Larvicidal, leishmanicidal, insecticidal and anthelmintic effects of Sterculia

The present study was conducted to evaluate the stem bark and leaf of Sterculia diversifolia for larvicidal, leishmanicidal, insecticidal and anthelmintic activities. In the larvicidal activity, dichloromethane and ethyl acetate fractions of both stem bark and leaf showed significant results, while the ethyl acetate fraction showed mild activity (IC50: 70.09 ± 0.09 μg/mL; 71.25 ± 0.01 μg/mL). In the insecticidal activity of the stem bark, n-hexane fraction showed mild activity (20-40% mortality) against all three subjects, while the insecticidal activity of leaf, methanolic extract and n-hexane fractions showed moderate activity (60% mortality) against Rhyzopertha dominica and Callosobruchus analis. In the anthelmintic activity of stem bark and leaf, ethyl acetate fraction exhibited activity in a dose-dependent manner giving time of paralysis and death shorter followed by dichloromethane fraction with 200 mg/mL concentration. Hence, it is clear that S. diversifolia stem bark and leaf possess larvicidal, leishmanicidal, insecticidal and anthelmintic agents. 
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Amongst medicinal plants, family Sterculiaceae is a source of bioactive compounds of different chemical classes such as alkaloids, glycosides, terpenoids, sterols, steroids, saponins, apigenins, flavonoids, polyphenols, essential oils, tannins, carbohydrates, proteins, triterpenes and reducing sugars (Khatiashvili et al., 2007;Ouédraogo et al., 2013). The genus Sterculia also possess various types of biological activities such as S. vallosa bears anthelmintic and leishmanicidal activities while S. guttata and S. foetida bears larvicidal and insecticidal activities respectively (Das et al., 2017;Alam et al., 2012;Katade et al., 2006;Rani et al., 2010). S. diversifolia has antibacterial, antifungal and antioxidant activity. In earlier study, few fatty acid constituents were isolated from the S. diversifolia (Salem et al., 2014). The present study was designed to investigate the larvicidal, leishmanicidal, insecticidal and anthelmintic activity of methanolic extract of S. diversifolia stem bark and leaf and their fractions.

Plant material
Plant materials (stem bark and leaf) were collected from the botanical garden of Pakistan Forest Institute (34°A bstract The present study was conducted to evaluate the stem bark and leaf of Sterculia diversifolia for larvicidal, leishmanicidal, insecticidal and anthelmintic activities. In the larvicidal activity, dichloromethane and ethyl acetate fractions of both stem bark and leaf showed significant results, while the ethyl acetate fraction showed mild activity (IC50: 70.09 ± 0.09 μg/mL; 71.25 ± 0.01 μg/mL). In the insecticidal activity of the stem bark, n-hexane fraction showed mild activity (20-40% mortality) against all three subjects, while the insecticidal activity of leaf, methanolic extract and n-hexane fractions showed moderate activity (60% mortality) against Rhyzopertha dominica and Callosobruchus analis. In the anthelmintic activity of stem bark and leaf, ethyl acetate fraction exhibited activity in a dose-dependent manner giving time of paralysis and death shorter followed by dichloromethane fraction with 200 mg/mL concentration. Hence, it is clear that S. diversifolia stem bark and leaf possess larvicidal, leishmanicidal, insecticidal and anthelmintic agents. 00'50.6"N 71°29'03.0"E), University of Peshawar, Pakistan, in September, 2014. The identification of the plant was done a taxonomist Mr. Ghulam Jelani at the Department of Botany, University of Peshawar. Under the reference No: Bot.20098, a specimen was deposited in the above-mentioned institute herbarium.

Extraction and fractionation
S. diversifolia stem bark (17 kg) and leaf (13 kg) were dried completely in shade at room temperature. These parts were then crushed to powder and macerated with 90%hydro-methanolic solvent for two weeks. After maceration, the filtration was conducted with Whatman filter paper No. 1. The obtained crude extracts were concentrated under a reduced pressure at 40°C using a rotary evaporator (R-1001-V, Zhengzhou Great Wall Scientific Industrial and Trade Co., China) (Rabbi et al., 2017). The methanolic extract of stem bark and leaf was obtained in 950 g and 1.2 kg quantity respectively. The extract was mixed with distilled water (2.5 L) and soaked overnight. Fractionation was conducted with various organic solvents i.e. n-hexane (3 × 5 L), dichloromethane (3 × 5 L), ethyl acetate (3 × 5 L) and n-butanol (3 × 5 L). The remaining was considered as watersoluble fraction i.e. aqueous fraction.

Larvicidal activity
The larvicidal activity of the crude methanolic extract and its fractions of both stem bark and leaf were screened according to the protocol recommended by the WHO (1981). Various concentrations (25, 50, 100, 250, 500 ppm) of the test samples were used. All the extracts were dissolved in respective solvents. A glass beaker of 500 mL was used containing 250 mL tap water. Early third instar of Aedes aegypti (25) was introduced to each of the test solutions as well as the control. In the case of control, methanol was only used. Six replicates were maintained at a time for each experiment. The LC50 was calculated using GraphPad prism 5 software (Bucker et al., 2013).

