Dose-mortality , Cytotoxicity and Repellent Activity of Abutilon hirtum ( Lam . ) Sweet against Callosobruchus chinensis ( L . ) , Artemia salina L . and Tribolium castaneum ( Hbst . )

Petroleum ether (Pet. ether), chloroform (CHCl3) and methanol (CH3OH) extracts of the aerial parts of Abutilon hirtum (Lam.) Sweet were subjected to dose-mortality against the stored grain pest Callosobruchus chinensis (L.), cytotoxicity against brine Shrimp Artemia salina L. nauplii and repellent activity against adult beetles of Tribolium castaneum (Hbst.). Against C. chinensis only CH3OH extract showed promising mortality and provided LD50 values 1.344, 1.294, 1.243 and 1.152 mg/cm after 6, 12, 18 and 24 h of exposure respectively, however, Pet. ether and CHCl3 extracts didn’t show mortality. Against A. salina nauplii Pet. ether, CHCl3 and CH3OH extracts showed cytotoxic effects; while Pet. ether extract gave LC50 values 2461.031, 642, 191.233, 94.618 ppm after 6, 12, 18 and 24 h of exposure respectively, and CHCl3 extract provided LC50 values 1336.124, 679.387, 276.961 and 199.988 ppm; and CH3OH offered 531.896, 212.840, 91.499 and 72.975 ppm after 6, 12, 18 and 24 h of exposure respectively. For repellency against T. castaneum adults CHCl3 extract showed significant result at 1% level of significance (P < 0.01), while the CH3OH extract showed moderate repellency at 5% level of significance (P < 0.05), but the Pet. ether extract didn’t show any significant repellent activity.


Introduction
As traditional medicine has remained the most inexpensive and easily approachable source of treatment in the primary health care system; and thus the local therapy is the only means of medical treatment for these communities [1].So, an increasing interest has been noticed in the study of medicinal plants and their traditional use in different parts of the world during the last few decades [2].The present investigation was attempted on Abutilon hirtum (Lam.)Sweet, is a perennial herb or shrub, 0.5-2.5 m in height.It is commonly known as "Indian mallow" or "Florida Keys Indian Mallow" and in Bengali it is known as "Jhampi"or "Belabenda" and distributed in tropical regions [3].Traditionally, the leaves used as demulcent, diuretic and in diarrhea [4].The decoction of the leaves used as a mouth wash and in bladder inflammations, wounds and treatment of ulcers, in addition to the fruits are eaten raw and the water extract of the bark is given to ease childbirth in Kenya [5,6].In Malaysia, A. hirtum is used as a poultice to ease the pain of kidney gravel and often mixed with glutinous rice and applied to ulcers.The leaves or flowers are applied to abscesses [7].The strategy for the present investigation was designed to carry on screening of the crude extracts of the test plant species on Brine shrimp A. salina L. nauplii and stored grain pest C. chinensis (L.) and adult flour beetles of T. castaneum (Hbst.)for the detection of biological activity and keeping an option to show the extent of activity by analyzing the data statistically that read on various parameters during the course of the investigation.C. chinensis (Coleoptera: Chryssomelidae) commonly known as pulse beetle found as pest to many stored legumes [8].The eggs are cemented to the surface of pulses and are smooth, domed structures with oval, flat bases.The larvae and pupae are only found in cells bored within the seeds of pulses [9].It takes 20-25 days for being adult from eggs [10,11].A. salina (Anostraca: Artemiidae) commonly known as Brine shrimp belongs to a genus of very primordial crustacean (Crayfish); Artemia is one of the standard organisms for testing the toxicity of chemicals [12].The females can produce eggs either as a result of mating or via parthenogenesis.Eggs hatch into nauplii that are about 0.5 mm in length.Eggs remain in a dormant state as cysts.These cysts can last for a number of years, and will hatch when they are placed in saltwater [13].T. castaneum (Family: Tenebrionidae) is a worldwide pest of stored products and of Indo-Australian in origin [14].The red flour beetle may produce an allergic response [15].Eggs are microscopic and slender larvae are creamy yellow to light brown in colour, while the adult is a small reddish-brown.Total life cycle contains subsequently for egg incubation 8.8 days, larval development 22-100 days depending on temperature, pupal development 4.5 days, and for reproductive maturation 4-5 days [16].

