Anti-hyperglycaemic activity of H. rosa-sinensis leaves is partly mediated by inhibition of

carbohydrate digestion and absorption, and enhancement of insulin secretion

Prawej Ansari1, Shofiul Azam2, JMA Hannan3, Peter R. Flatt1, Yasser H. A. Abdel Wahab1

1School of Biomedical Sciences, Ulster University, Coleraine, BT52 1SA, Co. Londonderry,

Northern Ireland, United Kingdom2Department of Integrated Bioscience, Graduate School, Konkuk University, Chungju-27478,

Republic of Korea3Department of Pharmacy, Independent University, Bangladesh (IUB) Bashundhara R/A,

Dhaka1229, Bangladesh

Corresponding Author,

Name: Yasser H. A. Abdel-Wahab

Mailing address: Ulster University, Co. Londonderry, Coleraine, BT52 1SA, NI, UK

Email id: y.abdel-wahab@ulster.ac.uk

Telephone no.: +44(0)2870124354

Shortened version of title: Anti-hyperglycaemic activity of H. rosa-sinensis

Keywords: Diabetes; plant therapies; glucose; insulin

Abstract

Ethnopharmacological relevance: Hibiscus rosa-sinensis (HRS) is a tropical flowery plant, widely

distributed in Asian region and an important traditional medicine used in many diseases including

cough, diarrhoea and diabetes.

Aim of this study: Hibiscus rosa-sinensis (HRS) leaves have been reported to possess anti-

hyperglycaemic activity, but little is known concerning the underlying mechanism. This study

investigated effects of ethanol extract of HRS on insulin release and glucose homeostasis in a type 2

diabetic rat model.

Materials & Methods: Effects of ethanol extract of grinded H. rosa-sinensis (HRS) leaves on insulin

release, membrane potential and intracellular calcium were determined using rat clonal β-cells

(BRIN-BD11 cells) and isolated mouse pancreatic islets. Effects on DPP-IV enzyme activity were

investigated in vitro. Acute effects of HRS on glucose tolerance, gut perfusion in situ, sucrose

content, intestinal disaccharidase activity and gut motility were measured. Streptozotocin induced

type 2 diabetic rats treated for 28 days with ethanol extract of HRS leaf (250 and 500mg/kg) were

used to assess glucose homeostasis.

Results: HRS, significantly increased insulin release from clonal rat BRIN-BD11 cells and this

action was confirmed using isolated mouse pancreas islets with stimulatory effects equivalent to

GLP-1. HRS induced membrane depolarization and increased intracellular Ca2+ in BRIN BD11 cells

and significantly inhibited DPP-IV enzyme activity in vitro. HRS administration in vivo improved

glucose tolerance in type 2 diabetic rats, inhibited both glucose absorption during gut perfusion and

postprandial hyperglycaemia and it reversibly increased unabsorbed sucrose passage through the

gut following sucrose ingestion. HRS decreased intestinal disaccharidase activity and increased

gastrointestinal motility in non-diabetic rats. In a chronic 28-day study with type 2 diabetic rats,

HRS, at 250 or 500 mg/kg, significantly decreased serum glucose, cholesterol, triglycerides and

increased circulating insulin, HDL cholesterol and hepatic glycogen without increasing body

weight.

Conclusion: These data suggest the antihyperglycaemic activity of HRS is mediated by inhibiting

carbohydrate digestion and absorption, while significantly enhancing insulin secretion in a dose

dependent manner. This suggests that HRS has potential as a novel antidiabetic therapy or a dietary

supplement for the treatment of type 2 diabetes.

Abbreviation

HRS: Hibiscus rosa-sinensis

EHRS: Ethanol extract of Hibiscus rosa-sinensis

DM: Diabetes mellitus

T2DM: Type 2 diabetes mellitus

OGTT: Oral glucose tolerance test

STZ: Streptozotocin

FBG: Fasting blood glucose

cAMP: 3', 5'-Cyclic adenosine monophosphate

KATP: Adenosine triphosphate-sensitive potassium

GLP-1: Glucagon-like peptide-1

GIP: Glucose-dependent insulinotropic polypeptide

SGLT2: Sodium-glucose transport protein 2

DPP-IV: Dipeptidyl peptidase-IV

LDL: Low-density lipoprotein

HDL: High-density lipoprotein

TG: Triglycerides

AMC: 7-amino-4-methylcoumarin

ELISA: Enzyme-linked immunosorbent assay

UARC: University Ayurvedic Research Centre

ICDRRB: International Center for Diarrheal Disease Research, Bangladesh

NaCl: Sodium chloride

NaOH: Sodium hydroxide

BaSO4: Barium sulfate

ANOVA: Analysis of variance

KOH: potassium hydroxide

KCl: potassium chloride

ACB: Acarbose

GOD-PAD: Glucose oxidase-phenol and 4-aminophenazone

LPM: Loperamide

TCA: Trichloroacetic acid

IBMX: Isobutyl-methyl xanthine

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1. Introduction

Diabetes mellitus (DM) is a chronic and complex metabolic group of disorders which is increasing

rapidly around the globe. Mortality attributable to diabetes in the year 2000 was estimated to be 2.9

million deaths, equivalent to 5.2% of all deaths (Roglic et al., 2005). Increasingly, diabetes is cited

as major global threat to public health (Booth et al., 2006; Roglic et al., 2005), with 246 million

individuals with this disorder around the world of which 80 percent reside in developing countries

(Dasappa et al., 2015). As a result, diabetes is ranked seventh among the leading causes of mortality

globally (Trivedi et al., 2004).

Type 2 DM is the most common form of diabetes, linked strongly with obesity, advanced age and

some hereditary disposition (Zimmet et al., 1990). Diet and exercise are the corner of stone therapy

but many classes of antidiabetic drugs including metformin, sulphonylureas, thiazolidinediones,

GLP-1 mimetics, DPP-IV inhibitors and SGLT2 inhibitors are needed to convey adequate diabetes

control in the majority of subjects. Despite this therapeutic arsenal, diabetes is not normalised and

complications persist as a major factor affecting quality of life and premature death. Also, in many

parts of the world, especially in the rural areas of developing countries, these drug options do not

exist necessitating reliance on traditional plant treatments. Study of such botanical remedies is very

limited and further studies are required to evaluate their efficacy, mechanism of action and

usefulness for isolation of new antidiabetic agents for future drug development.

H. rosa-sinensis L. (HRS; family; Malvaceae) is an extravagantly flowering, perennial, woody

decorative plant that is widely distributed in the tropical regions such as Asia. HRS is a well-known

traditional medicinal plant used in India and China from ancient time (Jadhav et al., 2009).

According to ethno-pharmacological survey, HRS has been reported to exhibit significant medicinal

properties such as wound healing, detoxifier, anti-fertility, anti-hypertensive, hypolipidemic, anti-

cancer, anti-asthmatic and cardio-protective effects (Goldberg et al., 2017; Jadhav et al., 2009;

Lingesh et al., 2019; Vincenta et al., 2016). Similarly, by surveying rural population of India, HRS

is also reported to be used as traditional medicine in the treatment of diabetes (Alam et al., 1990).

