Fazle Rabbi,Amir Zada,Amna Nisar,Achyut Adhikari,Irfan Ullah,Shafiq Ur Rahman

Fazle Rabbi,Department of Pharmacy,Abasyn University Peshawar,Peshawar 25000,Khyber Pakhtunkhwa,Pakistan

Amir Zada,Amna Nisar,Department of Pharmacy,University of Peshawar,Peshawar 25120,Khyber Pakhtunkhwa,Pakistan

Achyut Adhikari,Central Department of Chemistry,Tribhuvan University,Kritipur 44613,Kathmandu,Nepal

Irfan Ullah,Department of Pharmacy,Sarhad University of Science and Information Technology,Peshawar,25000,Khyber Pakhtunkhwa,Pakistan

Shafiq Ur Rahman,Department of Pharmacy,Shaheed Benazir Bhutto University,Sheringal,Dir (Upper),18000,Khyber Pakhtunkhwa,Pakistan

Abstract OBJECTIVE: To investigate Sterculia diversifolia G.Don for potential anti-diabetic activity in the in vivo mouse model of alloxan-induced hyperglycemia.METHODS: Sterculia diversifolia (S.diversifolia) was subjected to extraction and isolation techniques and structural characterization of the isolated compounds were performed using spectroscopic methods.The acute toxicity test was performed by orally administering S.diversifolia in doses of 500-2000 mg/kg.For the assessment of anti-hyperglycemic activity,S.diversifolia bark and leaves extracts were administered orally in doses of 50,100,and 200 mg/kg,along with metformin(150 mg/kg,i.p) as positive control,after confirmation of alloxan (150 mg/kg,i.p.) induced hyperglycemia in BALB/c mice.Serum biochemical parameters were monitored for the period of study.RESULTS: The phytochemical studies showed the presence of quercetin and kaempferol in S.diversifolia.The IC50 values in the in vivo acute toxicity study revealed the safety margin of S.diversifolia bark (1166.66 mg/kg) and leaves (683.34 mg/kg) extracts.A significant attenuation of alloxan induced hyperglycemia was produced by S.diversifolia extracts at 50 mg/kg (P ＜ 0.05),100 mg/kg(P ＜ 0.05,＜ 0.01),and 150 mg/kg (P ＜ 0.05,＜ 0.01,＜ 0.001) during 1-4 h,which was comparable to metformin (P ＜0.001).Significant (P ＜ 0.001) improvement appeared in blood hemoglobin,protein,cholesterol,triglycerides,urea,creatinine,HDL,and LDL of the stem bark and leaves extracts treated diabetic mice.CONCLUSION: These findings connote the usefulness of S.diversifolia as an anti-diabetic in traditional medicine and this might be attributed to the presence of quercetin and kaempferol,among other phytochemicals.

Keywords: Sterculia; hypoglycemic agents; quercetin;Kaempferols;biochemical parameters

