In vitro screening of peptidase inhibitory activity in some plants of North India

In the present study, trypsin and chymotrypsin inhibitory activity of some plants of different families was evaluated. A total of 55 plants were screened, out of which six showed the maximum trypsin inhibitory activity namely Acacia concinna, Caesalpinia bonducella, Lathyrus sativus, Mucuna pruriens, Psoralea corylifolia and Sapindus mukorossi. Results suggested that the plants showing trypsin inhibitory activity (TIA) also have chymotrypsin inhibitory activity (CIA). Both trypsin and chymotrypsin inhibitory activities were high in seeds compared to leaves followed by flowers. It was also observed that TIA was maximally present in Sapindaceae family whereas CIA was maximum in fabaceae family followed by others.


Introduction
The Peptidase inhibitors (PIs) are regulatory proteins that occur in seeds, leaves, flowers and tubers (Mayasa et al., 2016). On the basis of active amino acid present in their reactive site, these are distributed in different families like serine, cysteine, aspartic and metallocarboxy peptidase inhibitors (Pesoti et al., 2015). Generally plant serine PIs are grouped into Kunitz, Kazal, Streptomyces subtilisin inhibitor, Soybean trypsin and proteinase inhibitor, Potato I inhibitor, Potato II inhibitor, Ascaris trypsin inhibitor and other (Laskowski and Kato, 1980). PIs are specific in nature as they inhibit only peptidases leaving other proteins unaffected. Studies have shown that plant peptidase inhibitors are more beneficial over chemical PIs as they are safer and more specific in their action (Shamsi et al., 2016).
PIs serve various functions viz. signal initiation mediator, cellular event's transmission and termination processes like apoptosis, inflammatory response, blood coagulation, hormone response pathwayetc (Gomes et al., 2011). They regulate the development of insects, agricultural pests, plant and animal health (Shamsi et al., 2016). PIs accumulate in host plants in response to invading pathogens and thus have a defensive role to play in plants (Salzman et al., 2005). They affect the growth and development of insects which feed on various crops. Various peptidase inhibitors have been purified from plants. In the present study the trypsin and chymotrypsin inhibitory activity of plants belonging to different families of North India was screened. The knowledge gained from this study can be explored further for identifying the plants with anti-insect potential, isolation and characterization of peptidase inhibitors peptidase .

Collection of plant material
Leaves, seeds and flowers of the plants were taken according to their availability from the different states of North India. The plant material was identified from the Department of Botanical and Environmental Sciences, G.N.D.U., Amritsar and Herbal Health Research Consortium Pvt. Ltd., Amritsar, Punjab, India.

Preparation of extract
Collected plant material (seeds, leaves, flowers) was washed with distilled water and then treated with mercuric chloride to remove any bacterial or fungal contamination. The treated plant parts were then washed with distilled water, air dried and crushed in liquid nitrogen until finely grounded. The fine powder was dissolved in distilled water (1:5 w/ v) and stirred for 4 h on magnetic stirrer at 4 C. The suspension was then centrifuged at 8000 rpm for 30 min at 4 C and supernatant was collected. The supernatant was termed as Crude Extract (CE) and stored at -20 C till further use.

Trypsin and chymotrypsin inhibitory assay
Trypsin inhibitory activity was measured by following the protocol of Vasudev and Sohal (2016). Trypsin and chymotrypsin inhibitory activity was measured using BApNA (N-benzoyl DL-arginine p-nitroanilide) (prepared in 0.1mM DMSO) and casein (1%) respectively, as substrate. Twenty microlitres of enzyme (trypsin/chymotrypsin) was pre-incubated with 50μl of buffer (0.05M Tris HCl, pH -8.2) and 30μl of inhibitor at 37 C for 10 min. Then 100μl of substrate was added and the absorbance change was continuously monitored for 10 min at 410 nm at an interval of 1 min. The calibration curve was constructed using different concentrations of p-nitrophenol for expressing trypsin and chymotrypsin activity as μmol/min/mg protein. Formula used for calculation of inhibitory activity is given as:

Statistical analysis
All results were reported as means with the standard deviation. For each set of results, Analysis of variance (ANOVA) was applied using SPSS software followed by the Tukey's test.