This is in agreement with other studies which have showed the anti-diabetic potential of HRS in

animal models of diabetes (Afiune et al., 2017; Kumar et al., 2013). The imbalance between the

free radical production and antioxidant properties can lead to development of various pathological

conditions such as diabetes (Asmat et al., 2016). Previous studies have reported the antioxidant

properties of HRS (Ghaffar and El-Elaimy, 2012; Khan et al., 2014; Mak et al., 2013; Masaki et al.,

1995). HRS is reported to possess the following major phytoconstituents such as quercetin,

cyanidin, thiamine, niacin, ascorbic acid, genistic acid, lauric acid, margaric acid, hentriacontane

and calcium oxalate (Falade et al., 2009; Gomathi et al., 2008; Lim, 2014; Lingesh et al., 2019).

Accumulating evidence suggests that HRS may be useful for diabetes with tests in rodents revealing

potentially important antihyperglycaemic potential (Sachdewa and Khemani, 1999). In addition, the

authors proposed that the extract contained a sulphonylurea skeleton, within which the -SO2-NH-

CO group was modified by substitutions at S1 and S2. The flower extract resulted in LDL-lowering

activity and a reduction in blood sugar of up to 46% after 21 days of treatment (Sachdewa and

Khemani, 2003; Sachdewa et al., 2001a; Sachdewa et al., 2001b). Vimala et al., (Vimala et al.,

2008) and Moqbel et al., (Moqbel et al., 2011) also reported the insulin secretory activity of HRS in

a diabetic rat model. On the basis of these previous fragmented findings on HRS, the present study

was designed to comprehensively examine and evaluate the pancreatic and extra pancreatic actions

of HRS leaves using in vitro, in vivo and in situ analytical approaches.

2. Materials and Methods

2.1. Selection of plant materials and processing

HRS was obtained as a whole plant from the University Ayurvedic Research Centre (UARC),

Jahangirnagar University, Dhaka, Bangladesh. Prior to processing, a voucher specimen was

submitted to the Bangladesh National Herbarium, Mirpur, Dhaka. The specimen was identified by a

botanical taxonomist and was given an accession number 35204. The collected leaves of plant were

washed in tap water, dried by aeration at 40°C and ground into a fine powder. 100 gm of the

powder was dissolved in 1 L of ethanol, and placed on an orbital shaker (550 rpm) for 48 hours.

The mixture was then filtered through a fine muslin cloth to discard the granular, insoluble particles

and the remaining fine particles were forcibly sedimented by centrifugation (1500 rpm for 10 mins)

with the supernatant then filtered using a Whatman filter paper by vacuum evaporation. The filtrate

was concentrated using the Soxhlet apparatus (Electrothermal™ Soxhlet extractor, UK) and the

transformed sticky substance was subjected to freeze-drying at -55°C to obtain a fine powder

(EHRS), which was kept in sterile Scott bottles with silica gel sachets (desiccant) until used.

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2.2 In vitro insulin-releasing studies

Clonal BRIN-BD11 cells were incubated together with a range of EHRS concentrations (1.6 to

5000 µg/ml) and insulin secretion was determined after 20 min at 37oC (Abdel-Wahab et al., 2008;

Marenah et al., 2004; Mechkarska et al., 2011). Stimulatory concentrations of alanine and GLP-1

were used as positive controls. The origins, characteristics and use of this rat beta cell line for study

of insulin secretion and beta cell function have been described previously (McClenaghan et al.,

1996). Insulin release from isolated rat pancreatic islets was evaluated as described by Hannan et al.

(Hannan et al., 2007b). After test incubations, the supernatant samples were stored at -200C until

assay using Rat Insulin ELISA Kit (Crystal Chem™, USA).

2.3 Intracellular calcium ([Ca2+] i) and membrane potential studies

The effects of EHRS on membrane potential and [Ca2+]i in BRIN-BD11 cells were determined

fluorometrically as described previously (Abdel-Wahab et al., 2007; Miguel et al., 2004) using kits

from Molecular Devices (Sunnyvale, CA, USA).

2.4 DPP-IV activity in vitro

A fluorometric method was used to determine effects of EHRS extract on DPP-IV enzyme activity

(Duffy et al., 2007). The test was conducted by adding 30μl Tris-HCl with 10μl of a sample and

50μl of 200μM Gly-Pro-AMC (Sigma-Aldrich, Dorset, UK). The reaction was initiated via the

addition of 10μl of DPP-IV enzyme (Sigma-Aldrich, Dorset, UK). After 30 minutes, changes in

fluorescence were monitored using a FlexStation 3 (Molecular Devices, CA, USA) with an

excitation and emission at 370nm and 440nm with 2.5nm slit-width.

2.5 Induction of experimental diabetes

Male Long-Evans rats were purchased from the International Center for Diarrheal Disease

Research, Bangladesh (ICDDRB). The optimum conditions with room temperature of 22 ± 5°C,

humidity of 55-65% and 12 hr day/night cycle were maintained. Standard rat pellet diets and fresh

drinking water were provided ad libitum. The nutrient composition of diet was (38·5% fibre, 36·2%

carbohydrate, 20·9% protein and 4·4% fat with a metabolizable energy content of

11.8MJ/kg/2820kcal/kg) as used previously (Hannan et al., 2012b). A single intraperitoneal

streptozotocin injection (90 mg/kg b.w.) of neonatal rats at 2 days of age was used to induce T2DM

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in later life (Portha et al., 1989). There were no fatalities and all injected rats exhibited diabetes

symptoms by 14 weeks of age. At 12 weeks of age, an oral glucose tolerance test (OGTT; 2.5g/kg,

b.w.) was carried out and rats with blood glucose concentrations of 8-12 mmol/L were considered

to have T2DM. The “Revised guide for the care and use of laboratory animals by American

Physiological Society” were followed (Bayne, 1996).

2.6 Residual gut sucrose content

The effect of EHRS on sucrose absorption from the gastrointestinal tract was determined by

measuring the unabsorbed sucrose content after an oral sucrose load as described previously by

Hannan et al., (Hannan et al., 2007a). T2DM rats, fasted for 24 h, received a 50% of sucrose

solution by oral gavage (2.5 g/kg b.w.) followed by doses of EHRS (250 and 500 mg/kg) or an

equal volume of water as control. Blood samples were collected from the tip of the tail vein before

and after 30, 60, 120- and 240 min to determine the glucose levels. Rats were sacrificed at the

above time points and the gastrointestinal tract was excised into six segments: the stomach, the

upper 20 cm, middle and lower 20 cm of the small intestine, the caecum, and the large intestine to

measure unabsorbed sucrose content. Each segment was washed in acidified ice-cold saline (10 ml)

and then centrifuged at 3000 rpm (1000 g) for 10 min. The resulting supernatant was boiled with

sulphuric acid for 2 h to hydrolyse the sucrose following neutralization with NaOH. Blood glucose

concentrations and the amount of glucose liberated from residual sucrose in the GI tract were

measured.