1.INTRODUCTION

Recently,it is highly desirable to explore and discover economical,safe and effective therapeutic remedies possessing minimal adverse effects tendencies and bestow beneficial effects in diverse pathological conditions.1This can be achievable after thorough research on chemicals derived from both natural2-4and synthetic origin.5Diabetes mellitus is a chronic metabolic disorder characterized by marked increase in blood glucose,which is clinically diagnosed as hyperglycemia.It is one of the leading causes of disease associated morbidity and mortality throughout the world.The sustained hyperglycemia severely affects patient′s overall quality of life due to progressive worsening of physiological functions.6currently various drugs are clinically available to manage diabetes mellitus and associated symptoms.However,the therapeutic effectiveness of these drugs is greatly limited by the occurrence of dose related toxicities,and the overall economic burden associated with the management of disease state.7A lot of natural products have been investigated for their anti-hyperglycemic effects and other beneficial properties in diabetes mellitus and its associated symptoms.8It is suggested that natural products particularly herbal products can be effectively used in diabetes mellitus management due to their wide availability,cost effectiveness and low adverse effects propensities.9Sterculia diversifolia (S.diversifolia) belongs to genus Sterculia and family Sterculiaceae.10,11Different species of Sterculia were used traditionally for inflammation,gastrointestinal disorders,diabetes,skin diseases,microbial infection and many other conditions treatment.This genus is famous for a variety of medicinal uses and has been scientifically proven to exhibit diverse pharmacological effects including antidiabetic property.Various species of this genus including Sterculia villosa,Sterculia foetida,Sterculia lychnophora,Sterculia rupestris have been thoroughly investigated for attenuation of hyperglycemia in differentin vivoanimal models.12,13Preliminary phytochemical study on S.diversifolia revealed the presence of carbohydrates,coumarins,alkaloids,glycosides,saponins,tannins,flavonoids,steroids,terpenoids and vitamin-C.14Medicinally,S.diversifolia bears antimicrobial,antioxidant,cytotoxic,analgesic,anti-inflammatory,anti-pyretic,anti-glycation,immunomodulatory,insecticidal,larvicidal,leishmanicidal,anthelmintic,anticonvulsant and CNS depressant activities.10,11,14-16Considering the prominent anti-diabetic potential of the genus Sterculia,the present study investigated the antihyperglycemic potential of S.diversifolia in a refined mouse model of hyperglycemia.Additionally,S.diversifolia was subjected to column chromatographic techniques for isolation of secondary metabolites that might be involved in the anti-hyperglycemic effect of S.diversifolia.

2.MATERIALS AND METHODS

2.1.Chemicals and equipments

Alloxan monohydrate,metformin (Research Organics,St.Louis,MO,USA),commercial grade n-hexane,dichloromethane,ethyl acetate,n-butanol,and methanol(Musaji Adam and Sons,Karachi,Pakistan),glucometer(Optium Xceed,Abbot Laboratories,Alameda,CA,USA),rotary evaporator (Hahnshin Scientific Co.,Bucheon-si,South Korea),vortex mixture (Static mixer corporation,Chicago,IL,USA),weighing balance(Shimadzu Corporation,Kyoto,Japan),and grinder(Moulinex,Paris,France).

2.2.Plant material collection

The plant material of S.diversifolia (stem bark and leaves) was collected from Pakistan Forest Institute botanical garden,University of Peshawar,Peshawar,Pakistan,in September,2014.It was identified by a taxonomist (Mr.Ghulam Jelani) at the Department of Botany,University of Peshawar and a specimen was deposited in the Institution’s herbarium vide reference No.Bot.20098 (PUP).17

2.3.Extraction and fractionation

The stem bark (17 kg) and leaves (13 kg) of plant at room temperature were dried under shade.It was then crushed to powder using a grinder which was then macerated using CH3OH for 14 d (2 × 7 d),after which it was filtered (Whatman-1 filter paper).Rotary evaporator is used to concentrate extracts under reduced pressure at 40 ℃to obtain the crude methanolic extracts of stem bark(MESB) and leaves (MESL).15Obtained extracts (950 g for bark and 1200 g for leaves) were mixed with distilled water (2.5 L) and soaked (24 h).It was then extracted successively with n-hexane (3 × 2.5 L),dicloromethane(DCM) (3 × 2.5 L),ethyl acetate (3 × 2.5 L),and nbutanol (3 × 2.5 L) to obtain the respective solvents soluble fractions.The remaining portion was considered as aqueous fraction.

2.4.Isolation of compounds

The ethyl acetate fraction of both stems bark and leaves(20 g and 25 g respectively) were fractionated over a silica gel (60-120 mesh) column eluted with n-hexane,nhexane: ethylacetate,ethylacetate,ethylacetate: methanol and methanol with a graded increase in polarity.A total of seven (07) major fractions of stem bark extract and eight (08) fractions of leaves extract were collected.The fractions 1 and 5 were collected and rechromatogrphed using silica gel column and eluted with n-hexane: ethyl acetate.From the fraction 1,four subfractions were selected.The sub-fraction 2 and 3 of stem bark and leaves,respectively were mixed.These subfractions were once again fractionated over the silica gel column and eluted with increase of ethyl acetate content,so that sub-fractions having similar Rf value were collected to afford 200 mg and 335 mg quercetin (98%),43 mg and 33 mg kaempferol (97%) from the stem bark and leaves extracts,respectively.

