International Journal of Molecular Biology and Biochemistry

International Journal of Molecular Biology and Biochemistry

www.biologyjournals.net
Online ISSN: 2664-651X; Print ISSN: 2664-6501
Received: 01-07-2019; Accepted: 02-08-2019; Published: 09-08-2019
Volume 1; Issue 2; 2019; Page No. 01-05

Inhibitory effect of leaf and root bark of Calotropis procera and Parquetina nigrescens on larval midgut
enzyme activities of Callosobruchus maculatus
Alamuoye Nathaniel Olu
Department of Zoology, Obafemi Awolowo University, Ile Ife, Osun State, Nigeria
DOI: https://doi.org/10.33545/26646501.2019.v1.i2a.5
Abstract
The study evaluated the protein content and effect of leaf and root bark of Calotropis procera and Parquetina nigrescens on midgut
enzyme activities of the larvae of Callosobruchus maculatus with a view to solving the problem of cowpea infestation caused by C.
maculatus. Evaluation of inhibitory effect of the leaf and root bark of C. procera and P. nigrescens on total protease, trypsin,
chymotrypsin, α-amylase and lipase was carried out with 0.5% extract of each plant material as inhibitor source for the enzyme inhibitory
assays. Midgut enzyme activities of protease, lipase and alpha amylase and protein content were also quantified. The results showed that
the protein content in the midgut of the larvae of C. maculatus were 1.48±0.01, 1.51±0.01, 1.36±0.01 and 1.71±0.01 mg ml-1 in first,
second, third, and fourth instar larvae respectively and were significantly (P < 0.05) different. Protease activity was higher than both
alpha amylase and lipase activities in the insect midgut while inhibition of the extracts of leaf and root bark of C. procera and P.
nigrescens on enzyme activities varied across plant materials and stages of instar larva of C. maculatus.
Keywords: midgut, larvae, protease, lipase and alpha amylase

Introduction Bark of Calotropis procera and Parquetina nigrescens on midgut

For an efficient management of pest control, it is imperative to Enzyme activities of Callosobruchus maculatus
know the type of enzymes present in the gut of insects and pests.
Two major proteinase classes in the digestive systems of Materials and methods
phytophagous insects are the serine and cysteine proteinases [1]. Total protein determination of the midgut of C. maculatus
Lipase enzymes generally are defined as triacylglycerol Preparation of midgut tissue and homogenate
hydrolases that break carboxylester bonds in triacylglycerols, Larvae were dissected under microscope and the midgut
[2]
diacylglycerols, galactolipids and phospholipids . removed, placed in cold distilled water. A quantity of 0.1 g of
Triacylglycerols (TAGs) constitute a major lipid component in midgut tissue was homogenized with 1 ml of phosphate buffer
the diet of insects and their processes of digestion and absorption and centrifuged at 8000 g 4 °C for 10 minutes. The supernatant
are very similar to those in mammals [3]. After feeding, TAGs are was collected into a new centrifuge tube and kept on ice until use.
hydrolyzed in the midgut lumen and the products of digestion are
absorbed and used for the synthesis of complex lipids [4]. Insects Total protein determination
utilize enzymes to digest their food. One of these is alpa-amylase, Protein concentration of gut larvae was determined by the method
which cleaves α-1, 4 glycosidic bonds in starch, glycogen, of Bradford [9] using Bovine Serum Albumin (BSA) as the
oligosaccharides, and polysaccharides and thereby promotes standard (0.125, 0.25, 0.5, 0.75, 1.0, 1.5 and 2.0 mg mL-1).
larval carbon assimilation from the diets [5]. Some plant serine Protein reagent was prepared by dissolving 100mg of coomossie
proteinase inhibitors are bifunctional molecules and are able to brilliant blue G-250 in 50ml 95% ethanol. To this solution 100
inhibit trypsins as well as α-amylase [6]. Several functional ml 85% (w/v) phosphoric acid was added. The resulting solution
proteins such as lectin-like, cereal-type and Kunitz-like which are was diluted to a final volume of 1 liter. The solution was filtered
often found in the seeds of leguminous and graminaceous plant, through Whatman No.4 filter paper and stored in a reagent bottle
have been reported to inhibit a-amylase in insect midgut, thereby until use. An aliquot100 µl of the midgut homogenate were
blocking the main energy source for insect growth and piptted into a test tube, 6 ml coomassie blue reagent was added
development [7]. The binding of these α-amylase inhibitors and and the absorbance read at 595nm after two minutes and before 1
amylase forms a stable enzyme–inhibitor complex. This makes hour against blank. For preparation of standard curve, varying
insect midgut to secrete excessive digestive enzyme which often concentrations of bovine serum albumin (BSA) were piptted into
lead to insect death [8]. These inhibitors can affect the functional test tubes. The volume in the test tubes was adjusted to 1 ml with
properties of a-amylase such as decreasing the binding of a- phosphate buffer (0.1M, pH6.6). A volume of 5 ml of protein
amylase with its substrates, resulting in reduction or loss of reagent were added to the test tubes and the contents mixed. The
enzymatic activity [5]. absorbance at 595 nm was measured after 2 minutes and before
The present research assessed inhibitory effect of leaf and root 1hour against blank prepared from 1ml of phosphate buffer and
5ml protein reagent.

