Physiochemical, Insecticidal, and Antidiabetic Activities of Senna occidentalis Linn Root

The present study aimed to investigate the physiochemical activities of Senna occidentalis (Linn) roots and phytochemicals as insecticidal (ethyl acetate and methanol) and antidiabetic (ethanolic extract) activities. Physicochemical properties were carried out by using Association of Official Analytical Chemist methods; thin layer chromatography was carried out according to the Stahl method. Larvicidal activity and LD50 were studied against the third instar of Culex quinquefasciatus mosquito larvae to detect and extract toxicity. The ethanolic extracts of the roots were orally tested at the dose 200 mg/kg for the hypoglycemic effect on induced hyperglycemia in normal rats, assessed in the ethanol extract, and were compared with diabetic control and standards glibenclamide 10 mg/kg. Physiochemical parameters showed high rate in the nitrogen-free extract (69.6%), curd fiber (14.5%), crude proteins (8.15%), ether extract (3.75%), and both ash and moisture (2%), and high concentrations values were found in potassium (43 mg/l) followed by phosphorous (28.5 mg/l), calcium (15 mg/l), sodium (3.65 mg/l), and magnesium (0.145 mg/l). In this part, phytochemical compounds showed high amount of alkaloids, triterpene, flavonoids, tannins, sugars, and few amount of anthraquinone glycosides. Thin-layer chromatography (TLC) studies different colored phytochemical constituted with different Rf values. All the spots are colored under UV light, but some are localized colorless after spaying. The ethyl acetate (EtAc) extract showed eight spots, and the methanol (MeOH) extract showed thirteen spots. The larvicidal activity showed that the ethyl acetate extract was safe against mosquito larvae with an LD50 value 1412.54 (p < 0.05), and the methanol extract had moderate larvicidal activity against mosquito larvae with an LD50 value 257.54 (p < 0.05), while the ethanolic extract of Senna occidentalis (L.) causes a favorable hypoglycemic activity when compared to control significant reduction by [53.15%, 32.87%, and 20.94%], respectively, as well as standard glibenclamide. Based on the various data of the physicochemical parameters, TLC spots, and phytochemical compounds of Senna occidentalis root, they could be used as references standards for manufacturing units of Senna occidentalis root larvicidal and antidiabetic drugs.


Introduction
Standardization of medicinal plants is an essential issue to be considered as a therapeutic drug for safety in health care. Diabetes mellitus is a disease responsible for many deaths around the world [1,2]. Senna occidentalis (Linn), or Cassia senna or Cassia occidentalis, in Sudan, known as Soreib (Ar.), which belongs to the family "Caesalpinioideae," is widely distributed in all of Sudan. Senna occidentalis roots are used traditionally in treating of diabetes and other ailments [3]. However, its adequate validation as therapeutic, specifically antidiabetic and hepatoprotective effects, has not been established [4]. Senna occidentalis is prescribed for use only as a laxative and bowel cleanser for the treatment the colon and in surgery [5]. e seeds of Senna occidentalis are toxic for the children [6,7]. e various extracts of the whole plants were subjected to normal and alloxan-induced diabetic rats. Gidadoand his co-workers [4] and Verma et al. [8] studied the effect of Senna occidentalis leaves on diabetic rats. In traditional medicine, leaves were externally applied to wounds, sores, itch, cutaneous diseases, bone fracture, fever, ringworm, skin diseases, and throat infection and to cure sore eyes, hematuria, rheumatism, typhoid fever, and tuberculosis [9].
Insects, ticks, and mites are dangerous vectors of deadly pathogens and parasites, which may be considered as epidemics in the increasing world population of humans and animals [10][11][12][13][14]. Mosquitoes are the most important single group of insects in terms of public health importance, which transmit malaria, filariasis, dengue, and Japanese encephalitis [15,16]. Reduction of the source of infection is an essential step in the control of mosquito-borne diseases [17], although synthetic organic insecticides have been produced and used to eliminate mosquitoes. e treatment of the disease vectors using synthetic insecticides has failed due to their efficiency in attaining physiological resistance and effect on nontarget organisms [18].
e phytochemical compound present in other parts of Senna occidentalis has been isolated and such compounds are reviewed as sennoside, anthraquinone glycosides, fatty oils, flavonoids, glycosides, galactomannan, polysaccharides, and tannins [8]. e insecticidal activity of the extracts of this plant on vector mosquitoes is not sufficiently reported [19]. e objective of the present work was to determine and investigate the efficiency of the crude extract of the root of Senna occidentalis for larvicidal activity against the Culex quinquefasciatus mosquito larvae, as well as antidiabetic, with reference to physiochemical activities.