2.5.Experimental animals

Swiss-albino mice of either sex (4-5 weeks) were purchased from the Veterinary Research Institute Peshawar,Pakistan.They were kept in the Animal House of the Department of Pharmacy,University of Peshawar under standard laboratory conditions.The institutional ethical committee has approved the experimental procedures on these animals (reference number 03/EC-16/Pharm).

2.6.Acute toxicity test

Swiss Albino mice (n=6) of either sex were used.They were administered with extracts in different doses.Normal saline was given to control group at a dose of 10 mL/kg,post orally while the rest of groups were given bark and leaves extracts in doses of 500,1000 and 2000 mg/kgviaoral route.All the groups were observed for abnormal behavior during the first 4 h and mortality was observed after 24 h.18

2.7.Anti-diabetic activity

The overnight fasted Swiss albino mice weighing 25-30 g were intra-peritoneally administered with alloxan monohydrate (150 mg/kg).After 1 h,the mice were provided water and food.The blood glucose level (BGL)was monitored by a glucometer using the tail tip method.19After 72 h those mice were selected having BGL above 150 mg/dL and distributed into various groups (n=6).Group Ⅰ was given with 10 mL/kg normal saline,while Group Ⅱ was given with 150 mg/kg metformin.Group Ⅲ-Ⅴ were administered with MESB at 50,100 and 150 mg/kg respectively.Groups Ⅵ,Ⅶ,Ⅷwere administered with MESL at 50,100 and 150 mg/kg.All the treatments were administeredviaintra peritoneal route,while the BGL was measured at 0 h as pre-dose and after 1,2,3 and 4 has post-dose.

2.8.Biochemical analysis

The blood sugar level was measured by collecting the blood from the vein of mice tail using Accu-Chek Active test strips in Accu-Chek Active test meter.Total protein,Total cholesterol,triglycerides,serum urea,creatinine,HDL and LDL were assayed using the reported protocol.20

2.9.Statistical analysis

GraphPad Prism (Version 9.0.San Diego,CA,USA)software was used for the data evaluation using one way analysis of variance followed by Dunnett′s and Tukey′s Multiple Comparison tests.Each parameter mean ±standard error of mean were calculated while significance level was kept atP ＜0.05.

3.RESULTS

3.1.Characterization of the isolated compounds

Compound 1: purity (98%),yellow crystalline appearance,the spectrum of IR (KBr) showed absorption bands at 3350 (OH),1673 (C=O),1489 (aromatic) and 1167 cm-1(ether).UV λmax nm (MeOH): 258,266 sh,299 sh,360;+NaOMe: 272,327,416;+AlCl3: 275,303 sh,430;+AlCl3/ HCl: 271,300,364sh,402;+NaOAc/H3BO3: 262,298,387;+NaOAc: 270,325,393.1HNMR (400 MHz,DMSO d6) δ: 7.69 (dd,J=8.4 and 2.0 Hz,H-6 ′),6.98 (1H,d,J=8.4 Hz,H-5′),6.82 (d J=2.0 Hz,H-2’),6.26 (1H,d,J=2.0 Hz,H-6),6.52 (1H,s,H-8).13CNMR (400MHz,DMSO-d6) δ: 93.3 (CH,C-8),98.1 (CH,C-6),103.0 (C,C-10),115.6 (CH,C-2 ′,C-5′),119.9 (CH,C-6′),121.9 (C,C-1′),135.7 (C,C-3),145.0(C,C-3′),146.8 (C,C-2),147.8 (C,C-4′),155.7 (C,C-9),160.3 (C,C-5),165.5 (C,C-7),175.4 (C,C-4).

The spectrum of proton NMR exhibited five signals(aromatic).The aromatic proton doublet for protons H-6&H-8 at δ 6.26 &δ 6.50 were due to 5,7 substituted ring A meta coupled protons.Protons H-2′,H-5′ and H-6′showed peaks at δ 6.82,6.98 and δ7.69 exhibiting 3′,4′substituted ring B,ABX coupling system of a flavonol.The spectra of compound 1 were unambiguously matched with the previously reported compound quercetin.

