BIOACTIVE POTENTIALS OF PETUNIA INTEGRIFOLIA (HOOK.) SCHINZ & THELL. AGAINST CULEX QUINQUEFASCIATUS LARVAE, ARTEMIA SALINA NAUPLII AND POECILIA RETICULATA ADULTS

Petroleum ether (pet. ether), chloroform (CHCl 3 ) and methanol (CH 3 OH) extracts of Petunia integrifolia (Hook.) Schinz & Thell. Whole plant was subjected to assess cytotoxicity against the brine shrimp Artemia salina L . nauplii , larvicidal activity against the mosquito Culex quinquefasciatus Say larvae and piscicidal activity against the Poecilia reticulata Peters adults. The P. integrifolia extract in pet. ether showed the highest cytotoxic activity against A. salina nauplii by giving the minimum LC 50 value of 49.25 parts per million (ppm) at 48 h, while in case of CHCl 3 and CH 3 OH extracts, the values were 63.37 and 342.55 ppm, respectively. Against C. quinquefasciatus larvae the three extracts gave LC 50 values of 4.92, 5.51 and171.26 ppm, respectively all after 48 h of exposure. Against P. reticulata the LC 50 values were 21.70 ppm for pet. ether extract at 48 h, and 31.91 ppm for CHCl 3 extract after 42 h of exposure but CH 3 OH extract offered no mortality. According to the intensity of activity, therefore, the extracts could be arranged in the descending order of: pet. ether > CHCl 3 > CH 3 OH extracts; and the sensitivity of the test organisms to the extract of P. integrifolia could be arranged in the following descending order: C. quinquefasciatus larvae (in Pet. ether; LC 50 = 4.92 ppm) > P. reticulata (in pet ether; LC 50 = 21.70 ppm) > A. salina nauplii (in pet. ether; LC 50 = 49.25 ppm).


Introduction
Plants produce a wide variety of bioactive chemicals, making them a valuable source of various medications.Medicinal plants have been discovered to be effective in treating and managing a variety of health issues over time.Natural products are used to produce over half of all current clinical medications (Stuffness and Douros 1982), and they play an essential part in the pharmaceutical industry's drug development programs (Baker et al. 1995).Synthetic insecticides have recently become the most widely used agrochemicals, and they have taken a significant market share.However, plant-based pesticides have been used as a potential toxic source in agriculture for over 150 years (Oberemok et al. 2015).The plant species Petunia integrifolia (Hook.)Schinz and Thell have been found with some remarkable bioactive properties such as medicinal, traditional and pharmaceutical potentials (Rahman et al. 2008).The genus Petunia has been reported as an antibacterial agent against Streptococcus sp., Pseudomonas sp., and Salmonella sp.(Kumar 2015).Floral  Author for correspondence: n_islamm@yahoo.comdefensins of Petunia spp.inhibit the growth of the Fusarium oxysporum fungal strain (Lay et al. 2003).This plant can also be used as a mild anti-microbial and anti-oxidant medication (Rahman et al. 2008).Several steroidal chemicals such as petuniasterones found in Petunia species contribute to the plant's resistance to insect attack (Elliger and Waiss 1989) and directly repel aphids, tomato hornworm, asparagus beetles and leafhoppers in the crop field (Hummel et al. 2015).This herb is said to have been used as a hallucinogen in highland Ecuador that causes a sensation of flying, and a phenomenon sometimes attributed to the hallucinogenic tropane alkaloids, such as hyoscine (Butler et al. 1981, Schultes andHofmann 1980).Even though P. integrifolia was described for its biological activities, however, detailed information of biological activities of the plant is still scanty.That is why this plant species has been subjected to evaluate the cytotoxicity, larvicidal and piscicidal potentials against three model organisms viz., A. salina nauplii, C. quinquefasciatus larvae and P. reticulata adults.The brine shrimp lethality bioassay is an innovation in the bioassay for phytochemicals demonstrating cytotoxic effects and a wide range of therapeutic potentials e.g., anticancer, antiviral, pesticidal and anti-AIDS activity (Wu 2014).The test on brine shrimp nauplii was adopted as a viable instrument for assessing innovative pharmacological natural compounds (Hosain et al. 2011).The mosquito species C. quinquefasciatus is a vector for pathogens that cause fatal diseases in humans and animals, such as Japanese encephalitis and lymphatic filariasis (Benelli and Beier 2017).The guppy, P. reticulata, commonly known as million fish or rainbow fish, is a widely distributed tropical and popular freshwater aquarium fish.Guppies in the wild eat a wide range of foods, including benthic algae and mosquito larvae that help limit the spread of malaria.In evolutionary ecology, and behavioral research, P. reticulata is utilized as a model organism (Magurran 2005).However, recently the fish species has been reported to have some detrimental influences on local fish populations (El-Sabaawi et al. 2016, Vallie 2022).