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International Journal of Molecular Biology and Biochemistry

Determination of protease, lipase and alpha amylase enzymes lowest (42.3±0.10) in the root bark of C. procera. The percentage
activity of larval midgut of C. maculatus. inhibition on proteolytic activity of the third instar larval midgut
Quantification of the total protease, trypsin, chymotrypsin, lipase, of C. maculatus had the highest (85.6±0.10) in the plant extract
and alpha amylase activities of the midgut enzyme of C. from P. nigrescens leaf, followed by C. procera leaf which
maculatus were determined according to Cohesion Bioscience recorded 74.6±0.10 and the lowest (41.3±0.10) in C. procera root
assay kits bark. The percentage inhibition on proteolytic activity in the
midgut of the fourth instar larvae of C. maculatus had the highest
Effects of leaf and root bark of Calotropis procera and (86.4±0.10) in the plant extract from P. nigrescens leaf, followed
Parquetina nigrescens on the enzyme activity of the four by C. procera leaf (85.7±0.20) and the lowest (34.2±0.10) in the
instars larvae of C. maculatus root bark of C. procera. The percentage inhibition on the
For measurement of inhibitory effect of leaf and root bark of C. proteolytic activity of the first, second, third and fourth instar
procera and P. nigrescens on total protease, trypsin, larval midgut of C. maculatus was significantly different
chymotrypsin, α-amylase and lipase, 0.5% extract of each plant (P<0.05).
material was prepared as inhibitor source and used for the enzyme The inhibitory effect of crude extract of leaf and root bark of C.
inhibitory assays [10, 11], 70 µl of each extract of leaf and root bark procera and P. nigrescens on trypsin activity in the midgut of the
of C. procera and P. nigrescens were incubated at 700C for 20 larvae of C. maculatus as presented in the Table 3 showed that
mins to inactivates endogeneous enzymes and added to the inhibition on trypsin activity in the first instar of C. maculatus
reaction mixtures of total protease, trypsin, chymotrypsin, α- had the highest (56.7±1.00) in P. nigrescens root bark and the
amylase and lipase activities as described by Cohesion lowest (36.4±0.20) was in leaf of P. nigrescens. Inhibition
Biosciences assay kit. percentage on trypsin activity in the second instar larvae of C.
maculatus were in the range of 33.80±0.10 to 53.1±0.1. The leaf
Activity with plant extract of C. procera had the highest inhibition while the root bark of C.
% Inhibition = X 100
Activity without plant extract procera had the lowest inhibitory effects on trypsin activity. The
percentage inhibition on trypsin activity in the third instar larvae
Data analysis of C. maculatus had the highest (72.6±0.20) in C. Procera leaf,
Data obtained were subjected to analysis of variance (ANOVA) followed by the leaf of P. nigrescens (63.9±0.10) and the lowest
procedure of Minitab 16.1 [12]. Tukey’s Test at P = 0.05 was used (19.20±0.10) in C. Procera root bark. Similarly, the percentage
to compare means. inhibition on trypsin activity in the fourth instar larvae of C.
maculatus had the highest (74.3±0.10) in C. Procera leaf and the
Results lowest (11.7±0.10) was in root bark of C. procera. The
The results of the enzyme activity and protein content of the first, percentage inhibition on trypsin activity of the first, second, third
second, third and fourth instar larval midgut of C. maculatus are and fourth instar larvae of C. maculatus was significantly
