Insecticidal Activity of Hyoscyamus niger L. on Lucilia sericata Causing Myiasis

Background: Hyoscyamus niger L. (Solanaceae) generally known as henbane, is commonly distributed in Europe and Asia. In Turkey, henbane seeds have been used in folk medicine to remove worms from the eyes. The present study aimed to investigate the insecticidal activity of H. niger seeds. Methods: n-hexane, ethyl acetate, methanol and alkaloid extracts were prepared from the seeds of the plant and their insecticidal activities on Lucilia sericata larvae were evaluated. EC50 and EC90 values of the alkaloid extract were calculated and morphological abnormalities were investigated. Results: Alkaloid extract prepared from the seeds of this plant displayed significant insecticidal activity. EC50 values of H. niger seeds alkaloid extract were found to be 8.04, 8.49, 7.96 μg/mL against first, second and third instar, respectively. It was determined that malformations of larvae included damaged larvae with small size, contraction and weak cuticle. Furthermore, HPLC analysis was performed on alkaloid extract of H. niger seeds and main components of the extract were determined. It was determined that alkaloid extract mainly contain hyoscyamine and scopolamine. Conclusions: These results confirm the folkloric usage of the plant and suggest that the alkaloid content of the plant could be responsible for the insecticidal activity.


Introduction
Hyoscyamus genus has six species in the flora of Turkey, H. aureus, H. albus, H. leptoclyx, H. niger, H. pusillus and H. reticulatus [1]. The genus belongs to Solanaceae family. Hyoscyamus niger L. (black henbane) is the most popular species of Hyoscyamus genus. It has been used as a medicinal plant since ancient Greece [2]. The mature corolla of H. niger is lurid yellow, usually veined purple; the fruiting calyx is constricted at the middle; and the upper cauline leaves are amplexicaul [1]. H. niger leaves are used as an antispasmodic for overfed animals and the seeds are used for itching, reddening in eyes, and earache [3,4]. One of the most popular uses of H. niger in folk medicine is to expel worms in the mouth or eyes [3,[5][6][7][8][9]. Seeds are spread on dying embers and covered with a blanket. Eyes of the patient are exposed to the vapor under the blanket. After the vapor application, small white worms with black heads drop from the eyes or mouth [5,7,9,10]. According to phytochemical studies, all parts of the plant contain hyoscyamine and scopolamine [2,11]. In addition to these compounds, four lignanamides, a tyramine derivative, and ten other nonalkaloidal components were isolated from the seeds of H. niger. Among them, hyoscyamide, 1,24-tetracosanediol diferulate, and 1-O-(9Z,12Z-octadecadienoyl)-3-O-nonadecanoyl glycerol are new structures.
Lucilia sericata (Meigen) (Diptera: Calliphoridae) is a facultative ectoparasite used for necrotic wounds that conventional drugs can not treat [15] and is commonly known as the sheep blow fly or the green bottle fly. However, it can give rise to myiasis in humans. Myiasis is the invasion of animal and human tissues and organs by the larval stages of dipterous flies [16] causing either facultative or obligatory myiasis [17]. Myiasis usually occurs in regions, where people encounter animals [18]. Most patients live in economically disadvantaged, overcrowded, fly-infested areas [19]. Nosocomial myiasis in hospitalized patients happens with moderate incidence. Confined to bed patients with open wounds might have infestations if L. sericata are present [20,21].
In the light of these facts, this study evaluated the antilucidal activity of four extracts obtained from H. niger seeds, growing wild in Turkey and also HPLC analysis were conducted to identify the major components in the alkaloid extract of H. niger seeds which showed the highest inhibitory activity against L. sericata.

Results
The results indicated that while the alkaloid extract of H. niger showed high activity, the methanol extract showed moderate activity on the development of first instar of L. sericata. As the concentration increased, the H. niger alkaloid extract insecticidal effect increased. 100% larva mortality was reached after application of 32 µg/mL alkaloid extract. Larva mortality reached 60% and 81% at concentrations of 8 and 16 µg/mL, respectively ( Table 1).
The insecticidal activity of H. niger alkaloid extract was considerably active on the development of L. sericata second instar. The mortality rate was 100% at 32 µg/mL and 89% at 16 µg/mL in the alkaloid extract group. The mortality rate was 52% at 32 µg/mL in the methanol group ( Table 2). The 16 and 32 µg/mL concentrations of the alkaloid extract induced 100% mortality for the third instar (Table 3).    According to the HPLC studies, the amount of scopolamine and hyoscyamine were determined as 0.1578 (g/g) and 0.1256 (g/g), respectively (Figure 1).