2.7 Intestinal glucose absorption

The effect of EHRS on intestinal glucose absorption in nondiabetic rats fasted for 36h prior to

sodium pentobarbital anaesthesia induction (50 g /kg b.w.) was evaluated by an in situ intestinal

perfusion technique (Swintosky and Pogonwskawala, 1982). Ethanol extract of HRS (5 mg/ml, 10

mg/ml, equal to 0.25 g/kg, 0.5 g/kg) suspended in (Krebs-Ringer bicarbonate) buffer and

supplemented with glucose (54 g/l), was infused via the pylorus and the perfusate was collected

from a catheter inserted at the end of ileum. The control group was perfused only with Krebs’

solution supplemented with glucose. Perfusion at a constant rate of 0·5 ml/min was performed for

30 min at 370C. The results are shown as a percentage of glucose absorbed, the amount of glucose

before and after the perfusion of intestine was measured from solution.

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2.8 Intestinal disaccharidase activity and gastrointestinal motility

The activity of disaccharidase enzyme in intestine was evaluated using the technique as described

previously by Hannan et al. (Hannan et al., 2012a). Briefly, nondiabetic rats were fasted for 20 hr

followed administration of an oral dose of EHRS (250 and 500 mg/kg, b.w.) or water alone. The

rats were sacrificed after 1 hr and the small intestine was excised, sectioned longitudinally and

rinsed with ice-cold saline. The tissue was homogenized after adding saline (0.9% NaCl) and the

total volume was made up to 10ml. The homogenate aliquots were incubated at 37 o C with 40mM

sucrose solution for 60 min. Disaccharidase enzyme activity was measured from the amount of

glucose converted from sucrose as µmol/mg protein/h. The disaccharidase enzyme inhibitor,

acarbose (ACB), (200 mg/kg) was used as a positive control.

GI motility was assessed using BaSO4 milk as described previously by Chatterjee (Chatterjee,

1993). BaSO4 milk was prepared by adding BaSO4 as 10 % (w/v) in 0·5 % carboxy methyl cellulose

suspension. The EHRS was administered orally to normal rats previously fasted for 12h at the doses

of 250 and 500 mg/kg body weight. This was administered 1h before the oral administration of

BaSO4 milk (10 % (w/v) in 0·5 % carboxy methyl cellulose). Control rats received distilled water

(10ml/kg). After 15 min of oral administration of BaSO4, both treated and untreated rats were

sacrificed. The distance traversed by BaSO4 milk was measured and was expressed as a percentage

of the total length of the small intestine (pylorus to the ileocaecal junction). Loperamide (LPM) (5

mg/kg) and sennoside (10 mg/kg) were used as standard drugs controls.

2.9 Glucose tolerance and chronic effects in type 2 diabetic rats

To study the acute effects of EHRS on glucose tolerance, T2DM were fasted for 12hr. Blood

samples were collected from the tip of the tail prior to and at 30, 60, 120 and 180 min after oral

administration of glucose (2.5 g/kg b.w.) with or without extract (250 and 500mg/kg).

Glibenclamide (0.5mg/kg) was used as a positive control. For evaluation of chronic effects, EHRS

extract was administrated at 250 and 500 mg/kg doses by oral gavage twice daily for 28 days to

type 2 diabetic rats. Control rats received oral gavage of water only. Fluid and food intakes together

with body weight were measured at intervals of 7 days. Blood samples were obtained on days 0, 7,

14, 21, and 28, centrifuged to separate serum and stored at -200C until analysis. Blood glucose and

plasma insulin was measured by GOD-PAP method (Azad et al., 2017) (glucose kit, Randox™,

UK) and rat insulin ELISA kit (Crystal Chem™, USA) respectively. At the end of the chronic

5

study (day 28), the rats were sacrificed by cervical dislocation and the liver and pancreas were

isolated.

2.10 Liver glycogen content

The liver glycogen content was determined following the Anthrone method as described previously

(Vies, 1954). Briefly, the liver was weighed and finely homogenized with 10 ml of 5%

trichloroacetic acid (TCA). The precipitated proteins were filtered, and the clear filtrate was

analysed to measure glycogen content. 1 mL of the filtrate was mixed with 2 mL of 10N KOH and

boiled for 1 hr at 100 oC. After cooling, 1 mL of glacial acetic acid was added, and the solution was

made up to 10 mL by adding deionized water. Then 1 mL of this solution was mixed on ice with 2

mL of anthrone solution (100 mg anthrone dissolved in 50 mL of concentrated sulphuric acid), this

mixture was boiled for additional 10 minutes at 100 oC and then cooled. Aliquots were taken in a

microplate reader and the absorbance was measured at 490 nm.

2.11 Pancreatic insulin

Following 28 days of twice daily oral administration of EHRS (250 and 500 mg/kg), rats were

sacrificed and the pancreatic tissues were isolated, weighed, homogenized in 10 ml acid alcohol

solution (75% ethanol, 1.5% HCl 12mM, 23.5% distilled H2O) and centrifuged at 4 0C. The

supernatant was stored at -20 oC until use. Rat insulin ELISA kit (Crystal Chem™, USA) was used

to determine the pancreatic insulin content.

2.12 Statistical analysis

Statistical analyses tests were performed by using SPSS for windows (V.20). The results are

represented as mean±SEM. Data was analysed using repeated measures ANOVA and adjusted

using Bonferroni correction. P value of < 0.05 was considered significant.

3. Results

3.1. EHRS and insulin release

Ethanol extracts of HRS leaves (EHRS) induced a significant (p<0.01- p<0.001, 2-6-fold) dose-

dependent (1.6 to 5000µg/ml) increase in insulin release from clonal BRIN-BD11 cells at 5.6mM

glucose (Figure 1A). The positive control, alanine (10mM) caused a substantial increase in insulin

6

release (p<0.001; 5.5-fold). At higher concentrations (1000 – 5000 µg/ml), EHRS also induced

insulin release, however it was associated with the toxicity of the cells.

In the presence of 16.7 mM glucose (p<0.001), isobutyl-methyl xanthine (IBMX (p<0.05) and

tolbutamide (p<0.01, Figure 1C), a non-toxic dose of EHRS (200 μg/ml) significantly increased

insulin release. In contrast, diazoxide (p<0.01) and verapamil (p<0.01, Figure 1C) inhibited the

stimulatory effects of EHRS by 27%-33%. The extract retained insulin secretory activity in 30 mM

KCl-induced depolarized cells (p<0.001, Figure 1C). The removal of Ca2+ (Figure 1D) considerably

inhibited, but did not completely abolish, EHRS-enhanced insulin secretion.

With isolated rat islets, EHRS, at concentrations between 25-200 μg/ml, induced a 1.5-4.5-fold

increase of insulin release compared to16.7mM glucose (p<0.05-0.001, Figure 1B). In addition,

endogenous incretin hormone, GLP-1 (10-6 & 10-8 M)) and alanine (10mM) used as positive

controls, substantially increased insulin release (p<0.001, Figure 1B).

