Antidiabetic Potential and HPTLC Fingerprinting of Schleichera oleosa

Introduction: Schleichera oleosa (Lour) Oken., also known as Lac tree, is a traditional plant used to treat different ailments since ancient time. The folkloric use of this plant as hypoglycemics is still un-revealed scientifically. So, the present study includes the in-vivo evaluation of hypoglycemic activity with HPTLC fingerprinting, qualitative and quantitative phytochemical screening of the extracts of the plant. Methods: Ethanolic extract of the plant (SOE) was subjected for phytochemical screening and HPTLC finger printing (using CAMAG LINOMAT 5 instrument). For the evaluation of hypoglycemic activity Streptozotocin (STZ) induced diabetic rats were treated with glibenclamide (0.5mg/kg body weight) and ethanolic extract (100 mg/kg and 200 mg/kg body weight) of the plant. Acute and chronic studies were performed for the evaluation of blood glucose levels. Results: The presence of alkaloids, tannins, phenolics and flavonoids were confirmed in the preliminary phytochemical screening. Quantitative screening of total tannins (6.15±4.20), total flavonoids (25.13±3.21) and total phenolic compounds (48.09±2.58) were reported. HPTLC fingerprinting analysis of SOE affirmed the presence of quercetin. Conclusion: The positive outcome was observed in the results of hypoglycemic activity, as all the treatments significantly decreased blood glucose level. During the study, SOE and glibenclamide maintained the body weight of the rats while diabetic control reduced the body weight by 11.33%. In support of its traditional use Schleichera oleosa (Lour) Oken. was proved to be a potent antidiabetic herbal plant.


INTRODUCTION
Hyperglycemia is a result of high blood glucose in plasma and diabetes is the authentication mark of it. 1 Diabetes mellitus is a group of metabolic disorder mainly characterized by high blood glucose level in the blood (hyperglycemia), kidney (glycosuria), hyperlipidemia and ketonaemia. Type II diabetes also called as maturity onset diabetes mellitus, is caused due to abnormal function of glucoreceptors of β-cells of the pancreatic islet of Langerhans resulting in insufficient production of insulin. 2 The global scenario of diabetes is very alarming, as the counts of diabetic affected people will be more than 640 million by 2040. In India, the management of diabetes is very challenging mainly because of lacking public awareness and expensive cost of medications. 3 Diabetic-kidney disease, liver diseases and coronary artery diseases are the common and severe complications associated with diabetes. 4 The oral hypoglycemic drugs have potential adverse effects like low blood sugar, obesity, gastrointestinal disturbances, dizziness etc. 5 The choice of medicinal plants as an alternative approach for the management of diabetes is helpful to get rid of the diabetic complications. 6 Schleichera oleosa (Lour) Oken., belongs to Sapindaceae family, is a medicinal plant having the plethora of ailment treating properties. The plant possesses several biological effects like antiulcer, anticancer, anti microbial activities. Traditionally the plant is used as an antidiabetic. 7 Though the in-vitro study of the antidiabetic activity of Schleichera oleosa (Lour) Oken. has been reported recently by evaluating alpha-amylase inhibition, 8 scientific in-vivo evaluation has not been done yet. The present research deals with the in-vivo evaluation of hypoglycemic activity on streptozotocin induced diabetic rats and the High Performance Thin Layer Chromatography (HPTLC)-fingerprinting analysis of Schleichera oleosa (Lour) Oken. Crude extracts preparation The shade-dried cleaned leaves were powdered in grinder mixer, passed through sieve no. 40. The crude ethanolic (ethanol 95.5% v/v) extract of the powder was prepared by hot percolation process using Soxhlet apparatus at 70º-80ºC for 32 h followed by distillation and drying. The crude extracts (SOE) were kept at desiccators for further experiments.

Qualitative and quantitative Phytochemical screening
The qualitative identification of phytoconstituents of the ethanolic extract for alkaloids, tannins, flavonoids and phenolic compounds were performed by following standard methods. For alkaloids Mayer's, Dragendroff 's, Hager's and Wagner's tests were performed. Chloride test and Vaniline tests were performed to identify tannins. Flavonoids were identified by the treatment of extract with aqueous sodium hydroxide, concentrated sulphuric acid and Shinoda's test, whereas, ferric chloride, lead acetate and bromine water were used to assure the presence of phenolics. 9 The quantitative estimations of tannins (Folin-Ciocalteau assay), 10 flavonoids (Aluminium chloride colorimetric assay) 11 phenolic compounds (Folin-Ciocalteau reagent-15% sodium carbonate assay) 12 were examined by the standard methods with minor modifications in triplicates and the results were represented as mg of Tannic acid, Quercetin and Gallic acid equivalents per gm dry weight respectively.

