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IN VITRO SCREENING OF PROTEINASE INHIBITORS (TRYPSIN, CHYMOTRYPSIN AND

HELICOVERPA GUT PROTEINASE INHIBITORS) IN DIFFERENT PLANT TISSUE EXTRACTS
Padul M. V.*#, Patil M. T. #, Chougale A. D.##, Zambare V. P.###, Patil R. M.#, Ghule R. B.#, Naikwade S. V.#,
Garad A. S.#, Shaikh F. K.*, Gadge P. P.*, Shinde K. D.*,Dama L. B. ####, and Salve A. N.#####
*Department of Biochemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, M. S., India.
#
Post Graduate Department of Biochemistry, New Arts, Commerce and Science College, Ahmednagar. M. S., India.
##
Mass Spectrometry and Proteomics Group, Organic Chemistry Division, National Chemical Laboratory,
Pune- 411 008, M. S., India.
###
Center for Bioprocessing Research and Development, South Dakota School of Mines and Technology, Rapid City,
SD, 57701 USA.
####
Department of Zoology, D.B.F. Dayanand College of Arts and Science, Solapur-413002, (M.S.), India.
#####
Department of Botany, Government Institute of Science, Aurangabad, M. S., India.
(*#E-mail:manoharpadul@yahoo.co.in)
ABSTRACT
Most of the plant protection strategies are focused on selection and application of the natural proteinase inhibitors
(PIs) against insect pests. In addition, PIs also play a vital role in medicine for treatment of immunity related
diseases. PI activity exists mainly in seeds, leaves and flowers of plants. In search of novel PIs, 135 different plant
tissue extracts (leaf, flower and seed) were screened for their PI (trypsin, chymotrypsin and Helicoverpa gut
proteinase inhibitors) activities by using dot-blot assays. Most of the plant tissues screened revealed moderate PI
activity, few showed low PI activity and very few of them showed strong PI activity against trypsin, chymotrypsin
and Helicoverpa gut proteinases. The inhibitory potency of positive samples was further determined by solution
assays. Five plants namely Arachis hypogaea, Vigna sinensis, Dolichos lablab, Phaseolus aureus and Cassia siamea
showed higher activity which ranged from 22.91 to 58.33 %. Higher activities recorded in the seed as compare to leaf
and flower tissues. Dolichos lablab showed highest PI activity (58.33 %) followed by Cassia siamea (52.08 %). PI
activity was found to be distributed unequally in ammonium sulfate (NH2SO4) fractions.
KEY WORDS: Plants, Proteinase inhibitors, Trypsin, Chymotrypsin, Helicoverpa gut proteinases
INTRODUCTION
Proteolytic enzymes catalyzing the hydrolytic cleavage of specific peptide bonds in target proteins are called as
proteinases. These proteolytic enzymes are widely distributed in nearly all plants, animals and microorganisms
(Christeller, 2005; Joanitti et al., 2006). In higher organisms proteinases play key roles in many biological processes.
The proteolytic events catalyzed by these enzymes serve as mediators of signal initiation, transmission and termination
in many of the cellular events such as inflammation, apoptosis, blood clotting and hormone processing pathways
(Ivanov et al., 2006). But they may be potentially damaging when present in higher concentrations. For this reason their
activities need to be strictly regulated and controlled.
Several insect pests are found to be invading many economical agricultural crops resulting loss of yield (Pawar, 1998;
Rajapakse and Walter, 2007). These insect pests rely on a complex proteinases system present in their guts to digest
proteinaceous material present in seeds, leaves and flowers of these crops (Jongsma and Bolter, 1997, Harsulkar et al.,
1999). Essential amino acids obtained from proteolytic digestion are utilized for its growth and development. The
conventional crop protection method relies mainly on the use of chemical insecticide which has harmful effects on the
environment and human health. To design eco-friendly method, many researchers proposed insect-resistant plants
containing potent anti-proteinase gene (Jouanin et al., 1998, Lawrence and Koundal, 2002). These proteinase
inhibitors (PIs) on expression in transgenic plants block the activity of feeding insects proteinases resulting starvation
of insect and eventually death.
The plant PIs are generally small proteins, which regulate significant physiological processes, and are also induced
upon attack by insects or pathogens (Shewry, 2003; Schaller and Ryan, 1995; Xavier-Filho, 1992). Several plant
species have been reported to contain high levels of serine PIs and in leaves, flowers, seeds and tubers as their defense
tools against insects (Padul et al., 2012; Ryan 1990; Garcia-Olmedo et al., 1987). Among several insect pests of
agricultural crops, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), is one of the most devastating pests
worldwide (Sharma, 2001). It shatters the whole crop and invades more than 300 crop plants throughout the world
