Isolation, Purification, and Characterization of Homogenous Novel Bioactive Protein from Datura stramonium Stem Exhibited Larvicidal Activity against Anopheles stephensi

The insecticidal resistance of mosquitoes necessitates the development of a natural, safe, and plant-based method for vector control. Unfortunately, there are no effective vaccines or particular medications available to combat malaria; therefore, mosquitoes must be targeted directly. Previous studies have shown the health benefits of Datura stramonium, but its bioactive peptides or proteins are less explored. This is the first study on D. stramonium stem protein used for mosquito larval protein. The present study aimed to identify the purified mosquito larval protein from the crude extract of D. stramonium stem. Crude protein was isolated, precipitated, dialyzed, and purified by using ion-exchange chromatography, native PAGE, and HPLC. The highest larval mortality was observed at 5.5 mg/ml of crude protein concentration. Native PAGE was used for the analysis and purification of active proteins. The single homogeneous purified larvicidal protein appeared as a single band of 30 kDa by SDS-PAGE. The novel bioactive peptide was characterized by LC-MS/ESI-MS. The homology of the peptide was searched by the Mascot search engine. The database search revealed has not shown peptide similarity with D. stramonium protein, but homology with another plant Arabidopsis thaliana protein is probable for protein phosphatases. The lethal concentration of purified protein against 3rd instar larvae of Anopheles stephensi had LC50 and LC90 values of 25 μg/ml and 40 μg/ml. It has shown new insight into larvicidal activity and can be used as a new drug against malaria and other mosquito-borne diseases.


Introduction
Spiders, mites, crabs, centipedes, millipedes, lobsters, and insects are all members of the Arthropod phylum, which is the largest in the animal kingdom. Mosquitoes belong to the insect family, and they are a well-known vector for a variety of disease-causing infections [1]. WHO has classifed mosquitoes to be the number one public enemy [2]. Chikungunya, dengue fever, malaria, and flariasis are still major health problems in many countries. Mosquito-borne diseases are becoming epidemic diseases due to changing lifestyles and urbanization, resulting in the proliferation of larval habitats [3]. Humans sufer greatly as a result of these diseases. Malaria, leishmaniasis, yellow fever, Chagas disease, Japanese encephalitis, and trypanosomiasis are all vector-borne diseases that kill over 70,000 people each year [4]. Despite signifcant advances in the fght against malaria, an estimated 3.2 billion people, nearly half of the world's population, spread across 91 nations and territories remain at danger. Malaria claimed the lives of 409,000 people and sickened 229 million people in 2019 [5].
D. stramonium is a blooming plant that is tall, annual, and branched and belongs to the Solanaceae family. Datura is found in ten species, however, only two of them, Datura innoxia and D. stramonium, have known drug-like efects [6]. Te plant has a role as insect repellent, antioxidant, antimicrobial, anticancer, and anti-infammatory agents, and larvicidal and mosquito repellent and has anticholinergic activity [7]. Te plant contains alkaloids, steroids, glycosides, tannin, favonoids, saponin, atropine, phenol, protein, carbohydrates, and fat, according to the phytochemical analysis [8]. Datura stramonium ethanolic leaf extract possesses larvicidal and repellent actions against Anopheles stephensi and Culex quinquefasciatus [9]. Plants have been employed for the treatment of human diseases throughout the world since ancient times. Synthetic pesticides have the potential to afect water, soil, and the environment. Botanical pesticides are safe, less toxic, efective, afordable, and environmentally friendly [10]. Mosquitoes are efectively controlled by proteins derived from several plants [11][12][13]. Te various poisonous proteins (lectin, ricin, RIPs, alpha amylase inhibitors, PIs) are found in plants and have insecticidal activities against various insects [14]. A brief literature study indicated that there are not many studies regarding the isolated, characterization and purifcation of antilarvae proteins from plants so that the D. stramonium stem protein has to be characterized by its specifc larvicidal activity.
Te present study has been carried out to analyse the larvicidal potential of the stem of D. stramonium protein.
Te crude protein was isolated from the stem of D. stramonium by the TBS bufer and ammonium sulphate precipitation. Te protein extract was subjected to a mosquito larvicidal bioassay according to WHO guidelines. Te crude protein was subjected to DEAE-cellulose column chromatography and native PAGE for purifcation. HPLC and SDS-PAGE were also performed to check the purity of the extract. Te native PAGE purifed protein was trypsin digested and identifed by the LC/MS. Te peptides obtained through the LC/MS were searched and identifed with the help of the Mascot search engine.