HPTLC fingerprinting
The study of HPTLC fingerprinting was done according to the standard procedures. [13][14] The analysis was done by CAMAG LINOMAT 5 instrument (Camag; Muttenz, Switzerland), operated by WINCATS software. The ethanolic extract of Schleichera oleosa (Lour) Oken. was dissolved with HPTLC grade ethanol (100 mg in 1ml) followed by centrifugation. Quercetin was taken as standard. Sample solution and standard solution (each of 2µl) were sprayed on Silica gel 60F 254 TLC plate as bands of 5mm length 10 mm from the bottom edge, 10 mm from the side edge and 6 mm apart using 100µl Hamilton syringe. Saturation of the chamber was done with Toluene: Ethyl acetate: Formic acid (13:11:2), as the mobile phase for 25 min at room temperature. The chromatogram was developed by following ascending technique in CAMAG twin trough developer from the point of sample application to approximately 80 mm solvent front in given mobile phase followed by the drying of the plates in hot air to remove the solvent. The plates were kept in CAMAG REPRO-STAR 3 (photo-documentation chamber). The images were taken at the different wavelength of 254 nm and 366 nm. After derivatization, the plates were fixed in CAMAG TLC SCANNER 3 for densitometric scanning, done at 500nm. All the data like peak height, peak area, R f values, peak densitogram were denoted.

Hypoglycemic activity Experimental animals
For the experimental design healthy albino Wistar rats (160±20 g of body weight) were selected and the animals were kept in the animal house at the standard condition with commercial pellet diet and water. The animals were kept for 1 week before commencement of experiments and all the experiments were carried out by the approval of Institutional Animal Ethics Committee (IEAC) of Suresh Gyan Vihar University (Approval No: SGVU/PH/IAEC/2017/01).

Diabetes induction
Healthy selected rats were fasted overnight before induction of diabetes through Streptozotocin (STZ). Intraperitoneal injection of STZ at 50mg/kg body weight in 0.1M sodium citrate buffer was administered. Induction was confirmed after 48 h of administration. >200mg/DL value of fasting glucose level was confirmed as diabetic animals and screened for the further experiments. Glibenclamide (GLI) was used as standard drug.
The dose of the plant extract was selected by following the acute toxicity study. 15

Experimental design
The following five diabetic groups (n=5) were designed for the activity: Group I -Non-diabetic control (NC) Group II -Diabetic control (DC) Group III -Diabetic rats administered with GLI (0.5 mg/kg body weight) (DC+GLI) Group IV -Diabetic rats administered with SOE (DC+SOE) in the dose of 100mg/kg Group V -Diabetic rats administered with SOE (DC+SOE) in the dose of 200 mg/kg

Acute study
After the administration of GLI and SOE, the blood glucose level was noted at 0,2,4,6 and 24 h.

Chronic study
After daily dosing of GLI and SOE plant extracts, the body glucose of the rats was monitored for 28 days at the interval of 0, 7, 14, 21 and 28 days.

Blood glucose estimation
The blood samples were collected from all the rats through tail-vein and blood glucose levels were measured (in mg/dL) instantly by using glucometer (Accu-chek, Roche Diagnostic, Mannheim, Germany). 16

Body weight estimation
Body weight of the rats was recorded before (at 0 days) and after (at 28 days) of the study. The percentage change of the body weight was recorded and tabulated.

Statistical analysis
The results were expressed in ±SEM (n=5) and the comparison between the groups were determined followed by Tukey's multiple comparisons test, * p<0.01, ** p<0.001 were considered as significant. All the graphs were drawn in GraphPad Prism 7.4 software.

Qualitative and quantitative phytochemical screening
The ethanolic extract showed the presence of alkaloids, tannins, flavonoids and phenolic compounds, tabulated in Table 1. Quantitative estimations of total tannin, total flavonoids and total phenols were reported in Table 2 as 6.15±4.20 mg TAEg, 25.13±3.21 mg QEg, 48.09±2.58 mg GAEg dry weight respectively.

HPTLC fingerprinting for Quercetin
HPTLC fingerprinting of SOE was performed with the suitable solvent system and visualized under UV 254nm and 366 nm. The chromatogram and densitogram were given in Figure 1 (Figure 3). The R f values, peak areas, height has been tabulated in detail in Table 3.