Discussion
The current study describes the effects of the extracts prepared from H. niger seeds on L. sericata larvae which causes myiasis in humans. Myiasis is explained as the invasion of living vertebrates (animals and/or humans) by dipterous larvae.
In humans, dipterous larvae may feed on the host's alive or dead tissue as well as liquid body substances and give rise to broad infestations liable on the body location [22]. Wound myiasis results from flies of Calliphoridae which includes L. sericata. It can cause ocular myiasis and nasal myiasis [23][24][25]. There are three main methods for the treatment of myiasis: (I) the use of a toxic substance for larvae, (II) the production of confined hypoxia to power the appearance of the larva, and (III) the mechanical or medical elimination of the maggots [22]. H. niger seeds have been used to remove worms from eyes in folk medicine [5,7,9,10]. The presence of insecticidal activities of the oils obtained from Apium graveolens, Brassica compestris, Raphanus sativus and Trigonella foenum-graecum, was reported by Khater and Khater [21]. Their results suggested that oils obtained from those four plants might signify novel and safe possible insecticides for the control of blowflies. In the light of these facts, our study investigated the effects of H. niger seeds on L. sericata which cause myiasis. Behravan et al. [26] reported that H. niger flower extract could be used to fight Anopheles spp mosquito larvae. They found that the most active extract for destroying the mosquitoes Anopheles spp larvae was the henbane flower. Furthermore, Wang et al. [27] exhibited that the ethanol extract of H. niger seeds showed high insecticidal activity against the Aphis laburni Kaltenbach. After the alkaloid extract of H. niger seeds, morphological malformation of larvae involved contractile, small sized, and damaged larvae with weak cuticles. Similar results have been described following the treatment of anise, chamomile and rosemary oils as well lettuce against larvae of L. sericata [28]. These abnormalities also have been seen in Chrysomya albiceps which causes myiasis with the treatment of Punica granatum [29]. Moreover, Allium cepa, Nigella sativa and Sesamum indicum oils seriously affect pupation rates and appearance of adult Culex pipiens and Musca domestica [30]. Three endocrine glands are responsible for releasing neuro-hormones vital for growth, development and differentiation; the corpus cardiacum, corpus allatum, and prothoracic gland in larva insects. It has been presented that plant components cause advanced degeneration of all these endocrine glands in larvae [31]. This morphological degeneration indicates a comprehensive dysfunction of the neuroendocrine system.
Black henbane contains alkaloids such as hyoscyamine, atropine, tropane and scopolamine. Chromatographic analysis of the alkaloid percentages from H. niger identified in the leaves, roots and seeds are 0.17, 0.08 and 0.05, respectively [32,33]. Ghorbanpour et al. [2] found that the leaves of H. niger include hyoscyamine and scopolamine in the yield of 0.725 and 0.362 (g/g plant), respectively, and Ma et al. [12] isolated nonalkaloid compounds from H. niger seeds. On the other hand, our results exhibited that the amount of scopolamine and hyoscyamine were determined as 0.1578 (g/g) and 0.1256 (g/g), respectively, in the seeds of H. niger. Tropane alkaloids can induce antispasmodic effects of smooth muscle, reduction of bronchial hypersecretions, and relief of gastric pain [34]. In addition to alkaloids, henbane seeds contain withanolides, flavonoids, lignans, coumarinolignans, saponins, glycerides, glycosides and phenolics [32]. Begum [32] found that because of its nonalkaloidal constituents, henbane seeds possess antimicrobial, antidiarrheal, antispasmodic, anticonvulsant, anti-inflammatory, analgesic and antipyretic activities. Swathi et al. [35] reported that Datura stramonium, which belongs to Solanaceae family, has larvicidal and mosquito repellent activities. However, hyoscyamine and scopolamine possess central effects of anticholinergic toxicity including confusion, delirium, irritability, agitation, hallucinations (typically visual and/or tactile), seizures and mydriasis [36]. Hyoscyamine and scopolamine are major compounds of this plant. Therefore, the effects of H. niger seeds against L. sericata could be due to their hyoscyamine and scopolamine.

Plant Material
The plant, H. niger, was collected from Ankara in June 2015 and authenticated by Prof. Dr. Murat EKİCİ from Gazi University, Faculty of Science and Art, Department of Biology, Ankara. A voucher specimen (ANK10016) was deposited in the Ankara University, Herbarium of Faculty of Science, Ankara, Turkey.

Preparation of n-Hexane, Ethyl Acetate and Methanol Extracts
Plant material (500 g) was dried in the shade and the seeds were extracted with n-hexane

Preparation of Alkaloid Extract
For the extraction of the alkaloid extract, 10 L CH 2 CI 2 -MeOH-NH 4 OH (15:5:1) was added to H. niger seeds (100 g), sonicated for 10 min, then saved at room temperature for 1 h. Following filtration, the residue was washed twice with CH 2 CI 2 (1 L). The filtrate was evaporated to dryness. Five liters of CH 2 CI 2 and 2 L of 1 N H 2 SO 4 were added to the residue, then the solution was mixed. The CH 2 CI 2 phase was removed and the H 2 SO 4 phase was attuned to pH 10 with 28% NH 4 OH. Finally, alkaloids were extracted with once 2 L and twice with 1 L of CH 2 CI 2 . The collective extracts were filtered after adding anhydrous Na 2 SO 4 and then the residue was washed using 1 L of CH 2 CI 2 . The combined filtrates were vaporized to dryness at 40 • C [37]. The yield of the alkaloid extract was 5.28% [5.3 g].