Compound 2: purity (97%),yellow amorphous powder with an FTIR spectrum revealed broad absorption band at 3467/cm,which represents OH stretching.UV λ max(nm) (MeOH): 254 sh,268,322 sh,365;+AlCl3: 262 sh,270,352,426;+NaOMe: 275,320,416;+AlCl3/ HCl:260,271,350,426;+NaOAc/H3BO3: 269,295sh,320sh,370;+NaOAc: 275,300,385.1HNMR (400MHz,DMSO-d6) δ: 8.15 (2H,d,J=7.6 Hz,H-2′,H-6′),7.00(2H,d,J=8.4 Hz,H-3′,H-5′),6.53 (1H,d J=2.5 Hz,H-8),6.26 (1H,d,J=2.1 Hz,H-6).13CNMR (400MHz,CD3OD): δ: 93.3 (C-8),98.1 (C-6),103.5 (C-10),115.7(C-3′,C-5′),122.6(C-1′),129.5 (C-2′,C-6′),136.0 (C-3),157.1 (C-9),147 (C-2),161.3 (C-5),164.4 (C-7),159.4(C-4′) &176.2 (C-4).

Compound 2 at the aromatic region of proton NMR spectrum revealed proton doublet peaks (n=4).The proton doublets (aromatic) for H-6 and H-8 at δ 6.26 and δ 6.53 were due to 5,7 substituted ring A,meta coupled protons.The two proton doublets at δ 8.15 &δ 7.00 accounted for 4’ substituted ring B protons H-2’,H-6’and H-3’,H-5’ respectively.The spectra of compound 2 were in close agreement with kaempferol as reported in the literature.

On the basis of physical properties and spectral data of IR,1H-NMR,and13C-NMR,the isolated pure compounds (1 &2) were identified and confirmed as quercetin and kaempferol respectively as shown in Figure 1.

Figure 1 Chemical structure of isolated compounds

3.2.In vivo acute toxicity

Behavioral changes were not observed during the initial 4h of administration.After 24 h,66% mortality was observed up to a dose of 2000 mg/kg for MESB,while 100% mortality was noted for MESL up to a dose of 1000 mg/kg (Figure 2).The animals before death exhibited a typical lethargic effect followed by inactiveness and difficulty in locomotion.From the probit analysis,the LD50 values were calculated as 683.34 mg/kg for MESL and 1166.66 mg/kg for MESB as shown in Table 1.

Figure 2 In vivo acute toxicity test of Sterculia diversifolia

3.3.Anti-hyperglycemic activity

Administration of S.diversifolia bark and leaves extracts produced a significant effect on the alloxan induced hyperglycemia.A significant reduction in the blood glucose was observed in mice treated with graded doses(50,100 and 150 mg/kg) of bark extract and leaves respectively as compared to the glucose levels of the saline treated animals group.Metformin (positive control)administration at a dose of 150 mg/kg has a robust antihyperglycemic effect during the entire study duration i.e.1-4 h (P ＜0.001) as shown in Table 2.

3.4.Effect of treatments on biochemical parameters

Biochemical results demonstrated that negative control group presented significant increase (P ＜0.001) in all biochemical parameters.Standard drug,MESB and MESL treated groups showed results by significantly reducing (P ＜0.001) levels of total proteins,cholesterol,triglycerides,urea,HDL and LDL respectively.Treated groups also were shown significant improvement (P ＜0.001) in hemoglobin level (Table 3).

Table 1 Calculation of LD50for Sterculia diversifolia bark (MESB) and leaves (MESL) extracts

Table 2 Anti-hypergylcemic effect of Sterculia diversifolia bark (MESB) and leaves (MESL) extracts

Table 3 Effect of treatments on biochemical parameters of diabetes in alloxan-induced mice

4.DISCUSSION

In this study,S.diversifolia extracts were testedin vivofor acute toxicity to determine safe tolerable doses for thein vivoanti-diabetic study.Acute toxicity is the condition when immediate untoward changes appears due to exposure to substance(s).Acute toxicity test is performed to identify the minimum safe as well toxic dose for the substance or material.21In this study,the oral administration of S.diversifolia bark and leaves extracts were not well tolerated in all the administered doses,as mortality was observed.The animals exhibited behavioral signs of drowsiness,depression,reduced activity,clumping together,and difficulty in movement during the 24 h time period.