Mosquito
Rearing. Larvae of An. stephensi were procured from NIMR, New Delhi. Te culture of the mosquito was maintained at (26 ± 2)°C with a photoperiod of 12 : 12 h (light:dark) in the insectory of the Center for Biotechnology, M.D.U., Rohtak, and the larvae were fed with dog food. Te pupae were transferred in a small plastic bowl and kept in a mosquito cage for adult emergence. Te cotton pads were soaked in 10% aqueous glucose solution and kept in cages for mosquito feeding. Te rabbit was put in the cage for mosquito blood-feeding. For mosquito egg laying, a plastic bowel containing flter paper on the boundaries immersed with water was kept in the cage.

Protein Extraction from Stem.
Te collected plant part was frst washed with tap water and then with distilled water and kept for shaded dry at room temperature. Te sample was powdered with the help of an electric grinder and liquid nitrogen. Te total protein was extracted by the use of extraction bufer Tris-bufer saline (50 mM Tris-HCl (pH, 7.5), 150 mM NaCl, and polyvinyl pyrrolidine) with a slight modifcation in the ratio of the extraction bufer. Total protein was isolated in an extraction bufer in the ratio of 1 : 7 (w/v). Te sample was fltered with 2 ̶ 3 layers of muslin cloth and the fltrate sample was kept on a magnetic stirer at 4°C overnight. Te sample was centrifuged at 13,000 rpm for 30 minutes at 4°C. Te supernatant was collected and the pellet was discarded.

Protein Precipitation.
Te crude protein extract was obtained through the Tris-bufer saline and precipitated by diferent saturation percentages initially 20%, 30-40%, 50-60% ,70-80%, and 90-100% of ammonium sulphate. Te 80% saturated ammonium sulphate solution has given a good quality pellet after centrifugation at 13,000 for 30 min at 4°C. Te supernatant was precipitated with ammonium sulphate by 80% saturation, and the sample was kept at −20°C overnight to completely precipitate the protein. Te next morning sample was centrifuged at 14,000 rpm for 30 minutes at 4°C, the supernatant was discarded, and the pellet was washed 5 ̶ 6 times with acetone and the sample was dried. Te protein sample was dialyzed against distilled water for 24 h at 4°C. Te protein sample was kept at −20°C for further bioassay and purifcation.

Protein Quantifcation.
Te total protein concentration of the isolated sample was calculated by the standard Bradford method by UV spectrophotometer at 595 wavelength [15]. BSA was taken as a standard stock solution of 1 mg/ml.

Larvicidal Bioassay.
Larvicidal bioassay was conducted using the third instar larvae of An. stephensi according to the guidelines of the WHO [16]. Ten larvae of the 3 rd instar stage were placed in a plastic bowl containing water (99 ml) and test solution (1 ml), with a fnal volume of 100 ml. Six different concentrations of crude protein extract (0.172, 0.34, 0.68, 1.37, 2.75, and 5.5 mg/ml) and purifed protein (10,20,30,40,50, and 60 μg/ml) were prepared from the stock solution of protein extract with distilled water and used for the bioassay. Larvae mortality was monitored after 24, 48, and 72 hours. A control was set up with Tris-bufer saline. Te experiment was conducted in triplicate.

