Antioxidant effect of frankincense extract in the brain cortex of diabetic rats

Antioxidant effect of frankincense extract in the brain cortex of diabetic rats Journal of the Association of Arab Universities for Basic and Applied Sciences (2016) xxx, xxx–xxx University of Bahrain[.]

Antioxidant effect of frankincense extract in the

brain cortex of diabetic rats

a

Biochemical Technology Program, Department of Chemistry, Faculty of Applied Science, Thamar University, P.O

Box 87246, Thamar, Yemen

b

Department of Pharmacy, Faculty of Medicine and Health Sciences, Thamar University, P.O Box 87246, Thamar, Yemen

c

Department of Biology, Faculty of Science, Sana’a University, Sanaa, Yemen

d

Department of Pharmacy, Faculty of Medical Sciences, Al-Nasser University, Sanaa, Yemen

Received 9 April 2016; revised 19 July 2016; accepted 8 October 2016

KEYWORDS

Alloxan;

Diabetes mellitus;

Frankincense;

Thiols

Abstract The number of diabetes mellitus (DM) patients is one of the major concerns worldwide

As one of the main mechanisms of DM pathology is the involvement of oxidative stress, here we investigate the antioxidant capacities of frankincense (FRN) to treat or reduce the DM complica-tions in the brain cortices of DM rats Animals were segregated into four groups, the control group, FRN group given a dose of 500 mg of FRN/kg for 5 weeks, DM group given a single dose of 150/kg i.p of alloxan to induce diabetes and DM + FRN group given a single dose of 150/kg i.p to induce

DM then followed by FRN 500 mg/kg for 5 weeks The animals were sacrificed; their cerebral cor-tices were removed and used for biochemical and histopathological analyses

Alloxan treatment in the DM group showed significant reductions in catalase (CAT) activity and other non-enzymatic antioxidants i.e thiol groups, concomitant with decreases in the levels of pro-tein and albumin and increasing the level of uric acid However, FRN administration to DM ani-mals in DM + FRN group showed significant recovery of antioxidants, the thiol contents (total thiols, protein thiols and glutathione) of DM + FRN group have been increased as compared with

DM animals (p < 0.05) A recovery of CAT activity (p < 0.05) to almost the levels of control rats with the recovery in protein and albumin levels (p < 0.05) have been observed when FRN was administered The uric acid level increased in DM group, came back to the levels of control after administration of FRN (p < 0.05) We also observed that FRN reduces the histopathological dam-age caused by alloxan in DM + FRN group It is concluded that FRN shows a beneficial effects that can reduce the oxidative damage caused by alloxan induced DM in the cortex of rats

Ó 2016 University of Bahrain Publishing services by Elsevier B.V This is an open access article under the

CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ).

Abbreviations: CAT, catalase; DM, diabetes mellitus; FRN, frankincense; GSH, reduced glutathione; P-SH, Protein thiols; T-SH, total thiols

* Corresponding author.

E-mail address: angaz76@gmail.com (A Masoud).

Peer review under responsibility of University of Bahrain.

University of Bahrain

Journal of the Association of Arab Universities for

Basic and Applied Sciences www.elsevier.com/locate/jaaubas

www.sciencedirect.com

http://dx.doi.org/10.1016/j.jaubas.2016.10.003

1 Introduction

The increasing patient’s number of diabetes mellitus (DM),

expected to be 552 million by 2030, had lead the researchers

to evaluate the capacity of natural products to either reduce

or cure the complications of the disease (Whiting et al.,

2011) DM has been shown to be a risk factor in the brains,

causing cognitive impairments and dementia because the brain

is the main glucose consumers with a 60% use of the total

body’s glucose (Biessels et al., 2006; Exalto et al., 2012;

Gorelick et al., 2011; Wasserman, 2009) One of the main

fea-tures of DM is high glucose level, which advances to glycation

end-products and will subsequently produce reactive oxygen

species (ROS) which will result in cell damage and apoptosis

(Brownlee, 2005) Two types of DM are known with at least

80% of cases having type II DM, also called non-insulin

dependent DM, which can be induced in the animal models

by injection of chemical compounds that have similarities with

glucose in the body, such as alloxan yielding the main

charac-teristics of DM include body weight loss, hyperglycemia,

poly-uria and dysregulation of insulin levels (King, 2012) Because

of the lower side effects of the natural products, many

medic-inal plants have been used to cure different diseases

Frankin-cense (FRN) is one of those plants which is an aromatic resin

obtained from species of the Burseraceae family Boswellia

sacrahas been reported to possess a variety of

pharmacologi-cal effects including anti-hyperglycemia effect, antioxidant

effects, and anti-inflammatory effects (Akihisa et al., 2006;

