Draft - University of Toronto T-Space · protective effects in Alloxan induced Diabetic rats Journal: ... (coumarin), chlorogenic acid and caffeic acid (phenylpropanoids), apigenin,

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Chamomile and Oregano extracts synergistically exhibits

anti hyperglycemic, anti hyperlipidaemic and renal protective effects in Alloxan induced Diabetic rats

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2016-0189.R1

Manuscript Type: Article

Date Submitted by the Author: 30-Jul-2016

Complete List of Authors: Prasanna, Rajagopalan; Department of Clinical Laboratory Sciences,

College of Applied Medical Sciences, King Khalid University. Ashraf, Elbessoumy; Biochemistry Department, Faculty of Science, Alexandria University Essam, Mahmoud; Clinical Pathology Department Faculty of Veterinary Medicine, Zagazig University

Keyword: Diabetes Mellitus, Matricaria chamomilla, Origanum vulgare, Insulin- Diabetic complications, Synergism

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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Chamomile and Oregano extracts synergistically exhibits anti hyperglycemic, anti

hyperlipidaemic and renal protective effects in Alloxan induced Diabetic rats

Prasanna Rajagopalan

1*, Ashraf, A. Elbessoumy

2, Essam, A. Mahmoud

3

1 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia.

2Biochemistry Department, Faculty of Science, Alexandria University, Egypt

3 Clinical Pathology Department Faculty of Veterinary Medicine, Zagazig University, Egypt.

Running Title: Chamomile and Oregano synergistic action in diabetic rat models

Novelty & Impact Statements: The data of this study show for the first time that treatment with

a combined mixture of aqueous extracts of Chamomile and Oregano induce synergistic

ameliorative actions on certain diabetic symptoms in the Alloxan-rat model. The current

observations may provide support to the development and formulation of new natural dietary

supplements and pharmaceutical agents for the control and management of diabetes mellitus

complications.

*Corresponding author

Dr. Prasanna Rajagopalan Ph.D. F.I.C.S

Assistant Professor,

Department of Clinical Laboratory Sciences,

College of Applied Medical Sciences,

King Khalid University,

PO.Box # 641

Abha, Saudi Arabia.

Phone: +966540924232

Email- [email protected]; [email protected]

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Abstract

The bio-activities of separate Matricaria chamomilla (chamomile) and Origanum vulgare

(oregano) are well studied; however, the combined effects of both natural products in animal

diabetic models are not well characterized. In this study, Alloxan induced male albino rats were

treated with single dose aqueous suspension of chamomile or oregano at dose level of either 150

mg/kg or 300 mg/kg body weight or as equal parts as combination by stomach tube for 6

weeks. Post treatment, blood samples were assessed for diabetic, renal and lipid

profiles. Insulin, amylase activity and diabetic renal apoptosis were further evaluated. Treatment

with higher dose of the extracts (300 mg/kg) as individual or as mixture of low doses (150 mg/kg

of both the extracts) had significant weight gain, hypoglycemic effect (p

≤ 0.05) with decreased amylase activity and increased serum insulin levels. Restoration of renal

profile, lipid profile with increase in HDL-C (p ≤ 0.05) along with reversal of pro-apoptotic Bax

and anti –apoptotic Bcl-2 were well observed with 300 mg/kg mixture showing synergistic

activity of the extracts compared to individual low dose of 150 mg/kg. Collectively our results

indicate that, combination of chamomile and oregano extracts will form a new class of drugs to

treat diabetic complications.

Key words: Diabetes Mellitus, Matricaria chamomilla , Origanum vulgare, HbA1C, Insulin,

Lipid Profile, Synergism.

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1. Introduction

Diabetes mellitus is characterized by hyperglycemia, altered metabolism of lipids,

carbohydrates and protein which in turn leads to chronic complications including diabetic

nephropathy (Davis and Granner 1996). As diabetes mellitus cannot be cured, managing the

disease becomes the only choice of treatment. The plants with anti-diabetic activities provide

important sources for the development of new drugs in the treatment. Many herbs and spices

from traditional medicine have been used in most developing countries as a valuable alternative

for treating diabetes mellitus (Anderson 2004). The major advantages of herbal medicine seem

to be their efficacy, low incidence of side effects, and the low cost (Manal 2012).