(Rajapakse and Walter, 2007). Due to indiscriminate use of insecticides, it has developed high levels of resistance to
conventional insecticides (Kranthi et al. 2002). Almost 30 % of all pesticides used worldwide are directed against H.
armigera. Furthermore mounting evidence shows that H. armigera can adapt to the ingestion of PIs expressed in host
crop plants (Broadway and Villani, 1995; Johnston et al., 1991) Therefore, it is important to develop alternative
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methods of controlling this pest. Widespread applications of PIs in agriculture and medicine encourage us (leaves,
seeds and flowers) to find novel source. Keeping this goal in mind we screened 140 different plant tissue extracts for PI
activity and varied inhibition potential of these tissue extracts against trypsin, chymotrypsin and H. armigera gut
proteinases (HGP) were reported.
MATERIALS AND METHODS
Collection of material
Different plant tissues (seeds, leaves and flowers) were collected from different parts of Maharashtra (Ahmednagar,
Jalna and Nanded districts) in rainy and winter seasons during the two years 2008 and 2009. The collected tissues were
crushed in acetone by using tissue Homogenizer to make a fine powder and finally defatted with hexane. Helicoverpa
armigera larvae were collected from the chickpea field of the Mahatma Phule Agriculture University, Rahuri Dist.
Ahmednagar. Bovine trypsin, polyvinylpyrrolidone (PVP), chymotrypsin were obtained from Sisco Research
Laboratories, Pvt. Ltd., Bombay, India , azocasein was purchased from Sigma, USA. X-ray films used were from Agfa
Pvt Ltd Bombay India. Other chemicals used were of the highest purity available.
Extraction of proteinase inhibitors
Proteinase inhibitors in the leaf and flower were extracted in 10 volumes of distilled water containing 1%
polyvinylpyrollidone and kept for 48 hours at 15 0C. The suspension was centrifuged at 15,000 g for 30 minutes at 10 C
and the supernatant was used for further study. Similarly, proteinase inhibitors in the seeds were extracted in 6 volumes
of distilled water containing 1% polyvinylpyrollidone.
Extraction of Helicoverpa armigera guts proteinases (HGPs)
Mid gut tissue of fourth instar larvae was isolated from dissected larvae and stored frozen. The mid gut tissue was
homogenized in 0.1 M glycineNaOH buffer (1:6 w/v) pH 10 for 15 min at 10 0 C. The suspension was centrifuged at
15000 g for 10 min at 4 0C and the supernatant was used as a source of H. armigera gut proteinases (HGPs).
Preparation of trypsin and chymotrypsin solutions
Trypsin and chymotrypsin solutions (0.1mg/ml) were prepared in 0.1M Tris-HCl buffer pH 7.8
Dot-blot assay/ Spot test
Dot-Blot assay method for detection of PIs on X-ray film developed by Pichare and Kachole (1994) was used to detect
trypsin, chymotrypsin and HGP inhibitory activity from leaf, flower and seed tissues. The principle of this technique is
that the X-ray film is coated with gelatin and as we put a drop of proteinase on the film, it hydrolyzes gelatin and forms
clear transparent spot against a dark background, hence proteinases present in the sample are detected.
Whereas in the presence of inhibitor spot appeared as unhydrolysed gelatin against the background. Three varying
concentrations of enzyme and inhibitor (3:1, 1:1 and 1:3 v/v) were prepared. The volume of the reaction mixture was
adjusted with 0.1 M Tris-HCl buffer pH 7.8 for trypsin and chymotrypsin inhibitor activity while 0.1M Gly-NaOH
buffer pH 10 for HGP inhibitor activity, the final volume was made 20 l and then loaded on x-ray film. The film with
spots was incubated for 20 minutes. Depending upon the extent of gelatin hydrolysis, the film was washed with either
tap or warm water (45C). Hydrolysis of the gelatin was visually monitored. A spot appeared as unhydrolysed gelatin
against the background that indicates inhibition and also reveals the presence of inhibitory activity.
Proteinase inhibitor assay
Total proteinase inhibitor activity was measured by the azocasein assay (Brock et al., 1982). The reaction mixture was
made by keeping enzyme and inhibitor concentration constant (1:1 v/v) and total volume were made up to 200 l with
0.2M glycine-NaOH buffer pH 10. This reaction mixture was incubated at 37 C for 30 minutes. The reaction mixture
was added to preincubated 200 l azocasein (0.25%) solution (prepared in 0.1M Glycine NaOH buffer pH10) it was
allowed to keep at 37C for 30 minutes.
The reaction was terminated by adding 300 l 5% trichloroaceticacid (TCA). Controls of each vial were prepared by
adding TCA to azocasein solution before the addition of reaction mixture. After incubation at 37 C for 30 minutes,
tubes were centrifuged at 15,000 g for 10 minutes. 600 l NaOH (1N) was added to the supernatant and the residual
activity was estimated by measuring the optical density at 450 nm.