Banno et al., 2006; Gupta et al., 1998; Mothana, 2011;

Mothana et al., 2007; Masoud et al., 2014) FRN extract has

streptozotocin- and alloxan-induced DM (Azemi et al., 2012;

Kavitha et al., 2007; Masoud et al., 2014) The present study

has been designed to study the possible antioxidant effects of

FRN on rat’s brain cortices following DM development

2 Materials and methods

2.1 Preparation of the FRN extract

The freshly obtained plant from Hadramout Governorate,

Yemen was grinded, suspended in ethanol (96%), shacked

for 4 h and the extract was prepared as described previously

(Masoud et al., 2014; Chevrier et al., 2005) The selected dose

of 500 mg/kg was given orally as it is reported as a safe dose

(Devi et al., 2012)

2.2 DM Induction

DM rats were given a single dose (150 mg/kg

interaperi-toneally) of freshly prepared alloxan monohydrate

(Sigma-Aldrich, USA) dissolved in normal saline (0.9% w/v NaCl)

to induce DM The levels of blood glucose were estimated in

blood sample collected by tail tipping method using

Accu-Chek Glucometer (China) Animals of glucose levels greater

than 250 g/dl were selected for the study

2.3 Animals and treatments

Female Albino rats were obtained and housed in the animal

house unit, Sana’a University, Yemen They were divided

ran-domly into four groups having 6–8 animals The study was conducted for 5 weeks and followed the guidance of animal care and use, Sana’a University The doses were given as follows:

Control Group: These animals were given DMSO solution (n = 6)

DM group: These animals injected with a single dose of 150/

kg interaperitoneally of alloxan to induce diabetes (n = 8) FRN group: Animals of this group received ethanolic extract of FRN (500 mg/kg) dissolved in DMSO solution for

5 weeks (n = 6)

DM + FRN group: Following DM induction by a single dose of 150/kg interaperitoneally of alloxan, the animals were dosed ethanolic extract of FRN (500 mg/kg) dissolved in DMSO solution for 5 weeks (n = 8)

2.4 Sample preparation and isolation

At the end of the study (5 weeks of FRN administration) ani-mals were sacrificed by cervical dislocation, their brains were removed and the cortices were dissected and homogenized in phosphate buffered saline (pH 7.4) Part of the homogenates were centrifuged (3000g for 10 min) and the supernatants and rest of the homogenates were used for biochemical assays, also some cortices of each group were used for histopatholog-ical studies

2.5 Biochemical analyses 2.5.1 Total thiols The total thiol (T-SH) was spectrophotometrically quantified

in the cerebral cortex homogenate according to the method

of Ellman (1959) and as modified by Sedlak and Lindsay (1968) Tris–HCl (0.2 M) and EDTA (0.02 M, pH 8.2), homo-genate and 0.01 M DTNB (in methanol) were incubated for

15 min at room temperature then followed by centrifugation

at 1200g for 5 min The absorbance was read at 412 nm and the results were expressed as nmoles of T-SH/mg protein using molar extension coefficient of DTNB (13,600 cm 1M 1) 2.5.2 Glutathione contents

Glutathione (GSH) contents were measured in the cerebral

(1959) Sulphosalicylic acid (4% w/v) was used to precipitate proteins, followed by centrifugation at 1200g for 5 min 0.1 mM DTNB in 0.1 M phosphate buffer (pH 8.0) was added

to the supernatant and the absorbance was read at 412 nm after 2 min Results were expressed as nmoles of GSH/mg

(13,600 cm 1M 1)

2.5.3 Protein thiols Protein thiols (P-SH) were measured by subtracting the GSH from T-SH and the results were expressed as nmoles of P-SH/mg protein using molar extension coefficient of DTNB (13,600 cm 1M 1)

2.5.4 Catalase (CAT) activity The activity of CAT was measured in the supernatant as described by Luck (1971) The absorbance of a reaction

mixture (supernatant and 12.5 mM H2O2 in 0.067 M

phos-phate buffer (pH 7.0) was followed at 240 nm for 3 min

Results were expressed as lmoles of H2O2 decomposed/min/

mg protein using molar extinction coefficient of H2O2

(71 M 1cm 1)