Chamomile (Matricaria chamomilla L.), is one of the medicinal herbs known in ancient

Egypt, Greek and Rome (Najla et al.2012). It contains a large group of therapeutically interesting

and active compound classes like sesquiterpenes, flavonoids, coumarins, and polyacetylenes

(Ompal et al.2011). Eleven bioactive phenolic compounds, (Ompal et al.2011) such as herniarin

and umbelliferone (coumarin), chlorogenic acid and caffeic acid (phenylpropanoids), apigenin,

apigenin-7-O-glucoside, luteolin and luteolin-7-O-glucoside (flavones), quercetin and rutin

(flavonols), and naringenin (flavanone) are found in chamomile extract. Studies recorded that

chamomile ameliorates the hyperglycemia and diabetic complications via suppressing blood

glucose levels and increase the serum insulin and C-peptide levels in diabetic rats (Kato et

al.2008). The pharmacological activity of chamomile reveals its protective effect on pancreatic

beta cells in diminishing hyperglycemia-related oxidative stress (Zemestani et al 2016).

Oregano (Origanum vulgare), is a native plant widely distributed throughout the

Mediterranean, Euro-Siberian and Irano-Siberian regions. The main known active compounds

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are camphene, carvacol, gamma-terpiene, thymol,Terpinin-4-ol, myricine and Linalyl-acetate.

Oregano leaves are traditionally used as potent hypoglycemic agents in diabetes control and

treatment; (Jouad et al.2003) however it had no effect on the basal plasma insulin concentrations

in both normal and diabetic rats and the hypoglycemic effect is facilitated by inhibiting the

production of glucose in the liver.

Though rich background information on the anti-diabetic and medicinal properties of

these herbs are available, the combined aqueous extracts of these plans in managing DM and its

complications in alloxan induced diabetic animal models has not been studied. It is because of

the high degree of overlap between different signaling pathways and the redundancy within a

cell, agents with multiple mechanisms of action and particularly the use of combination

treatments have become increasingly important. In this study we therefore hypothesize on two

herbal extracts and their combination with different mechanisms in alloxan induced diabetes

mellitus and hyperlipidaemic model.

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2. Materials and Methods

2.1 Chemicals:

Kits for assessing blood (serum) glucose, glycated hemoglobin (HbA1C), total cholesterol

(TC), triglycerides (TG), high density lipoprotein- cholesterol (HDL-C), urea, creatinine, uric

acid, total protein and amylase were obtained from Human Co. Annexin V kit was purchased

from e-Bioscience USA. Bcl-2, Bax and β-actin antibodies were purchased from Cell Signaling,

USA. All other chemicals were purchased from Sigma.

2.1.1 Preparation of plant extracts:

Chamomile and oregano were obtained from local markets. Dry chamomile (flowers,

leaves, stems or all aerial parts) and oregano leaves extract has been prepared as describe

previously (Srivastava and Gupta 2009; Williamson et al 1996) to have a concentration of 200

mg/ml.

2.2 Experimental Animals:

All animal research was conducted in accordance and approval by the Institutional

Animal Care and Use Committee. Eighty male albino (Wistar) rats weighing 150-200 gram were

purchased from the animal house of National Research Center, Cairo-Egypt and housed for one

week under controlled condition and provided with standard diet and water ad libitum to allow

them acclimatize before conducting the study. Body weight and physical signs were monitored during

the experiments.

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2.2.1 Experimental Design:

Rats were divided into eight groups of ten rats per each group as per the below schematic figure.

2.2.2 Alloxan induced diabetic rats:

Group 1 and 2 were uninduced animals. Group 3 to 8 animals were induced with diabetes

mellitus by intra-peritoneal injection of 120mg/Kg body weight of alloxan dissolved in normal

saline. The development of diabetes was checked by measuring serum glucose level after four

days of alloxan injection. DM was confirmed by elevated fasting serum glucose over 300 mg/dl

(Misra and Fridovich 1972).

