanol-induced gastric lesions, which is predominant in the glandular part
of the stomach, has been reported to stimulate the formation of leukotriene C4
(LT4C4) resulting in the damage of rat
gastric mucosa 29.
Administration of Avocado fruit
extract enhanced the mucosal resistance and thus resulted in decrease in ulcer
index and gastric lesion surface. The antisecretory drug ranitidine, also
markedly inhibited the indomethacin-induced gastric lesions. These results
suggest that the anti-gastric lesion activity of the Avocado fruit
extract against indomethacin-induced gastric lesion might also be related its
antisecretory effect.
Ranitidine did not overcome the mucus
depletion induced by ethanol, since it acts via blocking of H2-receptors.
This is in accordance with the previous reports30. On
contrary, Avocado fruit extract administration resulted in decreased
ulcer index. The mucus depletion by the ethanol was overcome by Avocado
fruit extract which underline its cytoprotective nature.
It may also be proposed that the decrease in
gastric lesion severity by Avocado fruit may be attributed to its active
ingredients with gastric-protective nature. The phytochemical analysis revealed
the presence of tannins, saponins and flavanoids.These substances known to
affect the integrity of mucous membranes31. Tannins are known
to protect the outermost layer of mucosa and to render it less permeable and
more resistant to chemicals and mechanical injury or irritation and thus
prevent gastric lesion development32. Flavanoids have also
been reported to offer some protection in gastric lesion development by
increasing capillary resistance and improving microcirculation33.
Acidity plays an important role in the
pathogenesis of indomethacin induced gastropathy. Gastric mucosal damage
induced by indomethacin is amplified by the presence of high concentration of
acid into the gastric lumen34. Indomethacin induced damage to
rat gastric mucosa is markedly dependent on luminal pH35.
Gastric acidity may potentially facilitate indomethacin induced mucosal damage
by two mechanisms:(i) by enhancing gastric absorption of these drug or (ii) by
amplifying mucosal injury once mucosal defense have been impaired by the
decrease in prostaglandin synthesis34. Stimulation of mucus
secretions such as glycoprotein and mucin by Avocado fruit extract helps
in decreasing the volume and acidity of gastric juice. Further, hyposecretory
nature of Avocado fruit extract gastric lesion-induced rats may further
help in decreasing the volume, pH, and acidity of gastric juice towards near
normal levels. Thus, normalization of gastric juice acidity may indirectly help
in healing of gastric lesion Avocado treated gastric lesion rats.
Glycoproteins are the important constituents
of plasma membrane and specific intercellular organelles such as Golgi
complexes, lysosomes and secretory granules. Surface mucus cells and mucus neck
cells of gastric mucosa secrete mucus by exocytosis36. The
main components of gastric mucus are the acidity glycoprotein, sialic acid and
neutral mucopolysaccharides like total hexoses, hexosamine and fucose. These
glycoproteins are of importance for their specific properties such as gel
formation and viscosity. Glycoproteins are obligatory components of mucus and
their quantitative determination has been as a measure of mucus formation37.
A decrease in the synthesis of sulphated mucus glycoprotein has been implicated
in the etiology of peptic gastric lesion38.
The observed decease in the level of
glycoprotein moieties both in gastric juice of gastric lesion groups of rats
may be attributed to two factors namely (a) decreased activity of defense
mechanisms as s result of damage to the gastric mucosa and (b) disintegration
and degradation of glycoprotein moieties into their simpler components in the
process of gastric lesion induced by ulcerogens.
Two main features of the mucous layer are
its thickness and turnover rate. These two processes are great value in
protecting the mucosal layers from the offensive factors39.
The mucosal layer is a dynamic entity in which the surface cells are
continuously renewed40. The observed decease in the levels of
sialic acid and hexosamine levels may be attributed to the decreased production
or turnover of mucus. The mucus possessing fewer amounts of sialic acid and
hexosamine is prone for easy degradation41. Treatment with Avocado
fruit extract antagonizes the aggressive factors, which play a crucial role in
the pathogenesis of gastric lesions and augment defensive factors to protect
the gastric mucosa from gastric lesion. The increase in the levels of
glycoproteins, particularly sialic acid and hexosamine in the extract treated
groups indicate the increase in the production of mucus, thereby possibly
protecting the gastric mucosa in both gastric lesion models. The efficacy of
the extract was more or less same in both the models and showed promising anti-gastric
lesion activity more than that of ranitidine.
Tannins, one of the constituents of Avocado
extract, form a pellicle over the lining of gastric mucosa to resist the attack
of proteolytic enzymes42. Thus resistance to proteolysis may
help in the restoration of glycoprotein moiety of gastric mucosa in Avocado
treated gastric lesion rats. Further, flavanoids have been reported to be
present in Avocado fruit extract, flavanoids might play a role in
stabilizing the antioxidant status of the gastric mucosa, which may have
maintained its glycoprotein moiety. Thus, Avocado fruit extract may have
ameliorated glycoprotein abnormalities through its action on pepsin-mediated
proteolysis.
Thus, the present investigation establishes
the gastro-protective nature of Avocado fruit extract and the protective
effect may be mediated by defensive mucosal factors.
ACKNOWLEDGMENT:
The authors would like to thank Mr.G.Manoharan and P.
Selvarama Lakshmi, Lecturers in Mizan-Tepi University who made this research
work possible by assisting in calculating statistical data and computational
work.
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