3.2. EHRS and membrane potential and intracellular calcium ([Ca2+]i)

EHRS (200µg/ml) depolarized the membrane of BRIN-BD11 cells significantly (p<0.001, Figure

1E & G) in the presence of 5.6mM glucose. In addition, the extract also increased (p<0.001)

intracellular calcium concentration ([Ca2+]i) (Figure 1F & H). Similarly, the positive controls, KCl

(30mM) and alanine (10mM) caused significant (p<0.001, Figure 1E-H) increases in membrane

potential and intracellular calcium.

3.3. EHRS and DPP-IV activity in vitro

EHRS significantly inhibited the liberation of AMC (7-amino-4-methylcoumarin) in the dose-

dependent manner, giving a 60% reduction at a higher concentration of 5000 μg/ml (p<0.05-0.001;

Figure 2F). Sitagliptin, an established inhibitor, decreased the activity of DPP-IV 8% to 95%

(p<0.05; p< 0.01 and p<0.001; Figure 2 E) at doses between 16 x10-4 to 5μM.

3.4. EHRS and sucrose content in the gastrointestinal tract after oral sucrose loading

Oral administration of sucrose (2.5 g/kg) with EHRS (250 & 500 mg/kg) resulted in a significant

(p<0.05, p<0.01 & p<0.001) amount of unabsorbed sucrose in the stomach and upper intestine at 30

and 60 min, and in middle and lower intestine at 30, 60 and 120 min (Figure 3A-D). Moreover, a

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small amount of unabsorbed sucrose was still remaining in the caecum and large intestine at 120

min and 240 min (p<0.05 & p<0.01; Figure 3E-F). This emphasizes the rapid hydrolysis and

absorption of sucrose in the upper bowel (Figure 3).

3.5. EHRS and intestinal glucose absorption

Figures 4A and B show a significant (p<0.05, p<0.01 and p<0.001) reduction in absorption of

glucose in the rat intestine after supplementation of glucose with EHRS extract (250 and 500

mg/kg). However, 250mg/kg resulted in a significant reduction of glucose absorption only at 10 and

15 minutes (p<0.05 and p<0.01) whereas at 500mg/kg, it was consistently significant (p<0.01 and

p<0.001).

3.6. EHRS and intestinal disaccharidase enzyme activity and GI motility

Disaccharidase enzyme activity was significantly decreased by the EHRS extract at 250mg/kg

(p<0.05) and 500mg/kg (p<0.01; Figure 4C). In addition, a significant increase in gastrointestinal

motility was observed at 500mg/kg (p<0.05; Figure 4D), as compared to control. The established

drugs, loperamide (5mg/kg) and sennoside (10mg/kg) were used as positive control for GI motility.

3.7. EHRS and acute and chronic effects on glucose homeostasis in type 2 diabetic rats

Figures 2A-B and 2C-D show the effects of EHRS extracts (250 and 500 mg/kg) on glucose

tolerance and serum glucose after sucrose load. Oral glucose (2.5g/kg b.w.) with EHRS (250 and

500 mg/kg) improved glucose tolerance at 30, 60 and 120 min (p<0.05-0.01; Figure 2A-B) in

T2DM rats. Glibenclamide (a sulfonylurea drug) used as a positive control, significantly (p<0.05-

0.001; Figure 2A-B) lowered glucose concentrations. In addition, following an oral sucrose load,

EHRS (250 and 500 mg/kg) significantly (p<0.05-0.001; Figure 2C-D) decreased blood glucose at

30 and 60 min compared to control rats. The higher dose of 500 mg/kg was more potent than 250

mg/kg (p<0.05-0.01, Figure 2B & 2D) at different time points. Furthermore, EHRS administered

orally at the higher dose of 500mg/kg twice daily for 28 days, significantly decreased blood glucose

levels from day 7 and at 250mg/kg from day 14 (Figure 4E). EHRS also increased plasma insulin

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significantly on day 28 (p<0.05; Figure 4F). An established drug, glibenclimide (0.5mg/kg) acted as

positive control.

The twice daily oral administration of EHRS (250 and 500 mg/kg, b.w.) significantly increased

liver glycogen content (p<0.05), liver weight (p<0.05) and pancreas weight (p<0.05) at 21 and 28

days as shown in Table 1. Pancreatic insulin content was significantly increased on days 7 and 28

when EHRS was given at a dose of 250mg/kg (p<0.05) and on days 7, 21 and 28 (p<0.05, p<0.01)

at 500mg/kg dose. A significant (p<0.05) decrease in fluid intake was observed at 21 and 28 days.

Similarly, diminished in food intake was observed at 500mg/kg body weight on day 21 and 28.

However, no significant changes were observed in the body weight of the rats. The serum levels of

triglyceride, cholesterol and LDL were significantly reduced on 21 and 28 days at 500mg/kg dose

(p<0.05) and were comparable to the effect of reference drug, glibenclamide (p<0.05, day 21;

p<0.01 day 28). Furthermore, 500mg/kg dose significantly (p<0.05) increased high-density

lipoprotein (HDL) levels on day 28.

4. Discussion

The present study was designed to confirm the antidiabetic effects of H. rosa-sinensis and evaluate

the underlying mechanisms (Sachdewa et al., 2001a). To achieve this aim, we assessed a spectrum

of potential hyperglycaemic actions including the insulinotropic effects of ethanol extract of H.

rosa-sinensis (EHRS) on isolated mouse islets and clonal BRIN-BD11 cells. The results suggest

that the anti-hyperglycaemic activity of EHRS is partly mediated by actions within the GI tract as

well as enhancing insulin secretion from pancreatic β-cells (Hannan et al., 2007a).

EHRS concentration dependently enhanced insulin release from isolated islets and clonal BRIN-

BD11 cells at non-toxic concentrations affecting both basal and glucose induced insulin secretion in

the presence or absence of established modulators of β-cell function. The retention of insulin

secretory effects when tested with sulfonylurea (tolbutamide) or KCl (30 mM), indicates the

potential of EHRS to trigger multiple pathways such as cAMP or the phosphatidylinositol pathway,

or exert a targeted effect on exocytosis (Hannan et al., 2006). Contrary, diazoxide, a KATP-channel

opener (Wang et al., 2015) attenuated the insulin-releasing effects of EHRS. Thus, it is clear that the

insulinotropic action of EHRS requires closure of KATP channels. Furthermore, verapamil, an L-type

voltage-dependent Ca2+ channel blocker, (Weinhaus et al., 1995) reduced EHRS activity, supporting

the dependency on Ca2+ channel to induce insulin release. This was confirmed by the activity of

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EHRS to induce depolarization and increased intracellular Ca2+ in BRIN-BD11 cells. These

observations suggest that Ca2+ plays an important role in the in vivo insulin secretory actions of

EHRS. The cAMP phosphodiesterase inhibitor also greatly stimulated the insulin secretory effects

of EHRS illustrating the potential importance of adenylate cyclase pathway. Interestingly, H. rosa-

sinensis is used traditionally as folk medicine in asthma (Tomar et al., 2010), acting by stimulation

of cAMP in bronchial smooth muscle cells, facilitating airway relaxation and inhibiting replication

smooth muscle cells (Billington et al., 2013).