DEAE-Cellulose Column Chromatographic Separation.
Te protein was purifed by IEC (ion exchange chromatography). Te dialyzed protein sample showing relatively higher larvicidal activity was subjected to purifcation using ion-exchange chromatography by passing the protein through the DEAE-cellulose column. Lyophilized protein samples were dissolved in Tris-HCl (50 mM; pH 7.5) and loaded onto a DEAE-cellulose column at the fow rate of 0.5 ml/min. Te unbound proteins were eluted with Tris-HCl (50 mM; pH 7.5), and the bound proteins were desorbed with the same bufer with a gradient of NaCl (0.0-0.5 M). Te unbound and bound proteins were collected in sterile tubes. A diferent number of fractions were collected from all samples, and they were tested for larvicidal activity using the WHO protocol bioassay. Tose fractions that showed larvicidal activity were pooled together and dialyzed against a Tris-HCl bufer. Te dialyzed samples were lyophilized and stored at −20°C and further analyzed for the larvicidal test.
2.9. Preparative Native PAGE. Te protein fractions obtained from DEAE-cellulose column chromatography having the highest larvicidal activity were analyzed by preparative native PAGE [17]. Electrophoresis was carried out on Bio-Rad gel plates by using 5% stacking gel and 12% resolving gel. Te test samples were solubilized in sample bufer and loaded to the well of gel, and electric current of 50 V was frstly applied and then a supply of 150 V. When the dye reached the bottom, power supply was switched of, and the gel was put in Coomassie Brilliant Blue G-250 staining solution at 4°C overnight with shaking. In the morning, the gel was destained and we visualized the bands. Te standard protein marker was used to determine the molecular weight of the test samples. Te protein band of a small portion was cut out from the gel with the help of a sterile blade and kept in a destaining solution for complete removal of the dye. Te protein was eluted from the gel by grinding in a chilled mortar and pestle, and the gel slurry was tied to a dialysis membrane. Te dialysis membrane was immersed in a native PAGE bufer and run for 1 h. Te sample was collected from the membrane and centrifuged at 12,500 rpm for 30 min at 4°C. Te protein was present in the supernatant and used for purity and larvicidal bioassay.

HPLC Analysis.
Te homogeneity of the purifed protein was analyzed by using Agilent 1100 High Performance Liquid Chromatography (HPLC) (Agilent Technologies, USA) on a C18 column (USCFX03064 EC-C18, 2.7 μm, 3.0 × 100 mm, USA) as described by Schwarz [18]. Te 60 μg of the sample was prepared after mixing with its DDT, methanol, and MS grade water. Te sample was incubated for 30 minutes in the dark and loaded onto the HPLC. Te peak was observed at the retention time of min exactly coincided with that of the Tris-HCl bufer in which the protein was originally dissolved. Te chromatogram of the sample along with the blank was used for analysis.

Molecular Mass Determination by SDS-PAGE.
Te purifed protein was electrophoresed on SDS-PAGE gel along with a protein standard molecular weight marker (Bio-Rad, USA). Te molecular weight of the purifed protein was determined by staining with Coomassie Brilliant Blue and comparing the band along with a standard protein marker. 2.13. Protein Identifcation. By MASCOT search engine (Matrix Science), peptide match and identifcation of peptide were performed. MS and MS/MS data were submitted to the MASCOT search program (https://www.matrixscience. com/). Te search was also performed using the Swiss-Prot and NCBI database, restricted to Viridiplantae (green plants). Te search criteria were established considering carboxymethyl (C) modifcations as fxed efects and the alteration of the oxidation of the methionines as a variable efect. In trypsin hydrolysis, the possible loss of a cleavage site was considered, and the tolerance of the peptide and fragment masses was ±0.3 Da.
2.14. Statistical Analysis. Te data were subjected to probit analysis to calculate the LC 50 , LC 90 , 95% confdence limit, and R-square value. All the experiments were carried out in triplicate. Microsoft Excel version 2007 software was used for the statistical analysis. Te mortality rate was corrected with the help of Abbott's correction formula [19].  Table 1. But a higher percentage of yield was obtained in the extraction with Tris-bufer saline. Ten, the protein was isolated with the use of Tris-bufer saline extraction bufer from the stem of D. stramonium.