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RESULTS AND DISCUSSION

Plants have the capacity to synthesize certain biologically active substances, which play a major role in plant defense
against insect pests and microbial attacks. Some of these include defense proteins like proteinase inhibitors (PIs),
amylase inhibitors, lectins and class of pathogenesis-related proteins (Ryan, 1990; Tatyana et al., 1998; Padul et al.,
2012). These proteins are specifically produced in the plant upon biotic stresses and protect the plant tissue from the
damage (Ryan, 1990; Tatyana et al., 1998). PIs are the most exploited class of plant defense proteins for their use in
developing insect resistance in plants (Jouanin et al., 1998).
Furthermore PIs have a significant role in the regulation of many important physiological responses, such as digestion,
blood coagulation, fibrinolysis, complements cascade, phenoloxidase cascade, cell migration, release of hormones and
peptides (Macedo et al., 2011; Tang et al., 2008; Zou et al., 2005). HIV PIs were introduced in 1995 for the treatment
of HIV-infected patients. HIV PIs, including saquinavir, ritonavir, indinavir, nelfinavir, and amprenavir, rapidly and
profoundly reduce the viral load, as indicated by a decline in plasma HIV RNA concentrations within a few days after
the start of treatment (Wei et al., 1995, Neumann et al., 1995). These broad applications of PIs in both agriculture and
medicine prompted us to screen plants for their PIs activity.
In the present study 135 plant tissues extracts (leaves, seeds and flowers) were screened for proteinase (trypsin,
chymotrypsin and HGP) inhibitory activity by dot-blot assay. These activities were grouped under three categories as
low (A), moderate (B) and high (C) and presence and absence of inhibitory activity was denoted by a plus (+) and
minus (-) signs (Figures 1,2,3). Out of 101 samples of leaves of different plants, about 30 % samples showed negligible
inhibitory activity against trypsin, chymotrypsin and HGP (Table 1). Whereas most of the leaf samples showed low to
moderate inhibitory activities against tested proteinases. Among these, five leaf samples namely Arachis hypogaea,
Vigna sinensis, Dolichos lablab, Phaseolus aureus and Cassia siamea showed higher inhibitory activity against trypsin,
chymotrypsin and HGP on x-ray film by dot-blot assay. Among 25 seed extracts, maximum extracts showed moderate
to higher activity against trypsin, chymotrypsin and HGP (Table 2). All nine flower extracts used in this study showed
negligible activity towards all tested proteinases (Table 3).
Usually seed extracts of plants showed more inhibitory activity than leaf and flower tissues and this may be due to
higher accumulation of proteins in seeds than leaves and flowers (Ambekar et al., 1996; Giri and Kachole, 1998). But
in our study we found reasonable proteinase inhibitory activity in leaves of Arachis hypogaea, Vigna sinensis, Dolichos
lablab, Phaseolus aureus and Cassia siamea by dot-blot assay. Crude seed extracts of these plants also showed higher
inhibition to proteinase by dot-blot assay. These extracts possess activities in the range between 22.91 to 58.33 %
which are considered to have strong inhibitory activity. These seed extracts were subjected for ammonium sulfate
((NH4)2SO4) precipitation. The seed extracts of these plants were fractionated into three fractions (0-30 %, 30- 60 %
and 60-90 %). The percentage of inhibition to HGP obtained in crude and each fraction of ammonium sulfate is
depicted in table 4. A total 58.33 % inhibition is found in crude seed extracts of Dolichos lablab which is highest
among all positive seed extracts. On ammonium sulfate fractionation this activity distributed and highest activity found
in 30-90 % fraction. Low percent of inhibition was found in Phaseolus aureus (22.91 %) among all seed extracts.
Many studies report the PIs in the seeds where they are present for protection of seed protein. Synthesis of PIs in
chickpea begins at 24th day after flowering (DAF) and complete accumulation of PIs observed at 60 th DAF (Giri et
al.1998). Furthermore, studies of the synthesis of PIs and amylase inhibitors during seed development have been
reported in pigeon pea (Ambekar et al., 1996; Giri and Kachole, 1998). But very few reports are available on PIs from
leaves (Plunkett et al., 1982, Satheesh and Murugan, 2011).
Insects lay eggs on leaves, early growing larvae are found to feed on these tissues for its growth and development. On
later larval stage these insects invade seeds of plants resulting loss of yield. Hence control of these insects on leaves
may prove effective strategy. Recently PIs are detected in pigeonpea leaves and are found inducible (Padul et al.,
2012). In this study we also found good PI activity from the leaves of the few plants (Table 1).
Detailed biochemical characterization needed to understand the expressions and induction of PI activity in leaves.
Expression of this activity (from seeds and leaves) in other commercially important crops may prove an effective
strategy to control the attack of insect. Furthermore this inhibition potential may also utilize in medicine to block the
activity of over expressing trypsin and chymotrypsin like proteinases in immunological and other types of diseases.