Dipeptidyl peptidase IV (DPP-IV) inactivates the action of incretin hormones by N-terminal

cleavage giving rise to GLP-1 (9-36) and GIP (3-42), thus, serving to curtail insulin releasing

effects and modulation of blood glucose (Duffy et al., 2007). Therefore, inhibition of DPP-IV

enzyme activity is gaining increasing attention to enhance endogenous incretin action and treat

T2DM (Godinho et al., 2015). Incretin hormones control blood glucose by a dynamic pathway, they

increase pancreatic insulin secretion as well as reducing glucagon secretion (Singh and metabolism,

2014). In this study, EHRS significantly (P<0.05, P<0.01 and P<0.001) inhibited DPP-IV enzyme

and will therefore most likely enhance endogenous GLP-1 and GIP action. A previous study

showed that flavonol glycosides from the seeds of Lens culinaris Medikus (Fabaceae) inhibited

DPP-IV enzyme activity in the dose-dependent manner (Kim et al., 2018). Therefore, it seems

probable that EHRS may have such phytoconstituents that convey such enzyme inhibitory effects.

EHRS treatment significantly improved acute glucose tolerance in T2DM rats. The plant extract

also reduced glucose absorption significantly during gut perfusion and retained significant amount

of unabsorbed sucrose throughout the postprandial gut, suggesting that extract interferes with

postprandial glucose absorption from the intestine. Similarly, EHRS inhibited intestinal

disaccharidase enzyme activity and increased GI motility in the BaSO4 milk assay. This will result

in a short time for carbohydrates to absorption (Holgate and Read, 1983), resulting in a better

control over postprandial hyperglycaemia. The observed elevated sucrose content in the GI tract

implies impaired sucrose digestion. As a result, an increased amount of sucrose was passed to the

large intestine and caecum, and excreted without metabolism. EHRS reduced postprandial sucrose

absorption and enhanced GI motility, possibly, by forming glucose-fiber complexes that reduce post

prandial transit time or gastric emptying time. From previous literature it is well documented that

HRS is a fiber rich plant (Khristi and Patel, 2017). However, dietary fiber has multiple effects on GI

function, including altered GI transit time and increased GI content viscosity. This affects

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movement of food along the intestinal tract, nutrient contact with absorptive surface and modulation

of starch or glucose digestion (Grundy et al., 2016).

EHRS treatment of T2M rats for 28 days decreased non- fasting blood glucose and increased both

plasma and pancreatic insulin levels. Previous studies reported that EHRS extracts stimulate insulin

secretion (Moqbel et al., 2011; Vimala et al., 2008) are in agreement with present findings.

Consistent with this, liver glycogen content was also increased. Interestingly, both liver and

pancreas mass increased significantly after 28 days’ treatment. This may be due to regeneration of

pancreatic β-cell mass, decreased fat deposition or increased liver glycogen content (Hannan et al.,

2007b). EHRS also induced a remarkable reduction in food and fluid intake. However, no

significant changes in body weight were observed after 28 days of treatment suggesting a change in

energy expenditure that requires further investigation.

Dyslipidaemia is a hallmark of T2DM, associated with a marked by reduction in HDL and elevation

of LDL, total cholesterol, and triglycerides levels that leads to hyperglycaemia and hyperlipidaemia.

This promotes diabetes-induced cardiovascular abnormalities such as micro and macrovascular

secondary complications in T2DM (Gadi and Samaha, 2007). In the present study, administration of

500 mg/kg EHRS for 28-days significantly increased HDL whilst, decreasing LDL and TG levels.

Therefore, beneficial changes in lipid profile induced by EHRS treatment may improve diabetes-

induced complications.

5. Conclusions

In conclusion, this study demonstrated the mechanistic pathways of anti-hyperglycaemic action of

HRS via carbohydrate digestion, absorption and enhancing insulin secretion. HRS has the potential

to improve type 2 diabetes control, as it increases circulating insulin and improves hepatic glucose

utilization. However, further studies are warranted in human subjects to evaluate the effective dose

required to control the level of blood glucose in T2DM. This suggests that H. rosa-sinensis plant or

active phytoconstituents may provide basis for novel therapeutic agents for the management of type

2 diabetes and its complications.

Declaration of competing interest

The authors declare that there is no conflict of interest.

11

Acknowledgments

The present study was supported by the Ulster University Strategic Research Funding, North South

University, Dhaka, Bangladesh and award of Vice-Chancellor’s research studentship to PA.

Author contribution statement

YHAAW and PRF. were responsible for the conception and design of research and also contributed

equally to the supervision of the study. PA, JMAH and SA performed the experiments. PA and SA

analysed the data; PA and JMAH interpreted the results of experiments; PA prepared the figures

and drafted the manuscript; PRF, YHAAW and PA edited the revised manuscript; all authors

approved the final version.

References

Abdel-Wahab, Y.H., Marenah, L., Flatt, P.R., Conlon, J.M., 2007. Insulin releasing properties of the temporin family of antimicrobial peptides. Protein Peptide Let 14(7), 702-707.

Abdel-Wahab, Y.H., Power, G.J., Flatt, P.R., Woodhams, D.C., Rollins-Smith, L.A., Conlon, J.M., 2008. A peptide of the phylloseptin family from the skin of the frog Hylomantis lemur (Phyllomedusinae) with potent in vitro and in vivo insulin-releasing activity. Peptides 29(12), 2136-2143.

Afiune, L.A.F., Leal-Silva, T., Sinzato, Y.K., Moraes-Souza, R.Q., Soares, T.S., Campos, K.E., Fujiwara, R.T., Herrera, E., Damasceno, D.C., Volpato, G.T., 2017. Beneficial effects of Hibiscus rosa-sinensis L. flower aqueous extract in pregnant rats with diabetes. PLoS One 12(6), e0179785-e0179785.

Alam, M., Siddiqui, M., Husain, W., 1990. Treatment of diabetes through herbal drugs in rural India. Fitoterapia 61(3), 240-242.

Asmat, U., Abad, K., Ismail, K., 2016. Diabetes mellitus and oxidative stress-A concise review. Saudi Pharm J 24(5), 547-553.

Azad, S.B., Ansari, P., Azam, S., Hossain, S.M., Shahid, M.I.-B., Hasan, M., Hannan, J., 2017. Anti-hyperglycaemic activity of Moringa oleifera is partly mediated by carbohydrase inhibition and glucose-fibre binding. Biosci Rep 37(3), BSR20170059.

Bayne, K., 1996. Revised guide for the care and use of laboratory animals available. American Physiological Society. Physiologist 39, 199,208-211.

Billington, C.K., Ojo, O.O., Penn, R.B., Ito, S., 2013. cAMP regulation of airway smooth muscle function. Pulm Pharmacol Ther 26(1), 112-120.

Booth, G.L., Kapral, M.K., Fung, K., Tu, J.V., 2006. Relation between age and cardiovascular disease in men and women with diabetes compared with non-diabetic people: a population-based retrospective cohort study. Lancet (London, England) 368(9529), 29-36.