Plant Materials Collection and Identification.
Senna occidentalis roots were collected in September 2015 from Al-Debabat city, locality of Algoz, South Kordofan State, Sudan [20]. Samples were identified in the herbarium at the department of phytochemical and natural resource where herbarium no. k. 14/96 was deposited at the herbarium, National Center for Research Medicinal and Aromatic Plants Institute, Khartoum, Sudan.

Preparation of Plant Materials.
e roots part were airdried in shadow, powdered by using a locally made hammer mill, and then, the plant were weighted with electric balance and stored for further analysis.

Preparations of Crude
Extracts. 196 g of the dried plant was weighted and, then, extracted successively with n-hexane (for four hours), ethyl acetate, and methanol extracts (eighteen hours), and the single ethanolic extract was also extracted by using a shaker apparatus at room temperature. All extracts were air-dried and kept separately in universal bottles till used for subsequent experiments. eir physical appearance and percentage yields properties were tabulated in results. Percentage yields were calculated as follows: Weight of extract Weight of sample × 100. (1)

in Layer Chromatography.
e results of the in-Layer Chromatography (TLC) were carried out by using the standard method described by Galib et al. and Stalh [24,25].
2.6. Physiochemical Anaylsis. Physicochemical properties such as the proximate composition (moisture, crude protein, crude fiber, crude lipid, and nitrogen-free extract %) of the roots were investigated using the standard methods of the Association of Analytic Chemist (AOAC, 1990(AOAC, -2005 methods [26][27][28][29]. e mineral compositions of the root sodium and potassium contents were determined by using a flame photometer, while calcium and magnesium contents were determined by using an Atomic Absorption Spectrophotometer (1100 B Perkin-Elmer, Germany) and phosphorus was determined by the vandomolybdate standard colorimetric method [26].

Preparation and Hatching of Culex quinefasciatus.
Culex quinefasciatus egg rafts were collected from stagnant water in the Al kadarow area. e egg rafts were kept in an aluminum dish with 10.6 inches width and 1.6 inches depth containing distilled water till hatching, and the larvae were fed with fine powdered bread. Dead larvae were continuously removed to a void contamination of cultures with pathogens. e third instars larvae for experiments were used.

Experiment Toxicity Tests of Culex quinefasciatus.
e larvicidal activity of the Senna occidentalis root was determined to investigate lethality bioassay against Culex quinefasciatus mosquito third instar larvae of Senna occidentalis roots. e cytotoxicity bioassay experiment was performed according to modification procedure described by Nguta et al. [30]. irty larvae were selected and transferred into three test tubes by means of a 23 cm disposable Pasteur pipette from an aluminum dish, each containing 5 ml of distilled water. e samples for the experiment were prepared by dissolving 20 mg of each different extracts in each 5 mL of dimethyle sulphoxide (DMSO). Appropriate amounts of this DMSO solution (5 µL, 50 µL, and 500 µL) to give three different concentrations of ethyl acetate and methanol extracts (10 ppm, 100 ppm, and 1000 ppm ppm (or mg/L), respectively) were transferred into 100 ml test tubes (3 test tubes for each dose and 1 for control). ree replicates were prepared for each dose level and, then, dried further in vacuum for 1 h to remove DMSO completely. Control was prepared using only DMSO, and then, the thirty larvae were placed in each test tube. e surviving larvae were counted with the aid of a 3x magnifying glass, after 24 hours. e second count was performed after 72 hrs, and larvae were considered after dead when they fail to rise to the surface or settle motion less in the bottom of the test tube.

LD 50 Determination.
e lethal death fifty (LD 50 ) of plant extracts resulting in 50% mortality of the larvae of Culex quinefasciatus LD 50 and 95% confidence intervals were determined from the 72-hour counts, and the dose response data were transferred into LD 50 analyzed by Finney [31]. Probit Analysis computer program was used to determine lethal death fifty (LD 50 ) values. Potassium dichromate (K ₂ Cr ₂ O ₇ ) was used as a positive control with an LD50 value 1.7 μg/ml (toxic) and DEMSO (1000 μg/ml) as a negative control in the bioassay experiments (LD50 726129 × 10 11 μg/ml) according to the modification by Meyer et al.. LD 50 values below 250 μg/ml were considered as highly toxic, 250-499 μg/ml as moderately toxic, and 500-1000 μg/ml as lowly toxic. Values above 1000 μg/ml were regarded as safe or nontoxic according to McLaughlin and co-workers [32].