Screening of Larvicidal Protein from the Extract of Plant
Stem. Protein extracted from the stem of D. stramonium was screened for mosquito larvicidal activity using the WHO protocol against 3 rd instar larvae of An. stephensi. Te frst step of purifcation was done by precipitation with ammonium sulphate, and protein fractionated with 70-80% ammonium sulphate was taken for testing the larvicidal activity. Te partially purifed ammonium sulphate extract and dialyzed protein showed 80-90% mortality at the concentration of 5.5 mg/ml after 48 h. Table 2 shows mortality percentage at diferent concentrations after serial dilution from 0.172 to 5.5 mg/ml of crude protein extract and purifed protein at diferent concentrations (10 to 60) μg/ml. Larvae mortality at diferent concentrations of protein are shown graphically in Figures 1 and 2. Te LC 50 and LC 90 values of plant proteins for the third instar larvae of An. stephensi after 48 h of exposure are shown in Table 3. Te rate of mortality is directly proportional to increased concentration to dose. A correlation exists between the saturation of precipitation and the molecular mass of the proteins. Lesser saturation precipitates a higher molecular mass of protein and higher saturation precipitates the low molecular mass proteins. In the present study, higher larvicidal activity was observed at a crude protein extract concentration of 5.5 mg/ml. Te mortality rate was corrected with the help of Abbott's correction formula. Te LC 50 and LC 90 values of plant proteins for the third instar larvae of An. stephensi after 72 h of exposure are shown in Table 3.

Purifcation of Protein by DEAE-Cellulose Column
Chromatography. Te crude protein extract showing the highest mortality was used for further purifcation on DEAE-cellulose ion (anion) exchanger chromatography. A total of 35 fractions were collected of 1 ml sample each as shown in Figure 3, and each fraction was tested for larvicidal bioassay using the WHO protocol. Te percentage larval mortality of 20%, 10%, 25%, 10%, 30%, and 25% was observed in diferent eluted fraction numbers 6-11. Te active fractions 6-11 were pooled together and dialyzed against TBS (pH 7.5) and lyophilized. Te percentage of protein yield of chromatography samples was obtained at 4.2% as shown in Table 4. Te pooled active protein fraction A was showing 90% larvicidal potential at a concentration of 60 μg/ ml as shown in Figure 2. Te protein fraction A has a single   Tris-bufer saline 4800 96     Journal of Tropical Medicine peak as shown in Figure 3, which clearly reveals that the stem of D. stramonium has only one larvicidal protein. Further purifcation was performed to confrm the presence of homogenous protein from eluted fractions.

Purifcation of Larvicidal Protein by Preparative Native
Page. Te native page was done to analyse the presence of crude protein and purifed protein present in the stem of D. stramonium. Te single protein band was observed from the native page gel, as shown in Figure 4. Tis purifed single protein has larvicidal activity, and this band was eluted from the gel by the electrodialysis method. Te total percentage of yield is 2.2% obtained by preparative native PAGE, as shown in Table 4. Te purifed protein was used for the larvicidal bioassay against the larvae An. stephensi and further for molecular characterization. Te molecular mass of the purifed protein was calculated by extrapolating the mobility value (Rf value) of the purifed protein with the relative mobility values of the standard molecular mass protein ( Figure 4).

HPLC Analysis.
HPLC is a technique used to separate a single compound from a mixture of components based on peak analysis. Te homogeneity of the purifed protein was analyzed using HPLC and peak purity analysis, whereas the single peak observed belonged to the purifed protein with a high purity index. It showed only a single peak, confrming that the protein is 100% pure and without any impurity. Te chromatogram showed two peaks with one peak at a retention time of min representing the presence of purifed protein and another peak at a retention time of min representing the Tris-bufer saline in which the protein was dissolved. Te chromatogram clearly indicates the presence of a single peak that confrmed the protein is 100% pure ( Figure 5).

Mass Determination by SDS-PAGE.
SDS-PAGE reveals that a single homogenous band was appeared on the gel. Te molecular mass of the purifed protein was determined as 30 kDa (Figure 6).

LC-MS/MS Analysis.
Te purifed protein isolated from D. stramonium was digested with trypsin, and the peptides obtained were subjected to LC-MS/ESI-MS analysis. Te mass spectra (Figure 7) was searched by matching with the MASCOT search engine, and the peptide was identifed as shown in the Table 5. Te purifed peptide does not show similarity with the D. stramonium protein. Te purifed peptide (R.LVAKAAAR.A) showed similarity with a phosphatase protein of Arabidopsis thaliana as shown in Table 5. Te protein NCBI blast also showed the maximum similarity with the phosphatase protein of Arabidopsis thaliana. Te complete genome sequence of D. stramonium with annotated information is not available till now. Tis could be as certain as the reason for their lack of similarity.