Collection and preparation of plant materials
The whole plant P. integrifolia was collected from the Rajshahi University Campus.The plant materials were cut into small pieces and spread out to dry under shade at room temperature in separate wooden trays.Welldried materials were ground to powder with a blender, weighed, kept in conical flasks and then extracted with sufficient amounts of three solvents namely, petroleum ether (PE), chloroform (CF) and methanol (ME) at 50 g × 150 ml × 2 times for 48 h.Filtrations were done by Whatman filter paper Grade-40, and after evaporation, extracts left on the bottom of the round bottom flasks were collected in glass vials and kept in a cool place at 4ºC with appropriate labelling until used.

Hatching of brine shrimp cysts
Brine shrimp cysts were purchased from the Pet and Aquarium Market of Nilkhet, Dhaka, Bangladesh.They were hatched in an aquarium using 1 liter of 1 M NaCl brine solution (pH 8.5), incubated for 48 h under fluorescent light, and the nauplii were hatched within 24-35h at 25-30°C.After hatching, the nauplii were transferred to test tubes.Ten nauplii were transferred to each tube containing a required amount of water and 2 ml NaCl solution was added to each test tube to fill up to the previously marked line.

Hatching of mosquito larvae
C. quinquefasciatus rafts were collected from the drains of Rajshahi University Campus and reared under laboratory conditions.The eggs were kept in 500 ml beakers with 250 ml pond water for 24 h in a dark place in the laboratory, and the larvae were hatched within 16-20 h at room temperature.After hatching, the larvae were transferred to test tubes.Ten larvae were transferred to each tube containing a required amount of water.

Rearing of guppy fish
Adults of P. reticulata were collected from an aquarium shop in Rajshahi City.The collected fishes were then transferred to the laboratory, kept in an aquarium, and reared with the continuous oxygen supply through an aerator.Five fishes were transferred to 500 ml beaker for experimentation.

Preparation of test sample and cytotoxicity test
Test samples at different concentrations were considered as doses prepared in test tubes by adding a calculated amount of DMSO (dimethylsulfoxide).Then water was added to fill the pre-marked (up to 10 ml) test tubes with the help of a pipette.The nauplii were counted by visual inspection and were released in test tubes containing 10 ml of water, and the test tubes were kept at room temperature along with a control batch.Observation of mortality was made after 6, 12, 18, 24, 30, 36, 42 and 48 h of exposure.
Plant extract solutions at different concentrations (for all three extracts of P. integrifolia in pet.ether 300,250,200,150,100 and 50 ppm;in CHCl3 500,400,300,200, 100 and 50 ppm and CH3OH were 550, 500, 400 and 300 ppm in artificial seawater containing 1% DMSO (v/v) used for this assay.Ten nauplii were used in each of the test tubes and three replicates were used for each of the concentrations.Later on, 1 ml of NaCl solution was added to each test tube.After 6, 12, 18, 24, 30, 36, 42 and 48 h incubation, the number of survivors and the dead nauplii were counted using a simple microscope and the percentage of the mortality for each of the doses was calculated as compared with the control.DMSO served as the negative control and the final concentration of DMSO in the assay volume was always kept below 1% to prevent possible false effects originating from DMSO toxicity.LC50 value is the concentration of the sample required to kill 50% of the treated brine shrimp nauplii population.The LC50 value of <1.0 mg/ml was considered toxic, while >1.0 mg/ml deemed non-toxic (Meyer et al. 1982).

Preparation of test sample and larvicidal activity test
The extract of P. integrifolia was tested against C. quinquefasciatus larvae after 24 h of emergence throughout the lethality test.Test samples at different concentrations considered as doses were prepared in test tubes by adding a calculated amount of DMSO to make the extract hydrophilic before adding water in each of the test tubes.Then water was added to fill the pre-marked (up to 10 ml) test tubes with the help of a pipette.Ten freshly emerged larvae were added to each of the test tubes with different concentrations of the selected doses along with a control batch.The counting of larvae was made after 6 h of application with 6 h intervals up to 48 h through visual inspections.The doses were selected for the extract of P. integrifolia against the mosquito larvae in Pet. ether 50,25,15,10 and 5 ppm;in CHCl3 25,15,10 and 5 ppm and in CH3OH 400,300,200 and 100 ppm for the final experimentation.

Preparation of test sample and piscicidal activity test
For the piscicidal activity test, P. integrifolia extract was placed in a 500 ml beaker and made hydrophilic by adding a small amount of DMSO.Then water was added to fill the pre-marked beakers up to 300 ml.In each of the beakers, five guppies were released and exposed for 48 h.The dead fish were counted after 6 h of application with 6 h interval up to 48 h through visual inspections and the findings were compared to the control.The final doses for the piscicidal activity test against guppies were obtained: P. integrifolia extracts in Pet.ether 66.67, 50, 41.67 and 33.33 ppm; in CHCl3 66.67, 50 and 33.33 ppm.