Study of Hypoglycemic Effects on Induced Hyperglycemia in Rats as Experimental Animals.
Twenty-four healthy adult male albino strain rats weighing 150-250 g aged between 6-12 months ( Figure 1) were purchased from the department of animal house laboratory, Faculty of Veterinary Medicine, Khartoum, Sudan. e rats were kept in a well-aerated laboratory and were divided into four groups with six rats each in cages. e animals were fed freely on standard animal feeds with balanced diet and drinking water ad libitum during the experimentation period. e animals were exposed to a 12 h light-dark cycle.

Experimental Design of the Glucose Loading Induction
Model. Experimental rats were fasted from food for eighteen hours prior to experimentation and had water ad libitum. e first group (I) was administered with 10 ml/kg body weight of distilled water and considered as the normal control group (I). e second group (II) was administered with 2 g/kg body weight of glucose loading and considered as positive control. e third group (III) was administered with 10 mg/kg body weight of the hypoglycemic drug-glibenclamide and considered as the standard group. e last groups (IV) were treated with 200 mg/kg body weight the ethanolic extract of Senna occidentalis roots. All four groups of rats were, then, loaded with 50% glucose solution at a dose of 2 g/kg. e blood glucose level of all groups was monitored after 1, 2, and 4 hours after the glucose load according to Konuklugil et al. [33].

Determination of the Blood Glucose Level.
e fasting blood glucose levels were first determined at "0" minutes of initial fasting blood samples which were collecting from the overnight fasted rats, by drawing out about 1 ml of the blood samples from eye side (retro-orbital plexus) cervical dislocation by using heparnized-capillary tubes under mild diethyl ether inhalation anaesthesia on a four-hour interval (starting with the fasting plasma glucose levels of the four groups of rats were determined (0 time), then 1, 2, and 4 hours) for a period of one day. A manual digital spectrophotometer was used to analyze blood glucose levels throughout the experiment according to method described by Trinder [34].

Statistical Analysis of the Blood Glucose Level.
Results of the blood glucose level were expressed as mean ± standard error of the mean. e data were statistically analyzed using analysis of variance (ANOVA) with Duncan Multiple Range Test comparisons versus control groups. e values of p < 0.05 were considered as significant according to Duncan et al. [35].

e Physical Properties and Yields of Senna occidentalis
Root.
e results of physical properties such as consistency, color, and extractive value percentage yields of successively extracted in an order of nonpolar to polar solvent based on increasing degree of polarity are reported in Table 1. High values were found in polar solvent methanol (MeOH) followed by intermediate polarity ethyl acetate (EtAc) and nonpolar n-hexane (n-hex), respectively. Low n-hexane extractive value signifies the presences of amounts of fats, lipids, and some steroids in the plant materials and may indicate the addition of exhausted material, adulteration or incorrect processing during drying, or storage or formulating [1].

in-Layer Chromatography.
e results of the in-Layer Chromatography (TLC) separated the various phytochemical components in ethyl acetate and methanol extracts of Senna occidentalis root, by using solvent system toluene: ethyl acetate in the ratio (70 : 30) as described by Fried and Sherma [36] and Wagner and Bladt [37].  , S10 � 0.21 (light green fluorescent), S11 � 0.16 (arc light green fluorescent), and S12 � 0.08 (arc green fluorescent). ese colors indicated the presence of constituents may be with R f values 0.38 (white sky blue to white green fluorescent), 0.44 (red), 0.88 (violet) alkaloids (white blue fluorescent), phenolic compounds (blue to white green fluorescent), negligible amount of alkaloids (very weak intensity), or saponins with glycosides in the roots with R f (0.18, 0.32 (both orange) and a blue fluorescent spot R f (.0.16, 0.32, 0.72, and 0.97) respectively, and these could not be isolated; but, on spraying with anisaldehyde sulpharic acid reagent and heating plate (B) for ten minutes at 110°C, spots of purple color appearing at grey dark violet colors were detected with two R f values between 0.50 and 0.21 and constituents may be tannins or triterpine and phytosterol; Wagner and Bladt [37]. ese colorless ones in the same plate constituents may be oils ese results agreed with the chemical constituents reported by Wagner and Bladt [37] and Tsakala et al. [38].