Schematic diagram of experimental design

Male Albino rats

Control Groups Treatment Groups

Sham Uninduced control Diabetic control

(Group-1) (Group-2) (Group-3)

Chamomile group Oregano group Mixture group

150 mg/kg 300 mg/kg 150 mg/kg 300 mg/kg

(Group-4) (Group-6) (Group-5) (Group-7)

300 mg/ kg Equal mixture

(150 mg chamomile + 150 mg oregano)

(Group-8)

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2.2.3 Treatment procedure for DM induced rats:

Group 1 was sham control animals which were not disturbed. Group 2, 3 rats were

administered with vehicle without drug by stomach tube for 6 weeks. Group 4, 6 rats were given

aqueous suspension of chamomile at dose level of 150 mg/kg and 300 mg/kg body weight

respectively by stomach tube for 6 weeks. Group 5, 7 rats received aqueous suspension of

oregano at dose level of 150 mg/kg and 300 mg/kg body weight respectively by stomach tube for

6 weeks.

Group 8, rats were treated with an aqueous suspension of chamomile and oregano

extracts as combination of 50% each and together totalling 300 mg/Kg body weight. The group

received this at dose by same route and time period as that of individual treatments. No evidence

of side effects was observed after drug administration in all treated groups.

After 3 weeks of treatment, five rats from each group were selected; blood samples

collected by vein puncture using standard protocol (Parasuraman et al.2010) in plain centrifuge

tube and serum was separated for measurement of all biochemical parameters. The treatment was

continued with remaining animals for 3 more weeks. At the end of 6th

week, blood samples from

remaining 5 rats in each group were collected as before and serum was separated for analysis.

After blood collection at each period, the animals were sacrificed. The kidneys were rapidly

dissected and collected in RPMI1640 medium placed on ice. Kidney cells were isolated as

described in section 2.4. Prior to the blood collection procedure at the end of 3rd

and 6th

weeks,

the body weight of animals in each group was noted.

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2.3 Biochemical analysis:

Blood samples were assessed for several bio-indicators of diabetes among which are:

insulin, glucose, amylase, lipid, renal etc. and renal cellular apoptosis. Blood (Serum) glucose,

glycated hemoglobin (HbA1C), total cholesterol (TC), triglycerides (TG), high density

lipoprotein- cholesterol (HDL-C), urea, creatinine, uric acid, total protein and amylase were

estimated by kit protocols of Human Co. according to manufacturer’s instructions. The

calculation of serum very low density lipoprotein cholesterol (VLDL-c) and low density

lipoprotein cholesterol (LDL-c) were carried out according to the standard methods from the

equations: VLDL-c (mg/dl) = TG/5 and LDL-c (mg/dl) = TC – HDL – VLDL(Lee and Nieman

1996). Total protein was measured according to Bradford method (Bradford 1976). The serum

insulin level was assayed with an ELISA kit (Linco Research Inc., USA) according to the

manufacturer’s instructions.

2.4 Preparation of kidney cells for analysis:

The kidneys were washed and cut up with scissors into fine pieces, and minced with a

sterile blade to yield 2 × 2-mm pieces in sterile RPMI 1640 medium on ice. After through washes

with serum-free HBSS, to obtain single cell suspensions, cells were mixed with ultra-pure

collagenase III in medium 199 (200–250 units of collagenase per ml) and allowed to incubate at

37°C for 3–4 h. Further, cells were filtered through a 45-µl nylon mesh and washed with

RPMI/20% FBS media; then washed twice with HBSS. Cells were counted using hemocytometer

and transferred to a 5-ml tube, washed twice with HBSS with 2% heat-inactivated calf serum

(HICS; 5 min at 1,000 rpm) and re suspended in 100 µl (per 106 cells) of HBSS with 2% HICS.