2.5.5 Protein, albumin and uric acid assays

The biochemical tests include assays of total protein, albumin,

and uric acid (UA) were estimated following the instructions of

commercial kits provided by Spinreact, Spain

2.5.6 Histopathological studies

Histopathological studies were carried out by performing

rou-tine hematoxylin and eosin staining to evaluate the

morpho-logical and structural changes in the brain cortices and the

slides were examined under light microscope

2.5.7 Statistical analysis

Data were expressed as mean ± S.D and were analyzed by

one way ANOVA followed by Student–Newman–Keuls post

hoc test Differences between groups were considered

signifi-cant when P < 0.05 and all analyses were performed using

the sigma-stat software (version 3.5)

3 Results

Following DM development in rats, administration of FRN

extract showed improvement in the antioxidants parameters

assessed in the present study The typical signs of DM were

seen (e.g increase in blood sugar, excessive urine etc.) The

antioxidants of DM group showed sharp reduction in CAT

activity by 4.8-fold as compared to control (Fig 1), 3-fold

decreases in GSH content, 1.5-fold reduction and T-SH and

1.34-fold decreases in P-SH levels (Table 1) Treatment with

FRN increases significantly the activity of CAT in the brain

cortex of DM + FRN animals as compared to DM group

(p < 0.05,Fig 1) This improvement accompanied by increase

in the levels of thiol contents, where T-SH, GSH and P-SH

contents increased in the DM + FRN group as compared to

DM group (p < 0.05,Table 1) In the all above antioxidants

no significant changes were seen among control and FRN

group, however, the contents of all thiols were less in DM + FRN group as compared to both control and FRN groups

As a result of oxidative damage, proteins and albumin levels were affected in the brain cortices of DM group (decreased by 3.68 and 5.05 folds respectively, p < 0.05) when compared to control (Figs 2 and 3 respectively) However, administration of FRN extract brought back the levels of pro-tein and albumin to almost the similar values of control Our results showed 3.23-fold increase in UA in DM group compared to control, meanwhile, administration of FRN to

DM rats reduces this increase to levels similar to those seen

in control (p < 0.05,Fig 4)

To evaluate the morphological change in the brain cortices and comparing them with the biochemical findings, the histopathological observations showed irregular and large spaces around neuron cell body in the DM group with cell con-gestion, however, these changes were slight spaces around

histopathological changes were seen in both control and FRN groups

4 Discussion

The increasing evidences of oxidative stress which plays a major role in the pathogenesis of DM, had lead researchers looking for natural antioxidants that might play a role in reducing the damaged caused by DM Here, we report the pos-sible involvement of one of those natural antioxidants, FRN

Figure 1 CAT activity in the brain cortices of control, FRN,

DM DM + FRN groups Results expressed as mean ± S.D.,

p< 0.05 considered significant compared to control, n = 6 in

each group

Table 1 Thiol contents in the brain cortex of control, FRN,

DM, DM + FRN rats (nmoles/mg protein)

Control 5.77 ± 0.80 a 21.65 ± 2.34 a 27.43 ± 3.14 a

FRN 5.41 ± 0.06 a 23.40 ± 2.98 a 28.82 ± 3.05 a

DM 1.92 ± 0.16 b 16.14 ± 1.87 b 18.06 ± 2.04 b

DM + FRN 4.37 ± 0.16 c 19.86 ± 1.38 c 24.24 ± 1.54 c

Results are expressed as mean ± S.D.; n = 6 in each group Superscript alphabets (a, b and c) are significantly different from their corresponding group, p < 0.05.