Leishmanicidal activity
The crude extract of S. diversifolia (stem bark and leaf), and its subsequent fractions were screened for antileishmanial activity using Leishmania major. NNN biphasic medium was used in which promastigotes of Leishmania were grown. This medium was modified earlier and normal physiological saline was used for this purpose. For culturing parasite, the RPMI medium (1640 Sigma) was used. Inactivated fetal bovine serum (10%) was also supplemented to the medium. Leishmania major parasites were harvested at log phase of growth and centrifuged for 10 min at 3,000 rpm. Parasites were washed three times with normal saline maintaining the same experimental conditions. With the addition of freshly prepared culture medium, final density (106 cells/mL) of parasites was achieved. About 20 µL test sample and 180 µL medium were added to each well of 96-well microplate and then serially diluted. 100 µL prepared culture of Leishmania was added to all wells. One row containing only media and Leishmania culture was served as negative control while the second row served as positive control con-taining pentamidine and amphotericin B. The number of living parasites was counted microscopically after incubation for 72 hours at 22°C using Neubauer counting chamber. Ezfit 5.03 software was used for the determination of IC50 value (Ali et al., 2016).

Insecticidal activity
The crude methanolic extracts (stem bark and leaf) and their various fractions were studied for insecticidal activity against Tribolium castaneum, Rhyzopertha dominica and Callosobruchus analis. Insecticidal assay of crude methanolic extracts (stem bark, leaf) and their fractions were assessed by using the impregnated filter paper method (direct contact method). In this method, T. castaneum, R. dominica and C. analis were reared under

Principle
The anthelmintic assay of crude methanolic extract and various fractions of both stem bark and leaf were evaluated on adult earthworms (Pheretima posthuma) due to its anatomical and physiological resemblance with the intestinal roundworm parasites of human beings

Procedure
Step 1: Earthworms were collected from the moist soil and then washed with normal saline for the removal of the adhering dirt Step 2: Three earthworms were released in each glass beaker containing solution (50 mL) of reference drug albendazole and extract (50, 100 and 200 mg/mL each) in distilled water.
Step 3: Distilled water was served as negative control.
Step 4: The time taken to paralysis and death of earthworm was observed and noted.

Notes
The earthworms were authenticated by Mr. Abdur Rahim, an expert in animals taxonomy.
When the earthworms did not revive in the normal saline, it indicated water paralysis, while the earthworms lost their motility followed by fading away of their body color or even not moved when dipped in 50°C hot water concluded the death of animals.
controlled and specific conditions of humidity and temperature in the laboratory. Those insects were selected which are uniform in size and age. The test sample (200 mg) was dissolved in acetone (3 mL) to prepare the stock solution. In petri dish, filter paper (90 mm) was placed and then it was loaded with the test sample (1019.1 µg/cm 2 ). The volatile organic solvent was evaporated after 24 hours. Ten active insects were transferred to the next day to each petri dish with the help of a brush and incubated for 24 hours (27 ± 1°C) with 50% humidity in the growth chamber. Acetone and permethrin (239.50 µg/cm 2 ) were used as negative and positive control respectively. The percent mortality was determined by comparing the test sample with the positive control (Saeed et al., 2010). It was determined by counting the number of survived insects as below: Number of insects alive in test %Mortality= 100 -×100 Number of insects alive in control

Larvicidal activity
The maximum activity was shown by dichloromethane and ethyl acetate fractions of stem bark (Table I) (Table I).

Leishmanicidal activity
The leishmanicidal activity of the crude methanolic extract and its fractions of stem bark and leaf were evaluated against promastigotes of Leishmania major (Table II). In the leishmanicidal activity of stem bark, ethyl acetate fraction showed mild activity (IC50: 70.1 μg/mL) followed by n-hexane fraction (IC50: 77.3 μg/ mL). In the leishmanicidal activity of leaf, ethyl acetate fraction showed mild activity (IC50: 71.3 μg/mL) followed by dichloromethane fraction (IC50: 79.3 μg/mL).

Insecticidal activity
The insecticidal activity of crude methanolic extract and its fractions were studied against R. dominica, T. castaneum and C. analis. The results revealed that the crude methanolic extract stem bark showed low insecticidal activity (20%) against T. castaneum and inactive against rest of the insects (Table III). The n-hexane fraction showed good activity, 40% against R. dominica, 20% against T. castaneum and 30% against C. analis. Other fractions; dichloromethane, ethyl acetate, n-butanol and aqueous were found inactive against test insects. In case of leaf, crude methanolic extract showed good insecticidal activity (60%) against R. dominica and inactive against the rest of the insects. Other fractions; n-hexane, dichloromethane, ethyl acetate, n-butanol and aqueous showed 60, 10, 10, 20, 10% insecticidal activity respectively against C. analis, while were found inactive against R. dominica, T. castaneum.