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2.6 Annexin V Assay:

The assay was performed using Annexin V detection kit from e-Biosciences, USA as per

the manufacturer's instructions as follows. 0.5 X 106 of isolated kidney cells from control and

treated groups were washed twice with wash buffer (WB) and incubated with 0.25 µg/ml

Annexin V reagent in 1 X binding buffer for 15 minutes. After couple of washes with WB, cells

were re-suspended back in binding buffer containing 0.5 µg/ml propedium iodide and 10,000

events acquired on a guava easyCyte™ flow cytometer. Data were analyzed using InCyte

software-Millipore. Apoptotic cells were segregated with a quadri plot graph and total percentage

of apoptotic cells represented using Graphpad Prism software.

2.7 Western immunoblotting for apoptotic downstream signaling:

The isolated kidney cells were lysed in RIPA buffer containing 50 mMTris, 150 mM

NaCl, 0.5% deoxycholate, 0.1% SDS, 2 mM EDTA, 0.1% Triton X-100, 10% glycerol, 1 mM

phenyl methyl sulfonyl fluoride, 10 µg/ml aprotinin and 5 µg/ml pepstatin A. After the insoluble

materials were removed by centrifugation at 14,000 × g for 10 min at 4°C, protein were

quantified using Coomassie plus Protein Assay Reagent kit (Pierce; Rockford, IL, USA). Total

cellular proteins (20–40 µg) in the cell lysate were separated by 8–15% SDS polyacrylamide gel

electrophoresis. The proteins on the gel were then transferred onto a nitrocellulose membrane,

which was probed with the respective primary antibodies [(BCL-2; Santa Cruz Biotechnology);

BAX; Santa Cruz Biotechnology; anti-β-actin (Sigma-Aldrich, St Louis, MO, USA)] followed by

addition of the corresponding horseradish peroxidase (HRP) conjugated secondary antibodies.

The membrane was then stripped by stripping buffer containing 100 mM β-mercaptoethanol, 2%

SDS and 62.5 mMTris- HCl at 50°C for 30 min for quantification and normalization with β-actin

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(1:5000). Protein bands were visualized by using ECL reagents (Amersham Bioscience;

Piscataway, NJ, USA) and exposed to Kodak X-Omat Blue XB-1 films (Rochester, NY, USA).

Bands were quantified using Image J (Ver. 1.46, NIH) and normalized to actin.

2.8 Statistical analysis:

Statistical analyses were performed using Graphpad Prism 6.0 (La Jolla, USA). The

results are expressed as mean ± SE. The data was analyzed using one-way analysis of variance

(ANOVA) followed by Tukey’s procedure for multiple comparisons. Any value of p ≤ 0.05 was

considered significant. All experiments were conducted in duplicates to ensure consistency

(Tamhane and Dunlop 2000).

3. Results:

3.1 Anti hyperglycemic effects of chamomile, oregano and their combination:

Results illustrated in Figure -1 a, shows the changes in body weight, blood glucose levels,

HbA1C, insulin and amylase activity in all experimental groups. The body weight of the diabetic

control rats were significantly decreased as compared to the vehicle control group. Diabetic rats

treated with 300 mg/kg body weight of either oregano or chamomile extracts relatively

maintained the body gain compared with the diabetic group. While 150 mg/kg of either

chamomile or oregano treated groups did not show significant weight gain, the equal

combination of extracts treated group had significant results post treatment. In addition, the

combination treated group was observed to gain more weight than 300 mg/kg treated individual

extracts. These results were consistent at both 3 weeks and 6 weeks of treatment.

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The intra-peritoneal injection of alloxan effectively induced diabetes as reflected by

significant elevation of blood glucose, HbA1C, amylase activity and diminution of insulin level

when compared to vehicle control group (Fig. 1). Treatment of diabetic rats with 150 mg/kg or

300 mg/kg of chamomile extract showed significant increase in insulin, while oregano treatment

was ineffective at both doses (Fig. 1 b). The treatment of chamomile or oregano at 300 mg/kg

and 150 mg/kg reduced blood glucose (Fig. 1 c), HbA1C (Fig. 1 d) and amylase activity (Fig. 1 e)

when compared to diabetic control group. Our results revealed that the mixture of both extracts

significantly decreased the blood glucose when compared with the effect of 150 mg/kg each

extract alone on diabetic animals (Fig 1 c). The effect of glucose reduction by the mixture was

observed to be synergistic when compared with individual extracts (Table–1). On the same hand,

the mixture significantly decreased HbA1C and amylase activity (Fig. 1 d and e) and increased

insulin level (Fig. 1 b) when compared with the effect of 150 mg/kg chamomile or oregano

treated diabetic animals.