represented in the Table 1. Total protease activity of the instars different (P<0.05).
ranged between 1.21± 0.1 – 1.68± 0.01 µmol min-1 mg-1. Fourth The inhibitory effect of the crude extract of leaf and root bark of
instar larval midgut had the highest (0.83±0.01 µmol min-1 mg-1) C. procera and P. nigrescens on chymotrypsin activity of the
trypsin activity while the lowest (0.54±0.01 µmol min-1 mg-1) was larval midgut of C. maculatus as presented in the Table 4 showed
in third instar larval midgut and were significantly different (P < that the inhibition on chymotrypsin activity in the first instar had
0.05). Chymotrypsin activity had 0.43±0.01, 0.52±0.01, the highest (47.6±0.06) in root bark of P. nigrescens and the
0.31±0.01 and 0.66±0.02 µmol min-1 mg-1 in first, second, third, lowest (35.3±0.30) in root bark of C. procera. The percentage
and fourth instar larval midgut respectively and they were inhibition on chymotrypsin activity in the second instar larval
significantly different (P < 0.05). However, alpha amylase midgut of C. maculatus was highest (56.2±0.20) in C. procera
activity was 0.24±0.01, 0.31±0.01, 0.12±0.01 and 0.44±0.01 leaf, followed by the root bark of P. nigrescens (44.30±0.10), and
µmol min-1 mg-1,in first, second, third, and fourth instar larval the lowest (36.5±0.10) in root bark of C. procera. The percentage
midgut respectively and they were significantly (P < 0.05) inhibition on chymotrypsin activity in the third instar larval
different. Lipase activity had 0.35±0.01, 0.41±0.01, 0.21±0.01 midgut of C. maculatus had the highest (62.5±0.50) in C. procera
and 0.59±0.01 µmol min-1 mg-1 in first, second, third, and fourth leaf, followed by the leaf of P. nigrescens (53.1±0.00) and the
instar larval midgut respectively and they were significantly (P < lowest (22.3±0.17) in root bark of C. procera. In the fourth instar
0.05) different. The protein content in the midgut of the larvae of larval midgut of C. maculatus, percentage inhibition on
C. maculatus were 1.48±0.01, 1.51±0.01, 1.36±0.01 and chymotrypsin activity had the highest (77.3±0.20) in C. procera
1.71±0.01 mg ml-1 in first, second, third, and fourth instar larvae leaf and the lowest (17.5±0.20) in root bark of C. procera. The
respectively and were significantly (P < 0.05) different. percentage inhibition on chymotrypsin activity in the first,
Inhibitory effect of crude extracts of leaf and root bark of C. second, third and fourth instar larval midgut of C. maculatus was
procera and P. nigrescens on proteolytic activity of the larval significantly different (P<0.05).
midgut of C. maculatus are shown in Table 2. The percentage The inhibitory effect of crude extract of leaf and root bark of C.
inhibition on proteolytic activity of the first instar larval midgut procera and P. nigrescenson α- amylase activity of the larval
of C. maculatus was highest (75.6±0.10) in the plant extract from midgut of C. maculatus as presented in the Table 5 showed that
the leaf of P. nigrescens while that of C. procera leaf had the inhibition on α- amylase activity in the first the highest
lowest (34.2±0.10). Percentage inhibition on proteolytic activity (86.5±0.01) in C. procera leaf, followed by the root bark of P.
of the second instar larval midgut of C. maculatus had the highest nigrescens (80.2±0.10) and the lowest (71±0.20) in root bark of
(82.7±0.20) in the plant extract from P. nigrescens leaf and the C. procera. However, the percentage inhibition on α- amylase