Results
The presence or absence of trypsin and chymotrypsin inhibitory activity in the extract of 55 different plants is listed in Table 1.

Bignoneaceae family
In Bignoneaceae family five plants were evaluated for trypsin inhibitory activity (TIA) and chymotrypsin inhibitory activity (CIA), out of which leaves of Kigelia pinnata and seeds of Tecoma argentina and Tecoma stans gave~10% TIA and CIA. However, seeds of Tecoma grandiflora gave 7.92 AE 0.95% and 1.02 AE 1.69% and leaves of T. stans showed 6.24 AE 0.05% and 3.62 AE 0.55% of TIA and CIA, respectively (Table S1).

Combretaceae family
In Combretaceae family, Terminalia arjuna and Terminalia chebula showed neither trypsin nor chymotrypsin inhibitory activity.

Euphorbiaceae family
In Euphorbiaceae family, 32.16 AE 2.58% of TIA and 26.63 AE 1.04% of CIA was observed in the seeds of Phyllanthus embilica, whereas seeds of Ricinus communis showed 16.7 AE 0.58% of TIA and 10.26 AE 1.02% of CIA. None of the inhibitory activities were present in the leaves of Jatropha curcas.

Fabaceae family
Out of 31 plants of Fabaceae family, leaves of 19 and seeds of 18 plants were screened for their inhibitory activity. The maximum inhibitory activity was observed in leaves of the plants namely; Bauhinia variegata (

Laminiaceae family
In Laminiaceae family, Vitex negundo seeds did not show any activity.

Phytolaccaceae and Rhamnaceae family
The seeds of Rivina humilis from Phytolaccaceae and Ziziphus jujube from Rhamnaceae family showed neither trypsin nor chymotrypsin inhibitory activity.

Rutaceae family
The seeds and leaves of Aegle marmelos and leaves of Citrus medica, Murraya exotica, Murraya koengi from Rutaceae family showed neither trypsin nor chymotrypsin inhibitory activity.   3.11. Comparison of peptidase inhibitory activity in leaves, flowers, seeds and in different families Different parts of the plant, on comparison showed that seeds had the significant highest trypsin (F 6, 35 ¼ 121.35; P < 0.01) and chymotrypsin (F 6, 35 ¼ 198.86; P < 0.01) inhibitory activity followed by leaves and flowers (Figure 1). Among families, plants from Sapindaceae showed the maximum trypsin inhibitory activity followed by Putranjivaceae, Fabaceae, Euphorbiaceae, Rutaceae, Bignoniaceae, Sapotaceae and Malvaceae. Whereas, plants of Putranjivaceae showed the maximum chymotrypsin inhibitory activity followed by Sapindaceae, Fabaceae, Euphorbiaceae, Rutaceae, Bignoniaceae, Malvaceae and Sapotaceae. Plants belonging to Combretaceae, Lamineaceae, Phytolaccaceae, Rhamnaceae and Theaceae showed none of the activities (Figure 2). Also the plants which showed the TIA also possessed CIA.