3.2 Effects of extracts and their combination in renal function profile:

Figure 2 shows significant increase (p ≤ 0.05) in the levels of blood urea, creatinine and

uric acid, with a decrease in the total protein as markers of renal dysfunction in the diabetic

group compared to control group. Treatment of diabetic rats with 300 mg/kg of each extract

alone decreased blood urea, creatinine and uric acid levels and increased total protein levels

(Figure 2 a-d). The effect of oregano treatment was observed to be little higher than that of

chamomile on these parameters (Figure 2 a-d). While 150 mg/kg chamomile or oregano treated

groups did not respond to the reversal of urea, uric acid, creatinine or total protein as compared to

diabetic group, the mixture treatment almost renormalized these levels (Figure 2). The

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observations reveal that mixture of both extracts was more effective in the improvement of renal

parameters compared to the effect of both doses of each extract alone on diabetic animals.

3.3 Anti hyperlipidaemic activity of chamomile, oregano and their mixture:

The results of lipid profile are shown in Figure 3. Serum TC, TG LDL-c and VLDL-c levels

were significantly (P≤ 0.05) increased in diabetic group compared to the control, while HDL-c

levels were decreased (Fig 3 a-e). Treatment of diabetic rats with 300 mg/kg oregano or

chamomile extracts lowered the levels of total cholesterol (Fig. 3 a), triglycerides (Fig. 3 b),

LDL-c (Fig. 3 d), VLDL-c (Fig. 3 e) and elevated HDL-c (Fig. 3 c) compared to the diabetic

group. As observed in the kidney profile, the results of lipid profile also showed 150 mg/kg of

either extract treatment were trace effective, while the combination of extract was more effective

as anti- hyperlipidaemic compared with 300 mg/kg treatment of chamomile or oregano extracts

(Fig 3 a-e).

3.4 Renal protective effects of extracts and their combination in diabetic model:

To check if the extracts or their combination had a renal protective effect manifested by

decreasing apoptosis in kidney cells, we performed Annexin V assay using flow cytometry. As

observed in Fig. 4 a, kidney cells from diabetic control rats showed significant increase in

apoptotic cell population when compared with vehicle control group. 300 mg/kg chamomile or

oregano treatment decreased early and late phase apoptotic cells compared to diabetic control

(Fig 4 a). Apoptosis reducing effect was more prominent in mixture treated group when

compared with the diabetic control and single extract treated groups (Fig. 4 a, b).

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To substantiate Annexin V assay results, we next evaluated the pro and anti apoptotic

protein signaling of kidney cells by western blotting. The anti apoptotic Bcl-2 protein levels were

increased in 300mg/kg single extracts or mixture treated group compared with diabetic rats

(Figure 4 c). The Bax protein levels were decreased in all treatment groups compared

with diabetic control rats (Figure 4 c). As a consequence, the Bcl-2/BAX ratio significantly

increased in treatment groups with special reference to the mixture treated group (Fig. 4 d).

Sham animals did not have significant difference in any of the parameters tested when

compared to vehicle control group (data not shown).

4. Discussion

Plants and plant products both as extracts and derived compounds are gaining importance

as effective and versatile agents against various diseases (Qattan et al.2008). The demand for

herbal remedies is growing as various types of hypoglycemic agents produce number of side

effects (Mutalik et al.2003). Herbal formulations have been effectively prescribed for the

treatment of many diseases including diabetes mellitus. Before isolation of the active compounds

from the herbs, the crude extract must be tested for its effectiveness in animal models

(Karashima and Schally 1988). The hypoglycemic activity of some plants extracts have been

evaluated and confirmed in animals and in human beings (Sharma et al.2010). Also, complex

and interactive combination therapies have become more popular and are proved to be more

efficacious. The present study was therefore planned to elucidate the anti-hyperglycemic and anti

-hyperlipidaemic and renal protective properties of chamomile, oregano aqueous extracts and

their combination in alloxan induced-diabetic rats besides estimation of other related biochemical

profiles.