Statistical analysis
The lethality records of P. integrifolia on brine shrimp nauplii, mosquito larvae and guppy fishes were corrected by Abbott's formula: Pr = (Po -Pc / 100 -Pc) × 100, where Pr = corrected mortality (%), Po = observed mortality (%) and Pc = Per cent mortality in control (Abbot 1925).The data were then subjected to probit analysis (Finney 1947, Busvine 1971).The lethality relationship was expressed as a median lethal concentration (LC50) for the test organisms.

Discussion
The investigation on the bioactive potentials of P. integrifolia against three test organisms was found promising and the findings got strong support from the previous works.Mishra et al. (2021) reported wound healing potential of P. integrifolia flowers.Their study provides a scientific evaluation for the wound healing potential of the petroleum ether and water extracts of Petunia integrifolia flowers using excision and incision wound models in Swiss albino mice.Antioxidant activity was reported very recently by Petrova et al. (2020).The potential insecticidal activity of P. integrifolia was reported against stored product pests, e.g., Callosobruchus chinensis, Sitophilus oryzae and Tribolium castaneum (Islam et al. 2020).Petunia has been mentioned for the presence of acylsugars produced in the glandular trichomes of Solanaceae plants and linked to abiotic and biotic stress resistance (Nadakuduti et al. 2017).This plant species produces acylsugars to defend themselves against herbivores and various biological and environmental stresses (Nadakuduti et al. 2017).Kumar (2015) previously reported that P. axillaris possesses antibacterial activity to cure various human diseases and ailments.According to his findings, bacteria are susceptible to Petunia spp.extracts.The bacteria Staphylococcus aureus, Streptococcus sp., Pseudomonas aeroginosa, and Salmonella typhimurium were strongly inhibited by the P. axillaris leaf extracts in vitro.Thenmozhi and Sivaraj (2011) described Petunia as a medicinal as well as aesthetic plant.They reported that the leaf and callus extracts were potential for callus induction and antibacterial activity against Baccilus subtilis, Escherichia coli, Salmonella spp.and Streptococcus spp.P. hybrida was described for producing a high level of plant defensins, similar to ornamental tobacco Nicotiana alata (Lay et al. 2003).Broekaert et al. (1995) revealed that defensins inhibit fungus, oomycetes and positive bacteria.P. hybrida floral defensins suppress the growth of Botrytis cinerea and Fusarium oxysporum, and they may play a role in pathogen defense in floral tissues (Lay et al. 2003).
Petunia has been mentioned by Kays et al. (1994) for its poisonous potential and antibacterial and antifungal effects.They also revealed that the leaves of this plant produce an effective insecticide, which is widely employed as a natural insecticide and has all of the advantages of chemical compounds, such as quick action, broad spectrum insect activity, and rapid biodegradability.Sweet potato whiteflies are poisoned by a sticky secretion of sugar esters produced by the cultivar.Guerreiro (1984) reported some non-nitrogenous diterpene compounds as psychotropic substances rather than alkaloids in the related P. patagonica.Smoking P. violacea promotes similar effects of smoking Coriaria thymifolia, Butler et al. (1981).On the other hand, both plants are meant to make people feel like floating away from the ground or flying into the air.Tropane alkaloids are frequently blamed for this type of psychoactive experience.However, no alkaloids have been found in any section of P. violacea to date (Butler et al. 1981).Very recent investigation was done on this very well-known but less studied plant for the detection of nootrophic activity by Khan et al. (2022).

Conclusion
The results of this investigation depicted cytotoxic activity, coupled with larvicidal and piscicidal activities of P. integrifolia, which could be considered as further clarification of its potentials to be used in the pest control and pharmaceutical sectors.However, the genus Petunia has been reported as a source of antimicrobial, antibacterial, antifungal, and antioxidant agents.Several steroidal chemicals also found in Petunia species contribute to the plant's resistance to insect attack; and very recent finding broaden the horizon of Petunia potentials by adding nootropic activity; which triggers a hope for more potential leads in P. integrifolia.Thus, a thorough investigation on this plant is very much to be solicited.

Fig. 1
Fig. 1(i-vi): Regression lines of P. integrifolia extracts against A. salina nauplii at different hours of exposure.

Table 1 .
LC50 values of P. integrifolia extracts on A. salina nauplii, C. quinquefasciatus larvae and P. reticulata adults.
Regression lines of the LC50 values for P. integrifolia extracts at the minimum (12 h) and maximum (48 h) exposures in three test organisms are shown in the figures below.i) Pet.ether extract after 12 h ii) Pet.ether extract after 48 h iii) CHCl3 extract after 12 h iv) CHCl3 extract after 48 h v) CH3OH extract after 12 h vi) CH3OH extract after 48 h