Anthelmintic activity
In the anthelmintic activity, extract of S. diversifolia (stem bark and leaf) and its fractions demonstrated worms paralysis as well as death as presented in Table  IV. The extracts in higher concentrations produced much earlier paralytic effect and shorten the time to death for all worms. Ethyl acetate fraction exhibited anthelmintic activity in dose-dependent manner giving time of paralysis and death shorter followed by dichloromethane fraction with 200 mg/mL concentration. The crude methanolic extract and aqueous fraction exhibited moderate anthelmintic activity, paralyzed worm after 57.5 and 65.9 min, while killed after 72.
Positive control (permethrin) 100 100 100 100 100 100 Negative control -----activity was observed for ethyl acetate fraction (IC50 70.1 μg/mL and 71.3 μg/mL respectively) amongst test samples of Sterculia diversifolia stem bark and leaf showing almost similar results. The results of insecticidal activity reveal that methanolic extract of stem bark, n-hexane fraction showed mild to moderate activity (percent mortality) against all three insects followed by crude methanolic extract, while the insecticidal activity of leaf, none of the fraction showed insecticidal activity against all three insects, however, the methanolic extract showed 60% mortality against Rhyzopertha dominica while n-hexane fraction showed 60% activity against Callosobruchus analis. In anthelmintic activity of stem bark and leaf, ethyl acetate fraction exhibited activity in dose-dependent manner giving time of paralysis and death shorter followed by dichloromethane fraction with the highest concentration (200 mg/mL), showing almost comparable outcomes.
Crude extracts are a complex mixture of active compounds (Anees, 2008). Various extracts possess larvi-cidal activity against mosquitoes have the advantage in reducing resistance issue and also environmentfriendly. The disease can be easily controlled by controlling the vectors (e.g. parasitic vectors). The insect population can be reduced by controlling the mosquito larvae which is a result reduce disease burden (Ghosh et al., 2012). Throughout the world and especially in tropical countries, dengue fever is a measure of health problem. Dengue fever could be controlled by reducing Aedes aegypti production (Raveen et al., 2014). It is evident from the result that various concentrations of Sterculia diversifolia stem bark and leaf were the main cause of mortality in A. aegypti larvae as reported from plants Citrus grandis and Tinosphora rhumpii (Gutierrez et al., 2014) 1 .
Leishmaniasis, a common disease in the subtropical and tropical regions of the world, is an infection of the protozoal parasite of genus Leishmania (Monzote et al., 2014). Currently leishmaniasis is considered as a serious disease due to lack of availability of specific treatment; still some semi synthetic and synthetic drugs are implicated to treat leishmaniasis (Mears et al., 2015;Tiuman et al., 2011;Zucca et al., 2013). Drugs such as amphotericin B and pentamidine are also used in the management of leishmaniasis, lacking the desired efficacy (Aronson et al., 2016;Cunha et al., 2015;Sadeghi-Nejad et al., 2011). The extracts Achillea biebersteinii showed promising activity with potent leishmanicidal activities as compared to Sterculia diversifolia stem bark and leaf, which showed mild activity (Al -Sokari et al., 2015).
Insects control relies heavily on the use of synthetic insecticides; however, their widespread use has led to various problems such as the development of insect strains resistant to insecticides (Zia-Ul-Haq et al., 2012). The plant produces various secondary metabolites for defense purposes. These secondary metabolites protect as well as repel the harmful insects to control pests Pavela, 2016;Dubey et al., 2008). Monoterpenes, sesquiterpenes lactones and triterpenes are some of the secondary metabolites of various plants that possess insecticidal activity (Barney et al., 2005;Cespedes et al., 2015;Sosa and Tonn, 2008). Crude extracts of Lepidium sativum and Ipomoea hederacea showed the highest insecticidal activity against all three insects as compare to Sterculia diversifolia (Zia-Ul-Haq et al., 2012). Sterculia diversifolia stem bark, n-hexane fraction showed mild to moderate activity against all insects, while the MESD and n-hexane fraction of leaf showed highest activity against Rhyzopertha dominica and Callosobruchus analis respectively.
The anthelmintic potential of tested extract may be due to the presence of similar phytoconstitutes, which was evident. Studies have shown that phenolic and tannin possess plants to produce anthelmintic activities because tannins chemically belong to polyphenolic compounds. Phenolic and tannins are known to interfere with the generation of energy in helminth parasites by uncoup-ling the oxidative phosphorylation and also bind to free proteins in GIT of host animal or glycoprotein on the parasite cuticle, leading to death (Mali and Wadekar, 2008). It is possible that phenolic compounds and tannins also possessing in Sterculia diversifolia stem bark and leaf extracts may be responsible for the anthelmintic activity. Sterculia diversifolia stem bark and leaf shown moderate anthelmintic activity comparable to other medicinal plants e.g. Blumea lacera (Pattewar et al., 2012).
S. diversifolia stem bark and leaf possess various types of phytochemical constituents such as alkaloids, saponins, flavonoids, steroids, tannins etc. These constituents may carry larvicidal, leishmanicidal, insecticidal and anthelmintic potential, although further phytochemical screening is needed. Crude extracts require preliminary screening because it is a complex mixture of bioactive compounds. Hence, these plant extracts are an economical, safe, effective and environment-friendly alternative.