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Weight loss has been known to be one of the chief DM symptoms (Sellamuthu et

al.2009). Figure 1 a, shows body weight of the untreated diabetic rats significantly decreased as

compared to control and diabetic treated group. However, the diabetic rats treated with 300

mg/kg oregano/chamomile/ mixture relatively maintained the body gain with special reference to

the mixture treated group. Similar observation was obtained by other co workers, who reported

that the administration of plant extracts showed significant gain in body weight which may be

due to its protective effect in controlling muscle wasting, where loss of body weight associated

with diabetes could be a consequence of increased muscle wasting due to the loss of tissue

proteins (Kholoud and Manal 2012). It is postulated that the deficiency of insulin in the diabetic

rats lead to decrease in amino acids uptake by tissues with a consequent reduction in the level of

protein synthesis(Kato et al.2008). Therefore the increase in insulin levels observed (Fig. 1 b) can

also be allied to the weight gain observed in these groups (Fig. 1 a).

Diabetic rats treated with 300 mg/kg chamomile or oregano extracts showed significant

reduction of blood glucose, glycated hemoglobin (HbA1C) and amylase activity as compared to

diabetic group (Figure 1 b - d). Studies show that chamomile tea improves glycemic indices and

antioxidants status in patients with type 2 diabetes mellitus (Rafraf et al.2015). Chamomile

extracts are proven to lower the blood glucose (Manal 2012), HbA1C levels and amylase activity

(Darvishpadok et al.2012). Oregano extracts has been proven for its antihyperglycemic activity

(Vujicic et al.2015). Ethanolic extract of the plant is also shown to inhibit α-amylase activity in

vitro (Patrick et al.2007). Therefore the observed effects of chamomile or oregano stand well

with reported literature. However the efficacy was achieved with 300 mg/kg of chamomile or

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oregano treatment, where 150 mg/kg treatments were less effective. Interestingly, 300 mg/kg

dose, which contained equal mixture of chamomile and oregano extracts showed significant

decrease of blood glucose, HbA1C and amylase activity when compared with the effect of 150

mg/kg either extracts treated or the diabetic control group (Fig 1). The hypoglycemic activity

exhibited by the mixture was observed to be synergistic (Table-1). We have previously

established methods to calculate synergistic index either by calculating the ratio of expected

percentage to observed percentage or by arriving at ratio of average percentage to observed

percentage (Prasanna et al.2011). In the current study, both methods showed synergistic effect of

the mixture treatment in reducing the blood glucose levels of diabetic rats as observed in Table- 1

a and 1b.

Treatment of chamomile extract at 150mg/kg or 300 mg/kg body weight showed

significant insulin increase, with higher dose being more effective than lower one (Fig. 1 b). On

the other hand, oregano treatments at either dose did not have an effect on insulin secretion (Fig.

1 b). It has been observed that, oregano extract had no effect on the basal plasma insulin

concentrations in both normal and diabetic rats (Lemhadri et al.2004), while chamomile is

proven to significantly increase the serum insulin and C-peptide levels in diabetic rats (Kholoud

and Manal 2012). The observed effects of chamomile for its hypoglycemic action in diabetic rats

may be due to potentiating the effect of insulin in serum or by increasing either the pancreatic

secretion of insulin from the existing beta cells or its release from the bound form. Contrarily,

reports show the hypoglycemic activity of the oregano extract is facilitated by the inhibition of

hepatic glucose production and/or stimulation of glucose utilization by peripheral tissues,

especially muscle and adipose tissue (Lemhadri et al.2004). This action of oregano extract, when

combined with insulin secretion activity of chamomile, would have contributed to the observed

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anti-hypoglycemic effects in mixture treatment group where individual low doses were less

effective.