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Table 1: Dot-Blot assay for PI (CTI, TI and HGPI) activity of leaf sample of different plants. A = 3:1 (Enzyme
: leaf extracts v/v), B = 1:1 (Enzyme : leaf extracts v/v), C = 1:3 (Enzyme : leaf extracts v/v). Plus (+)
sign indicates the presence of PI activity and minus (-) indicates the absence of PI activity. CTI =
Chymotrypsin inhibitor, TI = Trypsin inhibitor and HGPI = Helicoverpa armigera gut proteinases
inhibitor.
Sample
No.

Common Name

Botanical Name

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53

Bhendi
Kupi
Chikku
Adulsa
Bel
Ghaipat
Maharukh
Shirish
Korphad
Dhamasa
Rajgira
Ramphal
Sitaphal
Groundnut
Kadulimb
Kanchan
Aapta (bhaw)
Shendri
Kagdiful
Kobi
Cauliflower
Palas
Tur
Kawali
Karadal
Halad
Karwand
Tarwad
Bahava /Amaltas
Suru
Sadaphuli
Harbhara
Limbu
Idlimbu
Coconut
Aalu
Cilindella
Gavti chaha
Lown
Kala dhatura
Pandhra dhatura
Hulaga
Didhawani
Money plant
Nilgiri
Nivdung
Dudhi
Avudumber
Pimpul
Kala lavi
Suryaphool
Jaswand
Undir kani