Figure 2 Protein levels in the brain cortices of control, FRN,

DM and DM + FRN groups Results expressed as mean ± S.D.,

p< 0.05 considered significant compared to control, n = 6 in each group

an aromatic resin from Boswellia species that used as

anti-inflammatory, antibacterial, antifungal and anticancer

(Banno et al., 2006; Chevrier et al., 2005; Weckesser et al.,

2007) and as antidiabetic induced by alloxan (Kavitha

et al., 2007; Masoud et al., 2014) or streptozotocin (Azemi

et al., 2012; Shehata et al., 2011) Our results showed that

administration of FRN (500 mg/kg for five weeks) to the

DM rats increases the antioxidant capacities in their brain

cortices, hence, reduces the oxidative stress caused by DM

It is well established that oxidative stress due to the

gener-ation of free radicals, glucose auto-oxidgener-ation and protein

gly-cation plays a role in the development of DM, where,

over-production of free radical disturbs the natural balance between

the production of free radicals and their antioxidant defense

system (Maiese, 2015; Bonnefont-Rousselot, 2004; Desco

et al., 2002; Lenzen, 2008; Muriach et al., 2014; Nowotny

et al., 2015; Yan, 2014; Yang et al., 2011) This was evident

from the data obtained here, where, CAT activity and thiol

contents were reduced significantly in the brain cortex of

DM rats Concomitantly, there were decreases in the levels

of protein and albumin accompanied by increase in UA

indi-cating disturbance in the antioxidant defense system in the

brain cortices of DM rats The histopathological changes

includes irregular and wide spaces around neuronal cell body

Figure 3 Albumin levels in the brain cortices of control, FRN,

DM and DM + FRN groups Results expressed as mean ± S.D.,

p< 0.05 considered significant compared to control, n = 6 in

each group

Figure 4 UA levels in the brain cortices of control, FRN, DM

and DM + FRN groups Results expressed as mean ± S.D.,

p< 0.05 considered significant compared to control, n = 6 in

each group

Figure 5 Histopathological changes in the brain cortices of (A) control (normal observation), (B) FRN (normal observation), (C)

DM (WS, wide space around the neuronal cell bodies) and (D) DM + FRN groups (SS, slight space around the neuronal cell bodies)

along with congestion seen in the brain cortex of DM rats

sup-port the biochemical findings However, FRN showed a relief

in the antioxidants following 500 mg/kg body weight

adminis-tration for 5 weeks We have seen that CAT activity, thiol

con-tents, protein level, albumin level and UA level came back to

almost the similar levels as seen in the control animals,

suggest-ing the potential antioxidant effect of this natural product We

reported previously that same dose of FRN helped rat’s red

blood cells (RBCs) to recover from oxidative stress (induced

by alloxan) by increasing the levels of antioxidant (Masoud

et al., 2014) Moreover, FRN showed that it possesses

antiox-idant effect (Azemi et al., 2012; Mothana, 2011; Mothana

et al., 2007, 2009)

One of the metabolic abnormalities of DM is

overproduc-tion of superoxide radical, which is converted to H2O2 by

the action of superoxide dismutase enzyme (Giacco and

Brownlee, 2010) The final product, H2O2, is then processed

by CAT, it is also well documented that the production of

xan-thine oxidase (XO) is increased, which is an enzyme plays

important role in the production of ROS in DM (Romagnoli

et al., 2010; Desco et al., 2002; Matsumoto et al., 2003) Here,

CAT activity was reduced in DM rats and the recovery

follow-ing administration of FRN could be attributed to the

antioxi-dant action of FRN which also helped in the recovery of other

non-enzymatic antioxidants Due to the presence of sulfhydryl

groups (-SH) in thiols including GSH, they are one of the

cel-lular non-enzymatic antioxidants acting as redox buffer

(Meister and Anderson, 1983) and quenching ROS and other

oxygen-centered free radicals (Kidd, 1997) This could explain

the reductions in thiols in our study, where, oxidative stress

caused by DM overwhelms the presence of natural

antioxi-dants in brain cortices; on the other hand, FRN helped cells

to recover its thiol contents Elevated UA levels might reflect

the increasing activity of XO, FRN administration in DM

+ FRN group back up the UA level to the similar levels found

in control animals The administration of FRN results in the

recovery of the antioxidant of brain cortices might be due to

the inhibition of the activity of XO which is responsible for

the production of ROS as other drugs reported to inhibit

XO (Romagnoli et al., 2010) We have reported that the

changes in CAT, thiols and UA have been recovered in the

RBCs of rats exposed to alloxan and treated with FRN extract

(Masoud et al., 2014) In addition to these changes, brain

cor-tices of DM rats showed histopathological changes, where,

irregular and large spaces around neuron cell body along with

cell congestion have been observed in DM group These

obser-vations were slight spaces around neuron cell body in DM

+ FRN group Our findings are consistent with different

stud-ies reported histopathological changes following DM

induc-tion (Malone et al., 2006; Edwards et al., 2010; Francis

et al., 2008), however, these findings in contrast to those

reported by Guven et al., who did not observe any changes

in neuron following 4 weeks of STZ-induced diabetes in rats

(Guven et al., 2009)

5 Conclusion

In Conclusion, our findings in favor of using FRN to reduce

the oxidative damage caused by DM Administration of

FRN extract to DM rats showed significant increase in the

antioxidant contents which was confirmed by

histopathologi-cal studies suggesting the antioxidant beneficiary of FRN as

a good candidate in the treatment of conditions that causes oxidative stress include DM with a suggestion for future study

to reduce the dose less than 500 mg/kg for long period Conflict of interest

No financial, personal or other conflict of interest

Acknowledgements The authors are greatly acknowledged Dr Ahmad Saif Muharram, Al-Nasser Board of Trustees Chairman, for his valuable assistance

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