Collection and preparation of test materials
The fresh materials of A. hirtum were collected from the bushy area of Station Bazaar (from both the sides of the railway lines), which is located just near the Rajshahi University Campus, Bangladesh and identified by the Department of Botany, Rajshahi University, where a voucher specimen is kept in the herbarium.The areal parts of the plants (leaves, stem, fruit and flower) were collected and chopped into small pieces, dried under shade and powered with the help of a hand grinder separately, weighed and placed in conical flasks to add solvents.The solvents Pet.ether, CHCl 3 and CH 3 OH (Merck, Germany) were used (200 g × 600 mL × 3 times) successively each of which kept for 48 h on a shaker.For each of the extracts filtration was done by filter paper at 24 h of time interval in the same flask followed by evaporation until the extract was left as a scum.The extracts was then removed to glass vials and preserved in a refrigerator at 4°C with proper labeling.

Collection and culture of test insects
The test insects of C. chinensis and T. castaneum of same age were used in this investigation which were received from the stock cultures of the Crop Protection Laboratory, Department of Zoology, Rajshahi University, Bangladesh. A. salina is a marine crustacean which is not easy to culture like C. chinensis and T. castaneum under laboratory conditions, but, they can be reared in a short edition.These nauplii are very easy to grow from its marketed cysts to carry on toxicity tests of certain materials.

Dose-mortality tests on C. chinensis
The concentrations used in this experiment were 1.070, 1.120, 1.171, 1.222 and 1.273 mg/cm 2 for Pet.ether, CHCl 3 and CH 3 OH extracts.For each of the doses 1 mL was dropped on a Petri dish (50 mm) containing pulse grains.Then the Petri dishes were airdried leaving the extract on it.After drying 10 beetles (3-5 days old) were released in each of the Petri dishes in 3 replicates.A control batch was also maintained with the same number of insects after preparing the Petri dish by applying and evaporating the solvent only.The treated beetles were placed in an incubator at the same temperature as reared in stock cultures and the mortality of the beetles was counted after 6, 12, 18 and 24 h of exposure.

Lethality test
Brine shrimp lethality bioassay is a recent development in the bioassay techniques for the detection of biological activity like cytotoxicity, as well as, a wide range of pharmacological activities (e.g.anticancer, antiviral, pesticidal, anti-AIDS, etc.) of the compounds.To carry on toxicity tests nauplii were grown from its marketed cysts.Test samples at different concentrations considered as doses were prepared in test tubes by addition of calculated amount of DMSO (dimethylsulfoxide) to make them hydrophilic before adding half of the required amount of water in each.Then additional amount of water were added to fill the pre-marked 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 along with a control batch were left for 30 h.Observation of mortality was made after 6, 12, 18, 24, 30 and 36 h of exposure.Doses for all three extracts Pet.ether, CHCl 3 , CH 3 OH were 200, 400, 800 ppm respectively.

Repellent activity
The method of repellency test used in this investigation was adopted from the method (No.3) of McDonald [17].The average of the counts was converted to percent repulsion (PR) using the formula, PR = (Nc-5) × 20; where, Nc was the average hourly observation of insects on the untreated half of the disc [18,19].A general concentration for each of the extracts (Pet.ether, CHCl 3 and CH 3 OH) was selected as a stock dose applied against T. castaneum adults and other successive doses were prepared by serial dilution to give 0.1571, 0.0785, 0.0392, 0.0196 and 0.0098 mg/cm 2 .Half filter paper discs (Whatman No. 40, 9cm diam.) were prepared and selected doses of all the extracts separately applied onto each of the half-discs and allowed to dry out as exposed in the air for 20 min (approx.).Each treated half-disc was then attached lengthwise, edge-to-edge, to a control half-disc with adhesive tape and placed in a Petri dish (9cm diam.).For each of the test samples three replicates were maintained.Being volatile the solvent was evaporated out within a few minutes.Then ten insects were released in the middle of each filter paper circles.Repellency was observed for one-hour interval and up to five successive hours of exposure, just by counting the number of insects from the non-treated part of the filter paper spread on the floor of the 90 mm Petri dish.The values in the recorded data were then calculated for percent repellency, which was again developed by arcsine transformation for the calculation of analysis of variance (ANOVA).