12

Chatterjee, T., 1993. Handbook of laboratory Mice and Rats. Department of Pharmaceutical Technology, Jadavpur University, India.

Dasappa, H., Fathima, F.N., Prabhakar, R., Sarin, S., 2015. Prevalence of diabetes and pre-diabetes and assessments of their risk factors in urban slums of Bangalore. J Family Med Prim Care 4(3), 399-404.

Duffy, N.A., Green, B.D., Irwin, N., Gault, V.A., McKillop, A.M., O'Harte, F.P., Flatt, P.R., 2007. Effects of antidiabetic drugs on dipeptidyl peptidase IV activity: nateglinide is an inhibitor of DPP IV and augments the antidiabetic activity of glucagon-like peptide-1. European journal of pharmacology 568(1-3), 278-286.

Falade, O.S., Aderogba, M.A., Kehinde, O., Akinpelu, B.A., Oyedapo, B., Adewusi, S.J.N.J.N.P., Medicine, 2009. Studies on the chemical constituents, antioxidants and membrane stability activities of Hibiscus rosa sinensis. 13, 58-64.

Gadi, R., Samaha, F.F., 2007. Dyslipidemia in type 2 diabetes mellitus. Curr Diabetes Rep 7(3), 228-234.

Ghaffar, F.R.A., El-Elaimy, I.A., 2012. In vitro, antioxidant and scavenging activities of Hibiscus rosa sinensis crude extract. J Appl Pharmace Sci 2(2), 51.

Godinho, R., Mega, C., Teixeira-de-Lemos, E., Carvalho, E., Teixeira, F., Fernandes, R., Reis, F., 2015. The place of dipeptidyl peptidase-4 inhibitors in type 2 diabetes therapeutics: a “me too” or “the special one” antidiabetic class? J Diabetes Res 2015, 806979.

Goldberg, K.H., Yin, A.C., Mupparapu, A., Retzbach, E.P., Goldberg, G.S., Yang, C.F.J.J.o.t., medicine, c., 2017. Components in aqueous Hibiscus rosa-sinensis flower extract inhibit in vitro melanoma cell growth. 7(1), 45-49.

Gomathi, N., Malarvili, T., Mahesh, R., Begum, V.H.J.P., 2008. Lipids lowering effect of Hibiscus rosa-sinensis flower petals on monosodium glutamate (MSG) induced obese rats. 1, 400-409.

Grundy, M.M.L., Edwards, C.H., Mackie, A.R., Gidley, M.J., Butterworth, P.J., Ellis, P.R., 2016. Re-evaluation of the mechanisms of dietary fibre and implications for macronutrient bioaccessibility, digestion and postprandial metabolism. British J Nutr 116(5), 816-833.

Hannan, J.M.A., Ali, L., Khaleque, J., Akhter, M., Flatt, P.R., Abdel-Wahab, Y.H., 2012a. Antihyperglycaemic activity of Asparagus racemosus roots is partly mediated by inhibition of carbohydrate digestion and absorption, and enhancement of cellular insulin action. British J Nutr 107(9), 1316-1323.

Hannan, J.M.A., Ali, L., Khaleque, J., Akhter, M., Flatt, P.R., Abdel-Wahab, Y.H., 2012b. Antihyperglycaemic activity of Asparagus racemosus roots is partly mediated by inhibition of carbohydrate digestion and absorption, and enhancement of cellular insulin action. The British journal of nutrition 107(9), 1316-1323.

Hannan, J.M.A., Ali, L., Rokeya, B., Khaleque, J., Akhter, M., Flatt, P., Abdel-Wahab, Y., 2007a. Soluble dietary fibre fraction of Trigonella foenum-graecum (fenugreek) seed improves glucose homeostasis in animal models of type 1 and type 2 diabetes by delaying carbohydrate digestion and absorption, and enhancing insulin action. British J Nutr 97(3), 514-521.

13

Hannan, J.M.A., Marenah, L., Ali, L., Rokeya, B., Flatt, P., Abdel-Wahab, Y., 2006. Ocimum sanctum leaf extracts stimulate insulin secretion from perfused pancreas, isolated islets and clonal pancreatic β-cells. The Journal of endocrinology 189(1), 127-136.

Hannan, J.M.A., Marenah, L., Ali, L., Rokeya, B., Flatt, P.R., Abdel-Wahab, Y.H., 2007b. Insulin secretory actions of extracts of Asparagus racemosus root in perfused pancreas, isolated islets and clonal pancreatic β-cells. The Journal of endocrinology 192(1), 159-168.

Holgate, A.M., Read, N.W., 1983. Relationship between small bowel transit time and absorption of a solid meal influence of metoclopramide, magnesium sulfate, and lactulose. Digestive Dis Sci 28(9), 812-819.

Jadhav, V., Thorat, R., Kadam, V., Sathe, N.J.J.P.R., 2009. Traditional medicinal uses of Hibiscus rosa-sinensis. 2(8), 1220-1222.

Khan, Z.A., Naqvi, S., Mukhtar, A., Hussain, Z., Shahzad, S.A., Mansha, A., Ahmad, M., Zahoor, A.F., Bukhari, I.H., Janjua, M., 2014. Antioxidant and antibacterial activities of Hibiscus rosa-sinensis Linn flower extracts. Pak J Pharm Sci 27(3), 469-474.

Khristi, V., Patel, V., 2017. Therapeutic potential of Hibiscus rosa sinensis: A review. Inter J Nutr Dietetics 4, 105-123.

Kim, B.-R., Kim, H., Choi, I., Kim, J.-B., Jin, C., Han, A.-R., 2018. DPP-IV inhibitory potentials of flavonol glycosides isolated from the seeds of Lens culinaris: In vitro and molecular docking analyses. Molecules 23(8), 1998.

Kumar, V., Mahdi, F., Khanna, A.K., Singh, R., Chander, R., Saxena, J.K., Mahdi, A.A., Singh, R.K., 2013. Antidyslipidemic and antioxidant activities of Hibiscus rosa sinensis root extract in alloxan induced diabetic rats. Indian J Clin Biochem 28(1), 46-50.

Lim, T., 2014. Hibiscus rosa-sinensis, Edible Medicinal and Non Medicinal Plants. Springer, pp. 306-323.

Lingesh, A., Paul, D., Naidu, V.G.M., Satheeshkumar, N., 2019. AMPK activating and anti adipogenic potential of Hibiscus rosa sinensis flower in 3T3-L1 cells. Journal of Ethnopharmacology 233, 123-130.

Mak, Y.W., Chuah, L.O., Ahmad, R., Bhat, R., 2013. Antioxidant and antibacterial activities of hibiscus (Hibiscus rosa-sinensis L.) and Cassia (Senna bicapsularis L.) flower extracts. J King Saud University-Sci 25(4), 275-282.