Reports indicate that, the treatment of diabetic rats with chamomile extract decreased

levels of both serum creatinine and blood urea to the normal level (Asghari et al.2015). The

decrease in blood urea and serum creatinine in chamomile treated diabetic rats may be due to the

antioxidant activity of chamomile (Najla et al.2012). Moreover, studies also reveal that, the oral

administration of the aqueous extract of oregano leaves improved the elevation in blood urea,

uric acid and creatinine levels in diabetic rats (Nema and Omimah 2013). Oregano extract is also

showed to have anti-urolithic activity both in vitro and in vivo models in addition to its

antioxidant, cell protective, antispasmodic and diuretic activities (Khan et al.2011). Our results

were in line with above literature where diabetic rat treated with 300 mg/kg each extract alone

decreased the levels of urea, uric acid, creatinine and increased total protein compared to diabetic

group. While 150 mg/kg individual treatments were less effective, the mixture of both extracts on

diabetic rat was more effective in the improvement of renal parameters compared to the effect of

300 mg/kg each extract on diabetic animals.

Diabetic rats treated with oregano and /or chamomile extracts indicated decrease of total

cholesterol, triglycerides and LDL-c with increase HDL-c compared to the diabetic rats. Oregano

is proven to be effective in reducing the serum lipid levels significantly in NIDDM rats (Pimple

et al.2012). There are studies to show aqueous chamomile extracts decreases TC, TG and LDL-c

with raise in HDL-c in diabetic rats (Asghari et al.2015; Najla et al.2012). The decrease in serum

levels of TC and TG may be attributed due to the uninhibited actions of lipolytic hormones on

the fat depots (Goodman and Gilman 1985) or to the increase in the metabolism of free fatty

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acids from the peripheral fat depots (Pari and Latha 2002). The results of lipid profile showed

that the treatment of diabetic rats with the mixture of both extracts were significantly effective as

anti-hyperlipidaemic compared to the effect of each extract alone on the diabetic animals.

Overall, above results could be translated into restoration of diabetes-induced lipo-toxicity by

either extracts or their combinations in diabetic rats.

Apoptosis is an important mechanism of cell death associated with diabetes induced renal

damage (Marcia et al.2015). Annexin V assay showed the decrease in apoptotic cell population

observed in extracts or mixture treatment (Fig. 4 a, b) confirmed the anti apoptotic effects of

chamomile, oregano and their combination in diabetes kidney cells. The event of apoptosis is

tightly regulated by anti apoptotic Bcl-2 protein and pro apoptotic Bax protein (Moretti et

al.2010). The ratio of Bcl-2/Bax is therefore critical in determining the susceptibility of cells to

induce apoptosis(Rasiova 2002). From fig 4 d, it is evident that up regulation of anti-apoptotic

Bcl-2 caused decrease in the pro-apoptotic Bcl-2/Bax ratio in all treatments, suggesting

chamomile, oregano and their mixture had a protective effect against diabetes induced renal

apoptosis . Collectively our results clearly show that both extracts and their combination play a

protective role in diabetes induced renal damage.

Diabetic lipotoxicity manifested by down regulation of fatty acid oxidation with altered

expression of its downstream lipogenic transcription factors is well linked with diabetic

nephropathy(Murea et al.2010). There are studies to show that lipid metabolism may serve as a

target for specific therapies of human diabetic nephropathy (Herman et al.2014; Kim et

al.2013). As both chamomile and oregano extracts have established anti oxidant activities, it is

noteworthy to have a protective effect on diabetes induced renal toxicity as observed in figure 4.

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These results also correlated well with anti hyperlipidaemic activity of the extracts and their

mixture observed in this study. There are many examples where synergy was observed, when

individual agents showed little or no effect (Khafif et al.1994; Suganuma et al.1999).

Collectively these results give us an insight on the combinational use of the plant

extracts, further the dose ratio and variations in batch to batch extraction process may alter drug

efficacy. However all these pitfalls can be overcome with cGMP facility standards, the dose for

the big animal’s especially non-human primates and clinical trials with humans may pose great

challenge with the concentration of the crude extracts.

5. Conclusion:

In conclusion, the present study clearly demonstrated that aqueous extracts of chamomile

andr oregano could be potentially useful in treating hyperglycemia and related diabetic

complications. In addition a mixture of Chamomile and Oregano extracts may produce a

therapeutic product with a potent anti-diabetic activity.