In the present study,the hypoglycemic action of S.diversifolia bark and leaves extracts was evaluated in the mouse model of alloxan-induced hyperglycemia.The antidiabetic effect of various compounds can be studied by using diabetogenic agents (alloxan,streptozotocin etc.).Keep in mind streptozotocin is more expensive than alloxan monohydrate.On this ground,investigators around the globe have a preference for the use of alloxan in experimental diabetes studies.22A significant decrease in blood glucose levels was observed at the time duration of second and third hours after treatment with MESB and MESL,respectively.However,the highest decline was recorded at the fourth hour for all the tested doses.The extracts exhibited an extended duration of hypoglycemic action and a beneficial effect was observed at a dose of 150 mg/kg.The protective effect against alloxan-induced hyperglycemia may signify augmented peripheral glucose utilization.It has been reported that the stimulation of β2 receptors by glycogenolysis (e.g.liver,skeletal muscles) and gluconeogenesis (e.g.liver) results in blood glucose level increase.23The suppression of glucose levels in the alloxan induced diabetic mice suggests that S.diversifolia possesses blood glucose lowering properties comparable to that of metformin in terms of degree and pattern of reduction in blood glucose.Kidney and liver damage are main untoward effects in severe diabetic patients as well as in severe alloxaninduced diabetes.In diabetic nephropathy,there will be a progressive damage like renal fibrin degradation and vein thrombosis.Glomeruli destruction causes significant increases in creatinine and blood which leads to chronic renal failure.Creatinine and urea are kidney function markers,indicating that the extracts were not nephrotoxic.20Similarly hyperglycemia is associated with dyslipidemia while under normal circumstances insulin activates the enzyme lipoprotein lipase whichhydrolyses triacylglycerols.24Dyslipidemia is characterized by increase in triacylglycerol and total cholesterol while decrease in HDL-cholesterol.This serum lipid profile (abnormal) progresses towards normal after therapy with MESB and MESL.Flavonoids may be the main cause of significantly increase LDL receptor mRNA levels,which,in a result,increase LDL hepatic uptake and degradation,causing a decrease in level of serum LDL.25Hence it can be concluded that MESB and MESL has revealed significant effect owing to its ability to reduce level of blood glucose,total protein,total cholesterol,Creatinine,urea and triglyceride and increase HDL level.

Flavonoids are vast polyphenolic phytochemicals group found in plants,fruits and vegetables which are known to possess potential antidiabetic properties.Quercetin has hypoglycemic effect by reducing blood glucose level and led to the recovery of cell proliferation in diabetic mice.26Quercetin alleviates both fasting and post-prandial hyperglycemia and the effect has been shown to be mediated in part by inhibiting α-glucosidase activity.27Kaempferol is able to diminish increase serum glucose levels and increases glucose uptake as efficiently as insulin.28Kaempferol has been shown to possess a potential antihyperglycemic property that is comparable to glibenclamide as it strongly corrected the levels of glycogen,and metabolic enzymes of carbohydrate (e.g.fructose 1,6-bisphosphatase etc.) and hepatic marker enzymes (e.g.alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase etc.).29Moreover,kaempferol derivative,kaempferol-3-O-(2 gal-rhamnosilobonoside) isolated from S.rupestris has been demonstrated to possess significant hypoglycemic effect in experimental animals.30

In conclusion,S.diversifolia bark and leaves extracts bears promising hypoglycemic activity with the tested doses that were easily tolerable as noted in thein vivoacute toxicity test.The extracts showed improvement in biochemical parameters studied such as hemoglobin,total proteins,cholesterol,triglycerides,urea,HDL and LDL etc.The antidiabetic property of S.diversifolia might results from its high contents of quercetin and kaempferol,which have established hyperglycemia attenuating activities in different animal models of hyperglycemia.These findings suggest that S.diversifolia like other species of its genus has the potential to be used as an antidiabetic agent for therapeutic management of diabetes mellitus.However further studies could be conducted to investigate the molecular intricacies involved by utilizing otherin vivoas well asin vitrostudies.