Results of Phytochemical Screening of Senna occidentalis
Root.
e general results of phytochemical screening are shown in Table 2. Tests supplementary Figure 1 showed the different testing of preliminary phytochemical screening of Senna occidentalis root; it gave the colors with few modifications according to the colors of extracts indicating the presence of the high concentrations of the various active phytoconstituents metabolites as follows: alkaloids (Dragendorff's Orange brown, Mayer's Yellow to creamshies,  Also, similar findings were reported in [8] in the other species of the same family. According to TLC results in Figure 3(a), tthyl acetate and methanol extracts of this plant, under 254 nm UV light, showed the presence of eight and twelve spots, respectively, where different R f values in colors may be flavanoids (orange to red yellow fluorescent), alkaloids (white blue fluorescent), and phenolic compounds (blue to white green fluorescent) and, after spraying with 1% anisaldhyde-sulphate reagent, showed few colored spots in the Et Ac extract and colorless spots in the MeOH extract with R f values; Figure 3(b) suggests the presence of many compounds in the extracts. e Et Ac and MeOH extracts were subjected to the phytochemical screening for the detection of various plant constituents. It was found that terpenoids, flavanoids, and phenolic compounds were present as the major active principle [36,37]. e active principles of many drugs found in plants are secondary metabolites, which are widely used in traditional medicine to treat various ailments. Galib et al. [24] mentioned that Senna occidentalis root could be used as larvicidal and antidiabetic.

Physiochemical Anaylsis.
e nutrition composition percentage of plant powder of S. occidentalis root, Table 3, was determined with the high percentage of physiochemical which was found to be in dry matter (98%) followed by the nitrogen-free extract (69.6%), curd fiber (14.5%), curd proteins (8.15%), ether extract (3.75%), and both ash and moisture (2%). Many plant proteins are low in one of the essential amino acids, but a combination of plant proteins such as grains with pulse or seeds may give a high-quality protein which is just as good as the protein from animal foods. Table 4 showed the mineral composition Senna occidentalis root, and high concentrations values were found in potassium (43 mg/l or ppm) followed by phosphorous (28.5 mg/l), calcium (15 mg/l), sodium (3.65 mg/l), and magnesium (0.145 mg/l). Similar findings were reported by Agbugui et al. [40] in the other species of the same family. He showed these elements from a part of the rigid body structure, soft tissue, and body fluids of most vertebrates, and Anhwange et al. [41] reported that, in general, minerals work in combination with each other and other nutrients; therefore, deficiency of any mineral may cause health problems. Sodium and potassium maintain water balance in cells, and they are important for nerve impulse transmissions and stimulation of normal movement of the intestinal tract. Glew et al. [42] reported that magnesium is essential because it maintains, repairs cells, and provides energy and its deficiency may result in vertigo, convulsions, nervousness, and heat palpitation. It also assists the muscles to keep reservoir of oxygen and increases the body's resistance to infection. Its deficiency results in anemia, tiredness, insomnia, and palpitations.

Cytotoxicity Bioassays (Larvicidal Activity) of Senna occidentalis Root.
e results of cytotoxicity bioassays with n-hexane, ethyl acetate, and methanol from the root extracts showed good larvicidal activities for all extracts; high mortality was observed with high concentration (1 g/L) representing 1000 ppm dose of methanol extracts, and low mortality was observed in low to moderate concentrations (0.01 and 0.1 g/L) representing 10 ppm and 100 ppm. 100% mortality was not obtained at all concentrations. In general, n-hexane and ethyl acetate were showed safe effects and methanol showed moderate effect; Table 5. Kumar et al. [43] showed the significant effect at other doses than that reported in this study, 100% mortality effect of petroleum ether and butanol of larvacidal activity of C. occidentalis against mosquito Culex quinquefasciatus at (0.2 and 0.3 g/L) representing 200 ppm and 300 ppm, respectively. is studies of larvicidal activity of Senna occidentalis root n-hexane, ethyl acetate, and methanol extracts had LD 50 values 1564721 ppm, 1412.54 ppm, and 1412.54 ppm, respectively, and were carried out against 3 rd instar of Culex quinquefasciatus mosquito larvae (Table 5), which revealed that the results of the lethal doses determined in both n-hexane and ethyl acetate extracts were larvicidal significant (p < 0.05) and highly effectively safe for mosquito larvae with increased LD 50 values of 1564721 and 1412.54, respectively, and this indicates that nonpolar (n-hexane) and intermediate polar ethyl acetate have more potent activity to larvae. e methanol extract showed least effective larvicidal significant (p < 0.05) moderate effect for mosquito larvae with an LD 50 value 257.54 ppm.
In Table 5, the aquatic immature larvae stage is recognized as the most vulnerable and best control strategy to effectively reduce mosquito population densities during infestations. Senna occidentalis root displayed good     larvicidal effect considered to larvae safe (nontoxic) control by the solvent DMSO with an LD 50 726129 × 10 11 at all concentrations according to the modification of Meyer et al. [44] and Duke et al. [45]. Yadav et al. [19] and Duke et al. [45] previously reported that the phytochemicals screening possess insecticidal activity which are present in plants of this genus, and it could be responsible to the larvicidal activity of Senna occidentalis root in n-hexane, ethyl acetate, and methanol extracts or it may be due to containing such phytochemicals and related compounds that display a role in plant defense against pests and insect and might have been responsible for larval deaths at high concentrations.