Abelmoscus esculantus
Acalipa indica
Acrus sapota
Adathoda vasaca
Aegle marmelos
Agave americana
Ailanthus excelsa
Albizia lebbeck
Aloe vera
Alternanthera sessilis
Amaranthus viridis
Annona reticulata
Annona squamosa
Arachis hypogaea
Azadirachta indica
Bauhinia purpurea
Bauhinia recemosa
Bixa orellena
Bougainvillea spectabilis
Brassica oleracea var. Capitata
Brassica oleracea,
Butea monosperma
Cajanus cajan
Calotropis procera
Canna indica
Carcuma longa
Carissa carandas
Cassia auriculata
Cassia fistula
Casuarina equisetifolia
Catharanthus roseus
Cicer aeriantum
Citrus limon
Citrus aurantium
Coccus nucifera
Colocasia esculenta
Cylindrella
Cymbopogon sp
Cynadon dictylon
Datura metal
Datura strominium
Dolichos lablab
Duranta erecta
Epipremnum aureum
Eucalyptus
Euphorbia antiquorum
Euphorbia laciniata
Ficus glomerular
Ficus religiosa
Gloriosa superba
Helianthus annus
Hibiscus rosa-sinensis
Ipomeia pentaphylla

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CTI
activity
A
B
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-

C
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

TI
activity
A B
+
+
+ +
+
+
+ +
+ +
+ +
+
+
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+
+ +
-

C
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

A
+
+
+
+
+
+
-

HGPI
activity
B C
+
+
+
+
+
+ +
+ +
+ +
+
+ +
+ +
+ +
+
+
+
+
+
+
+
+
+
+
+
+ +
+
+
+
+
+
+
+ +
+ +
+
+ +
+
+
+
+ +
+
+
+

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Table 1. Continued..

Vol. 1 No. 1 (2012)

Sample
No.

Common Name

Botanical Name

54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101

Ratale
Besharam
Mogara
Ghaneri
Mehendi
Subabul
Tomato
Aamba
Chafa
Lajulu
Gulbus
Bartodi
Kadipata
Neche
Tobacco
Ram tulas
Tulsi
Ambushi
Congress
Watana
Vilayati chinch
Nishigand
Ashoka
Karanja
Yedi babul
Badam
Madhumalati
Erandi
Gulab
Chandan
Bibba
Shewari
Bala
Bala
Mirchi
Wangi
Palak
Sporobola
Jamun
Chinch
Ticoma
Morphanki
Gulvel
Dagdi pala
Gehu
Mung
Chavali
Maka

Ipomoea batatas
Ipomoea carnea
Jasminium sambac
Lantana camera
Lawsonia inermis
Leucaena leucocephala
Lycopersicon esculentum
Mangifera indica
Michelia champaca
Mimosa pudica
Mirabilis jalapa
Morinda citrifolia
Murraya koenigii
Nephrolepis sp
Nicotiana tobbacum
Ocimum gratissimum
Ocimum tenuiflorum
Oxalis corniculata
Parthenium hysterophorus
Pisum sativum
Pithecellobium dulce
Polianthes tuberose
Polyalthia longifolia
Pongamia pinnata
Prosopis julifera
Prunus dulcis
Quisqualis indica
Ricinus communis
Rosa indica
Santalum album
Semicarpus anacardium
Sesbania sesban
Sida acuta
Sida cordata
Solanum capsicum
Solanum melongena
Spinacea oleracea
Sporobolus indicus
Syzygium cumini
Tamarindus indica
Tecoma stans
Thuja biota
Tinospora cordifolia
Tridax procumbens
Triticum aestivum
Vigna radiata
Vigna ungiculata
Zea mays

CTI
activity
A
B
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

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C
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

TI
activity
A B
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+
+
+
+
+ +
+ +
+ +
+ +
-

C
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

A
+
+
+
+
+
-

HGPI
activity
B C
+
+ +
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+ +
+ +
+ +
+ +
+
+
+
+
+
+
+ +
+
+
+
+ +
+
+
+
+ +
+ +
+

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Table 2: Dot-Blot assay for PI (CTI, TI and HGPI) activity of seed sample of different plants A = 3:1 (Enzyme
: seed extracts v/v), B = 1:1 (Enzyme : seed extracts v/v), C = 1:3 (Enzyme : seed extracts v/v). Plus (+)
sign indicates the presence of PI activity and minus (-) indicates the absence of PI activity. CTI =
Chymotrypsin inhibitor, TI = Trypsin inhibitor and HGPI = Helicoverpa armigera gut proteinases
inhibitor.
Sample
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25