Obtaining First, Second and Third Instars
The adult L. sericata flies were collected from the Tazlar village of Afyonkarahisar province using fly netting. Flies were reared in the laboratory according to El-Khateeb et al. [38]. Beef was used as bait for collecting flies in open area. In the laboratory, first stage larvae were obtained 8-12 h after hatching, according to temperature. Second stage larvae were obtained after 31 h and third stage larvae after 72 h according to temperature and humidity ( Figure 2) [39].

Mounting of L. sericata Larvae
For comprehensive morphological studies of normal larvae, the larvae were washed more than a few times with saline, placed in 5% caustic soda (NaOH) and incubated at room temperature for 1-2 h for the first instar or overnight for the second and third instar. The larvae were evacuated from their contents, washed with water, and dehydrated through ascending serial concentrations of ethanol (70, 80, 90 and 100%) for 1 h each. Lastly, they were cleared in clove oil and washed in xylene. The larvae were mounted in Canada balsam and incubated in an oven at 40 • C to dry for 24 h [40].

Identification of L. sericata Larvae and Adult
The identification was approved according to Holloway [41] and Zumpt [16]. Examination of posterior spiracles on a glass slide under light microscope (Olympus CX-21, Japan) showed two L. sericata (Figure 3).

Ingestion Assay
Early first, second and third instars of L. sericata were exposed to extracts H. niger at five different concentrations: 2, 4, 8, 16, 32 µg/mL. The processes were replicated four times for each concentration of extracts and for an untreated control group. Twenty-five larvae were used for each replicate (100 larvae were used for each concentration). Larvae were transported to a rearing plastic cup (100 cm 3 ) containing a piece of beef and the test materials and were exposed to the 72 h. The plastic cups were covered with clean gauze and protected by a rubber band. In the control groups, larvae were treated using distilled water and Tween-80. Larvae were preserved under laboratory circumstances at 27 ± 2 • C, 80 ± 5% relative humidity, and a 16:8 h light:dark cycle. Larva behavior (feeding and movement activity) was observed at 8, 12, 24, 36, 48 and 72 h (until 3rd instar). Larvae mortality amounts were determined till pupation. Larvae were measured alive if they displayed normal behavior when breathed upon or physically stimulated with wooden dowels; larvae unable of movement and not maintaining any signs of life were measured moribund or dead [42,43].

HPLC Conditions
HPLC studies were conducted using an Agilent Technologies HP 1100 series chromatograph equipped with a gradient pump, column oven, membrane degasser, UV detector and injector. Separation was conducted using a C-18 column (150 mm × 4.6 mm I.D., 5µm). The column was maintained at 25 • C and the mobile phase flow rate was 0.8 mL/min. Solvent A contained acetonitrile, and solvent B contained 15 mM ammonia water solution. The injection volume was 10 µL. The following gradient program was used as 0-6 min: isocratic at 10% A; 6-12 min: linear gradient from 10% to 40% A; 12-20 min: isocratic at 40% A; 20-25 min: linear gradient from 40% to 85% A; 25-30 min: linear gradient from 85% to 10% A. A 5 min delay was maintained for equilibration of the column and stabilization of the baseline. Total analysis time was 35 min. The peaks were documented at 205 nm. Quantitation of the alkaloids found in plant samples used a seven-point linear regression curve for scopolamine and hyoscyamine. The calculated mean amount of alkaloid (g/g) was based on the weight of the ground dry plants [44].

Statistical Analysis
Data were statistically examined using ANOVA to test the changes among the five concentrations of H. niger and control means. Duncan's test was used to separate means (p < 0.05) using GraphPad Prism 6.0. The percentage of mortalities caused from larvae treated using H. niger were corrected for natural mortality according to Abbot's formula [45].

Conclusions
In conclusion, H. niger seeds alkaloid extract was significantly effective against L. sericata. HPLC analysis revealed that hyoscyamine and scopolamine were predominant compounds in the alkaloid extract of H. niger. Therefore, these results confirmed the folkloric usage of this plant. Furthermore, it could be recommended the alkaloid content of the plant could be responsible for the insecticidal activity. In further studies, we are planning to test the larvicidal effects of hyoscyamine and scopolamine, which are the main constituents of the alkaloid extract of H. niger on L. sericata.