Effects of the Roots Supplement on the Blood Sugar Level (BSL).
In the present study, the effects of the ethanolic extract of Senna occidentalis (L.) roots were determined on blood glucose levels of all animals treated with the supplement and were seen to reduce significantly (p < 0.05) by about 58.25%, 48.59%, and 41.93%, respectively, when compared with that of the untreated diabetic control group (Table 6) and cause a favorable hypoglycemic activity when compared to fasting glucose levels within groups ( Figure 3) significant reduction by 53.15%, 20.94%, and 32.87%, respectively, but with standard glibenclamide 10 mg/kg significant reduction only between one hour with the value 37.10% and a significant increasing for the other 29.56% and 18.96%, respectively ( Figure 4). is study proved the use of Senna occidentalis roots in traditional medicine for the treatment of diabetes and showed that Senna occidentalis roots supplement has good potent hypoglycemic effects which may be due to its high contents of active phytochemical constituents such as flavonoids that was reported in most medicinal plants with hypoglycemic and antidiabetic properties. is finding agrees with those of Gidado, et al. [4] and Verma et al. [8].
e phytochemical profiling of Senna occidentalis root extracts presented in this study revealed a diverse range of the bioactive phenolics compound. Some of the identified bioactive phenolics were reported by several authors for antidiabetic activities; Valle et al. [46], Can et al. [47], and Mohammed et al. [48]. Besides, the larvicidal activity was carried out against Culex quinquefasciatus mosquito larvae, which showed that the methanol extract showed moderate effects and n-hexane and ethyl acetate extracts showed safe effects. e larvicidal activities clearly showed safe effects in this part at high dose required for insecticidal action (1000 ppm), but the methanol extract is lower than doses used in animal study (200 ppm) for diabetic action.
is was an attempt to identify the potent antidiabetic and pesticides compounds from Senna occidentalis root.
is plant is widely distributed, and it can be predicted that the potent antidiabetic and larvicidal activity of Senna occidentalis root is due to presence of compounds that have potential to be used in local communities for antidiabetic activity and also used as mosquito control during infestation seasons, thus promoting the use of natural antidiabetic and pesticides. It is concluded that Senna occidentalis (L.) in parts showed higher potent hypoglycemic activity in glucose-loaded diabetic rats than other plants. However, further effects can be investigated by the increase in the treating dose of the extract, and other pharmacological studies should be performed, specifically on dose 200 mg/kg, which was presented as the antidiabetic activity. Isolation and identification of more bioactive compounds were highly recommended. In vivo studies are needed to be conducted on these active ingredients to study the mechanism of action by which these compounds exert their effect as an antidiabetic activity. I  recommended studying the antidiabetic activity using alloxan-inducing diabetic type 2 in same dose 200 mg/kg and histopathological in animal by the same manner. Gidado et al. [4] and Verma et al. [8] they reported in their previous studied on Senna occidentalis leaf part and whole parts showed β-cell regenerating potential as depicted by the histopathological studies of the pancreatic tissue, even though some cytotoxic agents such as the cytotoxic saponins may be present in the supplement which is capable of causing damage to both the pancreas and liver and induced hepatotoxicity.

Data Availability
We have already included all data in the manuscript, the lab and data, phytochemical screening, cytotoxicity-larvicidal activity, antidiabetic activity, and nutrients and minerals.

Ethical Approval
Some were applicable to animal ethic clearance according to standard protocol with the rules of the Institutional Ethical Governing Committee of the Faculty of Medicine and the Sudanese Government National Council for Science and Technology, and other presented studies are purely based on chemical analysis in the lab, not in humans.