Common Name

Sadi babul
Rajgira
Groundnut
Bylait
Kadulimb
Tur
Cassia
Harbhara
Hulaga
Kal lavi
Shewaga
Rice
Ghewada
Bajara
Mug
Udid
Watana
Karanj
Erandi
Shewari
Jawar
Gehu
Mataki
Chawali
Maize

Botanical Name

Acacia arabica
Amaranthus viridis
Arachis hypogaea
Argemone mexicana
Azadirachta indica
Cajanus cajan
Cassia siamea
Cicer arietinum
Dolichos lablab
Gloriosa suparba
Moringa oleifera
Oryza sativa
Paracalyx scariosus
Penisehum typhoidesx
Phaseolus aureus
Phaseolus mungo
Pisum sativum
Pongamia pinnata
Ricinus communis
Sesbania sesban
Sorghum vulgarie
Triticum aestivum
Vigna aconitifolia
Vigna sinensis
Zea mays

CTI
activity
A B C
+ + +
- + +
+ + +
- +
- + +
+ + +
+ + +
+ + +
- + +
+ + +
- +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
- + +
- + +
- + +
+ + +
+ + +
- + +

TI
activity
A B C
+ + +
- +
+ + +
- +
- +
+ + +
+ + +
- + +
+ + +
+ + +
+ - + +
+ + +
+ + +
+ + +
- + +
+ + +
- +
- +
+ + +
-

HGPI
activity
A B C
- + +
- + +
+ + +
- + +
+ + +
+ + +
+ + +
+ + +
+ + +
- + +
- +
- +
- +
+ + +
- + +
- + +
- +
- +
- +
- +
- +
- + +
+ + +
- + +

Table 3. Dot-Blot assay for PI (CTI, TI and HGPI) activity of flower extracts of different plants A = 3:1 (Enzyme
: flower extracts v/v), B = 1:1 (Enzyme : flower extracts v/v), C = 1:3 (Enzyme : flower extracts v/v).
Plus (+) sign indicates the presence of PI activity and minus (-) indicates the absence of PI activity.
CTI = Chymotrypsin inhibitor, TI = Trypsin inhibitor and HGPI = Helicoverpa armigera gut
proteinases inhibitor.

Sample
No.

1
2
3
4
5
6
7
8
9

Vol. 1 No. 1 (2012)

Common Name

Botanical Name

Rui
Jaswand
Sadi babul
Gulab
Nishigandha
White kanher
Jui

Calatropis procera
Hibiscus rosasinesis
Acacia arabica
Rosa indica
Polianthes tuberosa
Nerium oleander
Jasminum auriculatum

Congress

Parthenium hysterophorus

Zendu

Togetus erecta

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CTI
activity
A
+
-

B
+
+
-

TI
activity
C
+
+
+
+
+

A
+
-

B
+
-

C
+
+
-

HGPI
activity
A
-

B
-

C
+
+
+

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Table 4: Percent (%) inhibition of Helicoverpa armigera gut proteinases (HGP) in crude seed extracts and
ammonium sulfate fractions (NH4)2SO4 of Arachis hypogea, Vigna sinesis, Dolichos lablab, Phaseolus aureus and
Casia sysemia.
Sr.No.

Seed Sample

Percent ( %)
inhibition
of HGP

Arachis hypogaea

47.91

(NH4)2SO4
fractions
a) 0-30 %
b) 30- 60%
c) 60-90 %

35.13
13.45
07.34

Vigna sinensis

37.50

(NH4)2SO4
fractions
a) 0-30 %
b) 30- 60%
c) 60-90 %

35.13
13.45
07.34

Dolichos lablab

58.33

(NH4)2SO4
fractions
a) 0-30 %
b) 30- 60%
c) 60-90 %

32.14
50.00
21.42

Phaseolus aureus

22.91

(NH4)2SO4
fractions
a) 0-30 %
b) 30- 60%
c) 60-90 %

07.14
15.00
05.42

Cassia siamea

52.08

(NH4)2SO4
fractions
a) 0-30 %
b) 30- 60%
c) 60-90 %

36.66
30.00
06.66

ACKNOWLEDGEMENT
MVP is grateful to BCUD, university of Pune for Financial assistance.
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