Discussion
Over the past few decades, there has been increasing interest in identifying, purifying and characterizing novel PIs. They are leading candidates with various applications across medicinal biotechnology and agriculture. Plants are excellent sources of PIs which help them to combat various diseases, insects, pests and herbivores (Ryan, 1990). TIA and CIA of various PIs are required for the control of various insect pests which act by the inhibition of their midgut peptidases. The PIs are present in different organisms viz. microorganisms, plants and animals. These are present in seeds, leaves and flowers of plants which inhibit the digestive enzymes of the insects/pests (Fan and Wu, 2005). A number of plant seeds of leguminaceae family have been identified for their peptidase inhibitor activity (Tamir et al., 1996). In this study 55 plants were studied which belonged to 13 different families viz. Bignoniaceae, Combreteaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Malvaceae, Putranjivaceae, Phytolaccaceae, Rhamnaceae, Rutaceae, Sapoteaceae, Sapindaceae and Theaceae.
Maximum trypsin and chymotrypsin inhibitory activity was reported from the seeds of S. mukorossi which belongs to sapindaceae family. Gandreddi et al. (2015) isolated and purified trypsin inhibitor from soap nut (Sapindus trifoliatus L. Var. Emarginatus) seeds and evaluated its role against larval gut peptidases of Helicoverpa armigera and Spodoptera frugiperda.
Maximum number of plants was screened from leguminaceae family, in which maximum peptidase inhibitory activity was observed from the seeds of M. pruriens followed by A. concinna, P. corylifolia, L. sativus and C. bonducella. There are previous reports on purification of trypsin inhibitors from the seeds of M. pruriens, P. corylifolia L. sativus and Trigonella foenum graecum respectively, which is in support of our results (Borde et al., 2012;Yang et al., 2006;Ramakrishna et al., 2010 andOddepally et al., 2013). Zhou et al. (2020) studied the X-ray structure of trypsin inhibitor purified from Cassia obtusifolia and gave inhibitory activity comparable with that of soybean trypsin inhibitor against midgut trypsin from Pieris rapae. Ferreira et al. (2019) purified two recombinant PIs namely, cruzipain inhibitor (rBbCI) and kallikrein inhibitor (rBbKI) from Bauhinia bauhinioides and analyzed its insecticidal activity against soldiers and workers of Nasutitermes. Ahmad et al. (2020) purified a trypsin inhibitor from Arachis hypogaea and evaluated its anticarcinogenic effect.
There are reports on the isolation and purification of trypsin inhibitors from several other families. Patriota et al. (2016) purified trypsin inhibitor from bignoniaceae family (Tecoma stans) which had an inhibitory effect on growth and promotes ATP depletion and lipid peroxidation in Candida albicans and Candida krusei. Shahid et al. (2008) purified a novel protein from Croton tiglium belonging to Euphorbiaceae family with antifungal and antibacterial activities. Similarly, Lone et al. (2017) and  Soomro et al. (2007) also isolated and characterized peptidase inhibitor from the seeds of a plant Ricinus communis of Euphorbiaceae family. Chaudhary et al. (2008) had purified and characterized trypsin inhibitor from the seeds of Putranjiva roxburghii of Putranjivaceae family by acid precipitation, cation-exchange and anion-exchange chromatography. Trypsin inhibitor was previously isolated by Shee and Sharma (2007) from the seeds of Murraya koenigii which belongs to rutaceae family. Rathinavelusamy et al. (2014) previously reported a protein with α-amylase inhibition potential and antidiabetic activity from the bark of Pterospermum acerifolium belonging to malvaceae family. Bijina et al. (2011) observed that the crude extract of leaves of Moringa oleifera showed maximum percent of inhibition (77%) followed by the seed extract (63%). Norioka et al. (1988) reported that the seeds of the leguminous species contained mainly the Kunitz family inhibitors and those of the more advanced ones had the Bowman-Birk family inhibitors. It was also reported by the same group that the Kunitz family inhibitors in leguminous seed have gradually been replaced by the Bowman-Birk family inhibitors in the process of evolution. Ryan (1990) had reported that PIs have high inhibitory activity against phytophagous insects as they possess alkaline guts where serine peptidases are dominantly required for digestion of food and therefore this can be effectively used as defense tool because of anti nutritional interaction.

Conclusion
The aim of this study was to screen Indian plants for the presnce of trypsin and chymotrypsin inhibitory activity where, A. concinna, C. bonducella, L. sativus, M. pruriens, P. corylifolia and S. mukorossi exhibited maximum PI activity. The present study has also revealed that seeds had maximum trypsin and chymotrypsin inhibitory activity as compared to leaves and flowers. Work is further being undertaken to explore the plants exhibiting maximum TIA and CIA for purification and evaluation of insecticidal activity.

Author contribution statement
Samiksha: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the manuscript. Satwinder Kaur Sohal: Conceived and designed the experiments; Analyzed and interpreted the data.