Discussion
Te search for larvae protein from botanical sources is a new approach to combat the vector control and the formulation of protein-based drugs. Plant proteins have been reported in many biological activities, including antibacterial, antilarvicidal, and antiviral properties [20][21][22]. Te mull protein was purifed from Myracrodruon urundeuva leaf, and it exhibited LC 50 value of 0.202 mg/ml against Aedes aegypti larvae [23]. Te type IInd RIP from the camphor seed (Cinnamomum camphora) has LC 50 value 168 ppm against the larvae of Culex pipines pallens [24]. Te biological efect of ApTI protein on Aedes aegypti larvae was decrease in survival rate after 96 h of treatment, from 93.08 ± 5.01% to 69.22 ± 10.88%, respectively. For 1 mg/ml ApTI, 100% mortality was observed, while the mortality of the control group reached a maximum of 10% [25]. One approach to reduce the mosquito population involves interrupting the mosquito's life cycle at the larval stage [26]. Synthetic larvicides in comparison to the natural larvicides are harmful to aquatic organisms and the environment due to their hydrophobic nature. Anopheles stephensi has been reported susceptible to temephos in India with LC 50 range of 0.008-0.015 ppm [27][28][29]. During the last three decades, temephos, an organophosphate compound, has been considered as a safe larvicide (LC 50 = 8600 mg/l) in vector control programs [30]. Tere are reports of resistance to some insecticides such as DDT, diledrin, and malathion in An. stephensi, as well as some indications of resistance to pyrethroids in the current years [31,32]. Another study on temephos reveals the LC 50 value of 0.0523 ppm and LC 90 value of 0.3822 ppm for the An. stephensi [33].   (minimum inhibitory concentration) of this purifed protein was 13 μg/ml and 15 μg/ml against both Gram-positive and Gram-negative bacteria [37]. Te protein purifed and identifed from the native Amazonian species exhibited antifungal activity [38]. In the present study, proteins have been isolated, purifed, and characterized from the stem of D. stramonium that exhibited larvicidal activity against An. stephensi. Te purifed protein showed 90% mortality at 40 μg/ml. A single band of 30 kDa was shown by SDS-PAGE gel. HPLC single peak result revealed that the protein was in a homogenous stage. Te purifed protein was subjected for trypsin digestion and identifed by LC-MS/MS. Te protein identity and homology were checked by the Mascot search engine. Te purifed protein does not show homology with the D. stramonium protein. Te homology of protein was matched with the protein phosphatase of plant Arabidopsis thaliana. Hence, the novel peptide was identifed in the stem of D. stramonium. Te LC 50 and LC 90 values of the purifed protein were 0.025 and 0.04 mg/ml. Plant protein phosphatases have catalytic activity by dephosphorylation of the phosphoprotein [39]. Phosphate groups are important in activating proteins so that the proteins can perform particular functions in cells. Plant protein phosphatases have a role in stress signalling and defence mechanism against pathogens, but the complete study of their mechanism is still unknown [40]. Larvicidal activity of isolated proteins could be because of dephosphorylation of mosquito enzymes responsible for metabolic activities, immunity, survival, etc.

Conclusion
In the previous study, D. stramonium plant extract had mosquito, larvicidal, and repellence activity against An. stephensi, Aedes aegypti, and Culex quinquefasciatus at low concentration of lethal dose. Tis is the frst report on the study of D. stramonium protein used against larvae of An. stephensi. Te present study showed that the purifed protein phosphatase of D. stramonium is able to kill the mosquito larvae of An. stephensi. Te mechanism of activity of the phosphatase protein may involve the dephosphorylation of mosquito enzymes that kill the larvae. Te purifed protein has a 90% mortality at a low dose of LC 50 25 μg/ml and LC 90 40 μg/ml. Te present study identifed a novel larvicidal protein of 30 kDa from D. stramonium stem. Tis protein can be used in the formulation of safe, natural, plant-based mosquitocides for the control of

Data Availability
Te data will be available on valid request to the corresponding author.

Conflicts of Interest
Te authors declare that they have no conficts of interest in the article.

Authors' Contributions
MK collected the data and wrote the original draft, and NS and SPS reviewed and edited the manuscript.