Hypoglycemic activity
Acute and chronic studies were performed to assess the hyperglycemic effect of SOE in two different doses which exhibited significant activity. To predict the acute effect, the blood glucose levels at the different time interval (0,2,4,6 and 24 h) were measured and reported in Figure 4. The reduction of blood glucose levels of glibenclamide on diabetic rats were highest (21.5%) than the higher dose of SOE (19.16%) and the lower dose of SOE (15.59%) in 24 h. For the long-term effect of the extract on hyperglycemia, the chronic study was conducted and the blood glucose levels were reported after the study of 28 days at 7 days interval ( Figure 5). Among the two extracts, high dose of SOE (200mg/kg) lowered the blood glucose more significantly (from 287±5.10 to 140±5.70 mg/dl) than low dose of SOE (100mg/kg) (from 295±4.70 to 180±3.54 mg/dl); however, the effect of glibenclamide was greatest among all (from 293±4.20 to 129±5.10 mg/dl). The preventing effect of body weight loss of SOE was conducted at first day and 28 th day after the study (Table 4). Glibenclamide, SOE (200mg/kg) and SOE (100mg/kg) showed 7.5, 10.37 and 11.39 % increase in body weight, whereas, diabetic control decreases the body weight by 11.33% ( Figure 6).

DISCUSSION
Binding of insulin at alpha-subunit of insulin receptor activates tyrosine kinase activity at beta-subunit of the receptor followed by cascade of phosphorylation and dephosphorylation reactions which causes the activation of secondary messengers, MAP (Mitogen-Activated Protein)      kinase and Glucose Transporters (GLUT) to maintain the normal blood glucose. The insufficient production of insulin fails to regulate normal blood glucose level and causes diabetes. The phytochemical screening of SOE affirmed the presence of tannins, flavonoids and phenolic compounds. Tannins increase the glucose uptake by activating GLUT-4, MAP kinase and secondary messengers like PI3K (Phosphoinositide 3-Kinase). 17 Stimulation of insulin production, preventing gluconeogenesis, enhancing insulin sensitivity and glucose uptakes are the few possible mechanisms of flavonoids in the regulations of diabetes. 18 α-amylase and α-glucosidase enzymes of the digestive tract can delay the absorption of glucose, so, their inhibition by phenolic compounds helps to control the post-prandial hyperglycemia. 19 HPTLC fingerprinting is an accurate and precise instrumental technique to identify the phytoconstituents and adulterants present in medicinal plants. [20][21] HPTLC serves as an important highly efficacious technique to establish the appropriate quality control parameters for the herbal extracts. 22 The present investigation of HPTLC fingerprinting of SOE affirmed the presence of quercetin along with twelve other compounds. Quercetin which is a part of the subclass of flavonoids groups of polyphenols, is an important aspect of research because of its versatile effect mainly on degenerative diseases by inhibiting lipid peroxidation. 23 The earlier studies reported that quercetin has significant hypoglycemic effect in STZ induced rats perhaps by increasing the insulin secretions. [24][25][26] Despite all the effective researches in synthetic chemistry for oral hypoglycemic drugs, the reliability on medicinal plants for the management of hyperglycemia is a long practice almost all over the world due to its affordability. 27 The use of medicinal herbs is the important aspect of an alternative approach to manage diabetes. 6 The presence of several active phytoconstituents in medicinal herbs serve a protective and therapeutic effect on diabetes mellitus by the reduction in insulin resistance, preventing glycogenolysis and gluconeogenesis. 28 The present work evaluated the hypoglycemic activity of SOE in STZ induced diabetic rats and the results were significant (* p<0.01, ** p<0.001 were considered as significant) as compared to diabetic control. STZ, obtained from a gram-positive bacterium, is uptake by pancreatic β-cells (via GLUT-2) to destroy them by several proposed mechanisms. 29 In case of acute study, the reduction of blood glucose of glibenclamide treated diabetic rats in first 6 h were found higher than SOE-high dose and SOE-low dose. The similar result was reported for Rhizophora mucornata in which high All values were expressed as ± Standard error of the mean (n=5) Figure 4: Acute hypoglycemic effect of SOE. All values were expressed as ± Standard error of the mean (n=5), followed by Tukey's multiple comparisons test, * p<0.01, ** p<0.001 were considered as significant. All values were expressed as ± Standard error of the mean (n=5), followed by Tukey's multiple comparisons test, * p<0.01, ** p<0.001 were considered as significant. dose (200mg/kg) of hydroalcoholic extract of the plant was found more potent than the low dose in first 6 h of the study. 16 For the daily treatment of SOE and glibenclamide up to 28days, it was found that the higher dose of SOE (51.21%) was more significant than SOE low dose (38.98%) in blood glucose lowering while compared to glibenclamide (55.97%). The similar study of the hypoglycemic effect of Kyllinga triceps plant extracts at 200mg/kg on STZ induced rats was reported as a significant (47.2% blood glucose reduction) than low dose. 30 The result of improvement in body weight of the rats for the daily treatment of SOE was important aspect in maintaining the body weight without altering the normal health, as obesity is the important issue for the management of type-2 diabetes. 31

CONCLUSION
In recent scientific research the alternative approach for the management of diabetes is an important exposure. The present study proved Schleichera oleosa (Lour) Oken. as a hypoglycemic agent. The presence of quercetin will help to isolate and characterize the active constituents responsible for the hypoglycemic activity and will be the future scope.