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activity in the second instar larval midgut of C. maculatus was The percentage inhibition of the crude extract of leaf and root
highest (84±2.00) in P. nigrescens root bark and the lowest bark of C. procera and P. nigrescens on lipase activity of the
(73.4±0.02) in the leaf of C. procera. The leaf of P. nigrescens larval midgut of C, maculatus as shown in the Table 6 revealed
had the highest (87.4±0.02) inhibition percentage on α- amylase that inhibition on lipase activity was high and ranged between
activity in the third instar larvae of C. maculatus while P. 76.45±0.01 to 96.00±1.00. The inhibitor from the leaf of P.
nigrescens root bark showed the highest (95.45±0.01) inhibition nigrescens had highest percentage inhibition on lipase activity
on α- amylase activity in the fourth instar larval midgut of C. while that of C. procera leaf was the lowest. The percentage
maculatus. The percentage inhibition on the activity of α- inhibition on lipase activity in all the four instars was
amylase in the first, second third and fourth instar larval midgut significantly different (P<0.05).
of C. maculatus was significantly different (P<0.05).

Table 1: Enzyme activity and protein content of the first, second, third and fourth instar larval midgut of C. maculatus.
Enzyme activity(µmol min-1 mg-1) and protein content (mg ml-1)
Instar larva
PA TrPA CTPA AA LA PC
First 1.42±0.01c 0.62±0.02c 0.43±0.01c 0.24±0.01c 0.35±0.01c 1.48±0.01c
Second 1.49±0.01b 0.71±0.01b 0.52±0.01b 0.31±0.01b 0.41±0.01b 1.51±0.01bc
Third 1.21±0.01d 0.54±0.01d 0.31±0.01d 0.12±0.01d 0.21±0.01d 1.56±0.01b
Fourth 1.68±0.01a 0.83±0.01a 0.66±0.02a 0.44±0.01a 0.59±0.01a 1.71±0.01a
Means in the same column with the same alphabets are not significantly different (P<0.05).
PA = Proteolytic activity, TrPA = Trypsin proteolytic activity, CTPA=Chymotryapsin proteolytic
activity, AA = Amylase activity, LA = Lipase activity, PC = Protein content

Table 2: Inhibitory effect of crude extract of leaf and root bark of C. procera and P. nigrescens on proteolytic activity of the larval midgut of C.
maculatus
% inhibition
Plant materials
First instar larvae Second instar larvae Third instar larvae Fourth instar larvae
C. procera leaf 34.20±0.10d 45.30±0.10c 74.60±0.10b 85.70±0.20b
c d d
C. procera root bark 56.40±0.10 42.30±0.10 41.30±0.20 34.20±0.10d
a a a
P. nigrescens leaf 75.60±0.10 82.70±0.20 85.60±0.10 86.40±0.10a
P. nigrescens root bark 66.50±0.10b 53.30±0.00b 51.20±0.10c 44.10±0.10c
Means in the same column with the same alphabets are not significantly different (P<0.05).

Table 3: Inhibitory effect of crude extract of leaf and root bark of C. procera and P. nigrescens on trypsin activity of the larval midgut of C.
maculatus
% inhibition
Plant materials
First instar larvae Second instar larvae Third instar larvae Fourth instar larvae
C. procera leaf 41.20±0.10c 53.10±0.10a 72.60±0.20a 74.30±0.10a
C. procera root bark 45.40±0.10b 33.80±0.10d 19.20±0.10d 11.70±0.10c
P. nigrescens leaf 36.40±0.20d 45.70±0.20b 63.90±0.10b 68.80±0.10b
P. nigrescens root bark 56.70±0.10a 42.10±0.10c 25.40±0.10c 12.20±0.10c
Means in the same column with the same alphabets are not significantly different (P<0.05).

Table 4: Inhibitory effect of crude extract of leaf and root bark of C. procera and P. nigrescens on chymotrypsin activity of the larval midgut of C.
maculatus
% inhibition
Plant materials
First instar larvae Second instar larvae Third instar larvae Fourth instar larvae
C. procera leaf 44.10±0.10b 56.20±0.20a 62.50±0.50a 77.30±0.20a
d c d
C. procera root bark 35.30±0.10 36.50±0.10 22.30±0.00 17.50±0.20d
c b b
P. nigrescens leaf 41.70±0.10 44.20±0.10 53.10±0.00 64.00±1.00b
P. nigrescens root bark 47.60±0.30a 44.30±0.10b 33.60±0.20c 29.00±1.00c
Means in the same column with the same alphabets are not significantly different (P<0.05).

Table 5: Inhibitory effect of crude extract of leaf and root bark of C. procera and P. nigrescens on α- amylase activity of the larval midgut of C.
maculatus
% inhibition
Plant materials
First instar larvae Second instar larvae Third instar larvae Fourth instar larvae
C. procera leaf 86.45±0.01a 73.35±0.02d 65.00±2.00d 60.15±0.01d
C. procera root bark 71.00±1.00d 76.10±0.00c 67.10±0.10c 86.30±0.10c
P. nigrescens leaf 75.25±0.01c 81.75±0.01b 87.35±0.02a 92.25±0.01b
P. nigrescens root bark 80.20±0.10b 84.00±2.00a 78.00±1.00b 95.45±0.01a
Means in the same column with the same alphabets are not significantly different (P<0.05).
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International Journal of Molecular Biology and Biochemistry

Table 6: Inhibitory effect of crude extract of leaf and root bark of C. procera and P. nigrescens on lipase activity of the larval midgut of C. maculatus
% inhibition
Plant materials
First instar larvae Second instar larvae Third instar larvae Fourth instar larvae
C. procera leaf 84.35±0.02c 92.30±0.10a 95.45±0.01a 96.00±1.00a
b c c
C. procera root bark 83.30±0.20 84.20±0.10 87.30±0.10 90.00±1.00c
P. nigrescens leaf 76.45±0.01d 82.15±0.03d 86.25±0.02d 88.15±0.02d
P. nigrescens root bark 91.15±0.03a 91.10±0.00b 93.10±0.10b 94.10±0.17b
Means in the same column with the same alphabets are not significantly different (P<0.05).

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