Marenah, L., McClean, S., Flatt, P., Orr, D., Shaw, C., Abdel-Wahab, Y., 2004. Novel insulin-releasing peptides in the skin of Phyllomedusa trinitatis frog include 28 amino acid peptide from dermaseptin BIV precursor. Pancreas 29(2), 110-115.

Masaki, H., Sakaki, S., Atsumi, T., Sakurai, H.J.B., Bulletin, P., 1995. Active-oxygen scavenging activity of plant extracts. 18(1), 162-166.

McClenaghan, N.H., Barnett, C.R., Ah-Sing, E., Abdel-Wahab, Y.H., O'Harte, F.P., Yoon, T.W., Swanston-Flatt, S.K., Flatt, P.R., 1996. Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion. Diabetes 45(8), 1132-1140.

14

Mechkarska, M., Ojo, O.O., Meetani, M.A., Coquet, L., Jouenne, T., Abdel-Wahab, Y.H., Flatt, P.R., King, J.D., Conlon, J.M., 2011. Peptidomic analysis of skin secretions from the bullfrog Lithobates catesbeianus (Ranidae) identifies multiple peptides with potent insulin-releasing activity. Peptides 32(2), 203-208.

Miguel, J.C., Patterson, S., Abdel-Wahab, Y.H., Mathias, P.C., Flatt, P.R., 2004. Time-correlation between membrane depolarization and intracellular calcium in insulin secreting BRIN-BD11 cells: studies using FLIPR. Cell Calcium 36(1), 43-50.

Moqbel, F.S., Naik, P.R., Najma, H.M., Selvaraj, S., 2011. Antidiabetic properties of Hibiscus rosa sinensis L. leaf extract fractions on nonobese diabetic (NOD) mouse. Indian J Experiment Biol 49(1), 24-29.

Portha, B., Blondel, O., Serradas, P., Giroix, M.H., Kergoat, M., Bailbe, D., 1989. The rat models of non-insulin dependent diabetes induced by neonatal streptozotocin. Diabetes Metabolism 15(2), 61-75.

Roglic, G., Unwin, N., Bennett, P.H., Mathers, C., Tuomilehto, J., Nag, S., Connolly, V., King, H., 2005. The burden of mortality attributable to diabetes: realistic estimates for the year 2000. Diabetes care 28(9), 2130-2135.

Sachdewa, A., Khemani, L., 1999. A preliminary investigation of the possible hypoglycemic activity of Hibiscus rosa-sinensis. Biomed Environmental Sci: BES 12(3), 222-226.

Sachdewa, A., Khemani, L., 2003. Effect of Hibiscus rosa sinensis Linn. ethanol flower extract on blood glucose and lipid profile in streptozotocin induced diabetes in rats. J Ethnopharmacol 89(1), 61-66.

Sachdewa, A., Nigam, R., Khemani, L., 2001a. Hypoglycemic effect of Hibiscus rosa sinensis L. leaf extract in glucose and streptozotocin induced hyperglycemic rats. Indian J Experiment Biol 39, 284-286.

Sachdewa, A., Raina, D., Srivastava, A., Khemani, L., 2001b. Effect of Aegle marmelos and Hibiscus rosa sinensis leaf extract on glucose tolerance in glucose induced hyperglycemic rats (Charles foster). J Environmental Biol 22(1), 53-57.

Singh, A.K., metabolism, 2014. Dipeptidyl peptidase-4 inhibitors: Novel mechanism of actions. Indian J Endocrinol 18(6), 753.

Swintosky, J.V., Pogonwskawala, E., 1982. The insitu rat gut technique. Pharm Inter 3(5), 163-167.

Tomar, V., Kannojia, P., Jain, K.N., Dubey, K.S., 2010. Anti-Noceceptive And Anti -Inflammatory Activity Of Leaves Of Hibiscus rosa-sinensis. Inter J Res Ayurveda Pharm 1(1), 201-205.

Trivedi, N., Mazumdar, B., Bhatt, J., Hemavathi, K., 2004. Effect of shilajit on blood glucose and lipid profile in alloxan-induced diabetic rats. Indian J pharmacol 36(6), 373.

Vies, J.J.B.J., 1954. Two methods for the determination of glycogen in liver. 57, 410-416.

Vimala, H., Naik, P., Chandavar, V., 2008. Insulin-secreting activity of Hibiscus rosa sinensis Linn, leaf extract in diabetes-induced Wistar rat. The Bioscan 3, 293.

15

Vincenta, K., Patel, V.J.I.J.o.N., Dietetics, 2016. Therapeutic Potential of Hibiscus Rosa Sinensis: A Review. 4(2), 105-123.

Wang, Y., Wang, S., Harvat, T., Kinzer, K., Zhang, L., Feng, F., Qi, M., Oberholzer, J., 2015. Diazoxide, a KATP Channel Opener, Prevents Ischemia–Reperfusion Injury in Rodent Pancreatic Islets. Cell Transplant 24(1), 25-36.

Weinhaus, A.J., Poronnik, P., Cook, D.I., Tuch, B.E., 1995. Insulin secretagogues, but not glucose, stimulate an increase in [Ca2+] i in the fetal rat β-cell. Diabetes 44(1), 118-124.

Zimmet, P., Dowse, G., Finch, C., Serjeantson, S., King, H., 1990. The epidemiology and natural history of NIDDM–lessons from the South Pacific. Diabetes/Metabolism Rev 6(2), 91-124.

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Figure Legends:

Figure 1: Dose-dependent effects ethanol extract of H. rosa-sinensis (EHRS) leaves on insulin release from (A) BRIN-BD11 cells, (B) Islets of Langerhans and, (C & D) insulin secretion in the presence of established stimulators or inhibitors, (E & G) membrane potential and (F & H) intracellular calcium in BRIN-BD11 cells expressed as RFU and respective area under the curve. Values are Mean±SEM for n=8 and 4 for insulin release and n=6 for membrane potential and intracellular calcium. *p<0.05, **p<0.01 and ***p<0.001 compared to 5.6 mM and 16.7 mM glucose alone. ϕp<0.05, ϕϕp<0.01 and ϕϕϕp<0.001 compared to 5.6 mM glucose in the presence of the extracts. Δp<0.05, ΔΔp<0.01, ΔΔΔp<0.001 compared to respective incubation in the absence of the extracts.

Figure 2: Effects of ethanol extract of H. rosa-sinensis (EHRS) on (A & B) glucose tolerance (GTT), (C & D) serum glucose after sucrose load (SGASL) in type 2 diabetic rats and (E & F) DPP-IV activity in vitro. Rats were fasted for 12 and 24hr and administered glucose or sucrose solution (2.5 g/kg body weight) by oral gavage in presence or absence of ethanol extract of H. rosa-sinensis (250 mg/kg and 500 mg/kg body weight) or Glibenclamide (GC) (0.5 mg/kg). Sitagliptin was used as established DPP-IV inhibitor. Values are Mean ± SEM represented by vertical bars (n= 6, for GTT and, SGASL and n=3 for DPP-IV activity). *p<0.05, **p<0.01 and ***p<0.001 compared to controls. Figure 3: Effects of ethanol extract of H. rosa-sinensis (EHRS) on (A-F) gastrointestinal sucrose content after oral sucrose loading in type 2 diabetic rats. Type 2 diabetic rats were fasted for 24 hours prior to the oral administration of sucrose solution (2.5 g/kg body weight) in the presence (treated group) or absence of (control group) ethanol extract of H. rosa-sinensis (250 mg/kg and 500 mg/kg b.w.). Values are Mean ± SEM represented by vertical bars (n = 6). *p<0.05 and **p<0.01 compared with type 2 diabetic control rats.