Dose mortality effect on C. chinensis
The results of dose mortality effects of CH 3 OH extracts of A. hirtum against C. chinensis are represented in Table 1.The CH 3 OH extract offered mortality by giving LD 50 values ranged between 1.152 to 1.344 mg/cm 2 against C. chinensis for 24 and 6 h respectively.

Repellent effect on T. castaneum
The CHCl 3 extract showed the significant repellent activity at 1% level of significance (P < 0.01) and the CH 3 OH extracts showed the significant repellent activity at 5% level of significance (P < 0.05), while the Pet.ether extracts did not show any significant repellent activity against the adult beetles of T. castaneum (Table 3 and 4).

Discussion
Now the environmental safety of an insecticide is considered to be of paramount importance.An insecticide does not have to cause high mortality on target organisms in order to be acceptable [23].The findings of the present investigation receive supports from the experiment done on A. hirtum and its related species by previous researchers.Works on A. hirtum extracts for evaluating cytotoxicity, insect mortality and repellency is scanty, and however there are some evidences of research that have been done to find out the medicinal, phytochemical, pharmacognostical and structural properties.It has been already proved that A. hirtum is highly effective in medicinal and traditional usage for the treatment of various human ailments [24].The leaf extract of A. hirtum (Lam.)Sweet can positively be used in the treatments provided by the Herbal Medicine Industry [25].Also leaf extract of A. hirtum possesses significant hepatoprotective activity [26].Its related species A. indicum was found to be effective as the anti-snake venom and anti-ulcer agents [27].And many important phytoconstituents like β-sitosterol and tocopherol oil were isolated from this plant [28,29].Methanolic petroleum ether, diethyl ether and ethyl acetate extract of A. hirtum showed potent antiarthritic activity [30].An another study suggested that the hydro ethanolic extract of A. hirtum possessed antioxidant and anticataract activity which might be due to the presence of phytochemicals such as tannins, phenols, flavonoids and alkaloids [31].After studying the previous works and analyzing the present outcome of this investigation it is clear that A. hirtum contains compounds that have insect mortality, cytotoxicity and repellent activity.

Conclusion
By analyzing the results of dose mortality, cytotoxicity and repellent activity tests of A. hirtum extracts against A. salina, C. chinensis and T. castaneum it is possible to come to a conclusion that A. hirtum has some bioactive potentials could be used in strategic control of certain pest population in the field, in the storage and in the aquatic media.

Table 1 .
LD 50 values of CH 3 OH extracts of A. hirtum against C. chinensis.

. Cytotoxicity test on A. salina
The results of the lethality bioassay of Pet.ether, CHCl 3 and CH 3 OH extract of A. hirtum against the brine Shrimp A. salina L. nauplii are represented in Table2.All the extracts offered promising cytotoxic activity.The LC 50 values for Pet.ether extract were 2461.031,642, 191.233 and 94.618 ppm; for CHCl 3 extract were 1336.124,679.387, 276.961 and 199.988 ppm; for CH 3 OH extract were 531.896, 212.840, 91.499 and 72.975 ppm all after 6, 12, 18 and 24 h of exposures respectively.

Table 2 .
LC 50 values of Pet.ether, CHCl 3 and CH 3 OH extracts of A. hirtum against A. salina.

Table 3 .
ANOVA result of repellency of T. castaneum by A. hirtum extract.

Table 4 .
Repellent activity of the Pet.ether, CHCl 3 and CH 3 OH extracts of A. hirtum against T. castaneum.