Funding Organization: Not Applicable.

Acknowledgement: We profoundly thank Dr. Harish C Chandramoorthy, Department of

Microbiology and Parasitology, College of Medicine, KKU, Abha for his immense help in

analyzing the results and writing the manuscript. We also thank Dr.Narasimhan Guruswamy,

Department of Pharmacology, College of Pharmacy, KKU, Abha for his help rendered during

final preparation of the manuscript.

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Figure legends:

Figure 1: Effect of chamomile, oregano and their mixture on (a) body weight, (b) insulin, (c) –

blood glucose, (d) HbA1C and (e) serum α-Amylase in alloxan- induced diabetic rats for 3 and 6

weeks treatment . Results expressed as mean ± SD (n=5). * p ≤ 0.05 Vs uninduced control rats;

** p ≤ 0.05 Vs diabetic control rats.

Figure 2: Renal function profile of alloxan induced diabetic rats on treatment with chamomile,

oregano, individually or as mixture. (a) Blood urea nitrogen, (b) Creatinine, (c) Uric Acid (d)

Total protein levels. Results expressed as mean ± SD (n=5). * p ≤ 0.05 Vs uninduced control

animals; ** p ≤ 0.05 Vs diabetic control animals

Figure 3: Anti hyperlipidaemic effects of chamomile, oregano extracts either individual or

mixture. (a) Total cholesterol [TC], (b) triglycerides [TG], (c) high density lipoproteins [HDL],

(d) low density lipoproteins [LDL], (e) very low density lipoproteins [VLDL] in alloxan-

induced diabetic rats. Results expressed as mean ± SD (n=5). * p ≤ 0.05 Vs uninduced control

rats; ** p ≤ 0.05 Vs diabetic control rats.

Figure 4: Apoptotic profile of isolated rat kidney cells with or without treatment.

(a) representative histograms showing flow cytometric enumeration of the apoptotic cells by

Annexin V staining, (b) decrease of total apoptosis in kidney cells with chamomile, oregano

individual or combined treatment, (c) representative western blots of anti-apoptotic Bcl-2 and

pro-apoptotic BAX expression in rat kidney cells. (d) Quantitative analyses of the Bcl-2/BAX

ratio. Results expressed as mean ± SD (n=5). * p ≤ 0.05 Vs uninduced control rats; ** p ≤ 0.05

Vs diabetic control rats.

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Figure-1

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Table-1: Hypoglycaemic effect of chamomile, oregano and their combination in 3 and 6 weeks

treatments. Data are represented as mean ± SD (n=5 per group).

a Expected value of chamomile and oregano combination = ([observed chamomile treatment

value]/ [control value]) × ([observed oregano treatment value]/ [control value]) × (control value).

b Ratio = (expected value/observed value).

Table-1 a

Treatment

Insulin reduction

(Normalised with

DC)

Observed Values (%)

Expected Value

(%)a

Rat

io b

3

week

s

Diabetic Control

(DC) 100.000

150 mg/kg

chamomile 88.892

150 mg/kg oregano 82.075

Mixture 44.461 72.95

1.6

4

6

week

s

Diabetic Control

(DC) 100.000

150 mg/kg

chamomile 80.856


Mixture 35.453 62.45

1.7

6

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A ratio of > 1 indicates a synergistic effect, a ratio of 1 indicates additive effect and a ratio of < 1

indicates less than additive effect.

a Average value of chamomile and oregano combination

b Ratio = (average value / observed value).

A ratio of > 1 indicates a synergistic effect, a ratio of 1 indicates additive effect and a ratio of < 1

indicates less than additive effect.

Table-1 b

Treatment

Insulin reduction

(Normalised

with DC)

Observed (%) Average(%)a

Rati

o b

3

weeks

Diabetic Control

(DC) 100.000

150 mg/kg

chamomile 88.892 85.48


Mixture 44.461 1.92

6

weeks

Diabetic Control

(DC) 100.000

70.04 150 mg/kg

chamomile 80.856


Mixture 35.453 1.97

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