Figure 4: Effects of ethanol extract of H. rosa-sinensis (EHRS) on (A & B) intestinal glucose absorption, (C) disaccharidase enzyme activity and, (D) gastrointestinal motility (by BaSO4

traversed) in non-diabetic rats and (E) fasting blood glucose and (F) plasma insulin level in type 2 diabetic rats after 28 days of EHRS administration. Rats were fasted for 36hrs and intestine was perfused with glucose (54 g/l) in the presence (treated group) or absence of (control group) ethanol extract of H. rosa-sinensis (5 mg/ml and 10 mg/ml). BaSO4 administered at 60 min following oral dosing of H. rosa-sinensis. Acarbose (200 mg/kg), Loperamide (5 mg/kg) and Sennoside (10 mg/kg) were used as positive controls for disaccharidase activity and gastrointestinal motility respectively. Values are Mean ± SEM represented by vertical bars (n = 8). *p<0.05, **p<0.01 and ***p<0.001 compared with controls.

17

Table 1: Chronic effects of H. rosa-sinensis (EHRS) on pancreatic insulin content and other parameters in type 2 diabetic rats after 28 days treatment.

Days Treatmentgroup

Body wt. (gm)

Food intake (gm)

Fluid intake

(ml)

Liver wt.

(g/100g BW)

Liver glycogen

(g/100g tissue)

Pancreasewt. (g/100g

BW)

Pancreatic insulin content (nmol/g)

HDL (mg/100ml)

LDL (mg/100ml)

TG (mg/100ml)

Cholesterol (mg/100ml)

0 days Control 151±2.5 33±1.3 58±3.5 3.5±0.5 1.6±0.05 450.0±5.33 0.65±0.02 60.2±2.4 42.0±2.3 72.2±2.4 68.2±2.2250mg/kg 150.±2.5 32±2.5 64±3.5 3.5±0.7 1.7±0.07 444.0±6.77 0.69±0.01 31.6±1.4 55.6±3.5 75.6±2.5 70.3±2.3500mg/kg 153±3.6 32±1.6 60±4.4 3.5±0.3 1.6±0.05 455.0±6.67 0.58±0.02 33.9±1.6 49.7±3.6 74.0±2.5 67.4±2.55GC (0.5mg/kg) 152±3.3 32±2.0 56±2.5 3.6±0.7 1.6±0.07 465.0±5.77 0.68±0.02 48.6±2.3 53.3±1.5 71.1±2.6 60.2±2.7

7 days Control 153±3.5 32±2.5 60±3.6 3.5±0.5 1.6±0.07 465.0±4.78 0.84±0.05 56.3±2.3 45.3±2.4 77.2±2.5 75.8±2.3250mg/kg 150±2.6 29±2.0 58±2.6 3.5±0.6 1.8±0.03 450.0±6.67 0.93±0.07 35.7±1.6 53.4±3.5 78.7±2.3 71.8±2.9500mg/kg 153±3.3 28±1.5 57±1.5 3.5±0.6 1.8±0.03 474.0±4.67 1.15±0.05* 39.6±1.7 47.5±2.7 67.4±2.4 66.3±2.7

GC(0.5mg/kg) 152±2.6 26±1.5 57±2.5 3.6±0.7 1.6±0.05 480.0±5.55 1.28±0.02* 53.3±2.0 48.7±3.3 65.3±2.7 58.2±2.814 days Control 153±2.5 32±1.6 62±2.5 3.6±0.3 1.5±0.03 468.0±6.87 0.78±0.04 49.9±2.8 50.0±3.4 81.2±2.5 82.3±2.5

250mg/kg 150±2.6 29±2.2 58±2.6 3.6±0.6 1.7±0.07 470.0±5.45 0.92±0.02 38.8±1.5 44.3±2.8 75.2±2.3 67.2±2.3500mg/kg 153±2.5 27±1.4 56±3.5 3.6±0.2 1.7±0.03 472.0±5.67 0.95±0.03 42.9±2.2 42.8±2.4 59.4±2.2 60.8±2.8GC (0.5mg/kg) 152±2.6 26±2.5 55±2.5 3.6±0.35 1.8±0.05 484.0±6.67 1.05±0.03 55.6±2.6 38.8±1.5 56.1±2.3 56.1±2.7

21 days Control 153±3.6 32±1.5 64±1.6 3.8±0.7 1.5±0.03 480.0±5.55 0.82±0.02 45.3±2.3 56.3±2.7 84.1±2.7 89.2±2.5250mg/kg 151±2.7 24±2.5 58±2.* 3.8±0.8* 2.0±0.0* 520.0±6.7* 0.94±0.02 41.0±2.5 43.8±2.5 71.2±2.5 64.2±2.8500mg/kg 153±3.5 24±1.5* 56±3.5* 3.9±0.2* 2.2±0.0* 525.0±6.6* 0.98±0.04* 44.1±3.3 39.7±3.5* 56.4±2.4* 58.2±2.7*GC (0.5mg/kg) 152±4.5 22±1.7* 53±2.5* 3.8±0.4* 2.4±0.07* 560±6.7* 1.1±0.07* 63.9±2.8* 35.8±1.3* 54.6±2.4* 54.6±2.4*

28 days Control 154±2.7 32±1.4 65±3.6 3.9±0.7 1.4±0.03 495±6.7 0.75±0.03 42.1±3.6 60.2±3.4 86.1±2.8 91.7±2.8250mg/kg 153±3.5 23 ±1.3 58±2.5* 4.0±0.4* 2.1±0.03* 550±5.7* 1.03±0.05* 43.4±2.5 37.9±2.7 67.7±2.9 61.1±2.3500mg/kg 153±3.5 22±1.6* 54±3.5* 4.2±0.3* 2.3±0.07* 535±5.7* 1.11±0.05** 49.7±3.0* 34.0±2.3* 57.4±2.4* 56.9±2.3*GC (0.5mg/kg) 152±2.6 20±1.5* 50±2.5* 4.1±0.3* 2.6±0.05* 573±4.8* 1.26±0.03** 67.7±2.7*

*31.6±1.4** 51.0±2.5** 54.5±2.2**

Ethanol extract of H. rosa-sinensis (250 mg/kg and 500 mg/kg body weight) or Glibenclamide (GC) (0.5 mg/kg) or saline (control) were administered orally to T2DM rats for a period of 28 days. Values are Mean ± SEM (n = 8). *p<0.05, **p<0.01 and ***p<0.001 compared with type 2 diabetes controls.

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Figure 1.

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Figure 2

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Figure 3.

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Figure 4.

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