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Matricaria Chamomilla extract demonstrates antioxidant properties against elevated rat brain oxidative status induced by amnestic dose of scopolamine
Biomedicine & Aging Pathology 4 (2014) 355–360
Available online at
ScienceDirect www.sciencedirect.com
Original article
Matricaria Chamomilla extract demonstrates antioxidant properties against elevated rat brain oxidative status induced by amnestic dose of scopolamine Zahra Alibabaei , Zahra Rabiei , Samira Rahnama , Shiva Mokhtari , Mahmoud Rafieian-kopaei ∗ Medical Plants Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran
a r t i c l e
i n f o
Article history: Received 12 April 2014 Accepted 10 July 2014 Available online 15 August 2014 Keywords: Matricaria Chamomilla Alzheimer’s disease Scopolamine
a b s t r a c t Alzheimer’s disease (AD) is clinically characterized by progressive loss of cognitive abilities and usually is accompany with elevated oxidative stress. Chamomile is a plant with antioxidant activity with is currently used in Iranian folk medicine as sedative, analgesic, antipyretic and antispasmodic agent. The present study was investigated the effect of Matricaria chamomilla (MC) on learning and memory functions in scopolamine-induced memory deficit in rats. Memory enhancing activity in scopolamine-induced amnesic rats was investigated by assessing the Morris water maze and passive avoidance paradigm. Forty-two male Wistar rats were divided into 6 equal groups as bellow: 1 – control (received water), 2 – SCOP (received scopolamine 1 mg/kg for 15 days), 3 and 4 – SCOP + MC (received scopolamine and MC extract 200 and 500 mg/kg b.w. per day for 15 days), 5 and 6 – intact groups (received MC extract 200 and 500 mg/kg b.w. per day for 15 days). M. Chamomilla ethanolic extract produced significant memory enhancing activity when evaluated by Morris water maze and passive avoidance paradigm models. Our results suggest that M. chamomilla ethanolic extract has repairing effects on memory deficit and might be beneficial in patients with Alzheimer’s disease and behavioral disorders. The memory enhancing activity of the extract may be attributed to the free radical scavenging activity, which would have been afforded by the active constituents present in the extract. © 2014 Published by Elsevier Masson SAS.
1. Introduction Alzheimer disease (AD) is the most common form of dementia. There is no cure for the disease and it worsens as it progresses. Most often, AD is diagnosed in people over 65 years of age although the less-prevalent early-onset Alzheimer’s can occur much earlier [1]. Because of this problem, alternative and complementary therapies are needed. Several studies have shown the neuroprotective and cognition-enhancing properties of herb extracts and other natural products using different animal models [2–5]. Scopolamine, a blocker of muscarinic acetylcholine receptor that has been used to induce experimental models of Alzheimer’s disease [6]. Scopolamine significantly increases AChE and malondialdehyde (MDA) levels in the cortex and hippocampus [7]. The
∗ Corresponding author. Tel.: +983813346692. E-mail address: rafi[email protected] (M. Rafieian-kopaei). http://dx.doi.org/10.1016/j.biomag.2014.07.003 2210-5220/© 2014 Published by Elsevier Masson SAS.
elevation of brain oxidative stress after administration of scopolamine further substantiates the value of scopolamine-induced amnesia as an animal model [8]. Chamomile is perennial flowering herb that grows in widespread regions, including Europe, Africa, and Asia. Chamomile is widely distributed in Iran and is used in Iranian traditional medicine for various conditions. One of the most important flavonoid of Chamomile is apigenin, which has mild relaxing property [9]. Previous studies showed that streptozotocin resulted in oxidative stress and affected the antioxidant status. Treatment with ethanolic extract of Chamomile significantly reduced postprandial hyperglycemia and oxidative stress, and augmented the antioxidant capacity. Treatment with ethanolic extract of Chamomile significantly increased the blood glutathione level [10]. In the present study, we investigated the behavioral recovery following chronic exposure to ethanolic extract of Matricaria chamomilla on scopolamine-induced dementia in rat model.
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2. Materials and methods
2.6. Experimental protocol
2.1. Preparation of the extract
• Group 1: received vehicle (distilled water) for 15 days consecutively. • Group 2: received scopolamine 1 mg/kg intraperitoneally (i.p) for 15 days consecutively (SCOP group). • Groups 3 and 4: received simultaneously scopolamine 1 mg/kg and M. Chamomilla ethanolic extract with doses of 200 and 500 mg/kg for 15 days (SCOP + 200, 500 MC). • Groups 5 and 6: intact groups that received only M. Chamomilla ethanolic extract with doses of 200 and 500 mg/kg for 15 days (intact 200, 500 MC).
Air-dried M. chamomilla was pulverized with a blender and the extract was prepared by adding ethanol (70%) on the plant powder. The pooled extract was filtered after 48 hours. The filtrate was concentrated using a rotary vacuum evaporator under reduced pressure until dryness. The extract was stored at 4 ◦ C, and reconstructed with distilled water to obtain different doses when needed to treat the animals.
2.2. DPPH free radical scavenging assay Antioxidant activity of the M. chamomilla extract was measured using the DPPH assay based on the scavenging ability to 2, 2diphenyl-1-picrylhydrazyl (DPPH) stable radical [11]. Butylated hydroxytoluene (BHT) was used as a positive control. The samples in different concentrations were mixed with DPPH solution and ethanol. After vortexing, the tubes were left in the dark at room temperature after which the absorbance was measured at 517 nm using a UV–vis spectrophotometer (Biochrom Ltd, England). Each measurement was performed in triplicate under identical conditions. Antioxidant activities were expressed as the IC50 values (the concentration of antioxidant required to cause 50% reduction in the original concentration of DPPH). Inhibition of free radical by DPPH (%) was calculated as follows:
I (%) = 100 × Acontrol − Asample /Acontrol
Animals were randomly divided into 6 groups with 7 in each group. 2.7. Water maze test A circular water pool with 183 cm in diameter and 60 cm in deep was used for a water maze test. The entire inside of the pool was painted black, and the pool was filled with muddy water containing ink at 22 ◦ C. The tank was placed in a dimly lit, soundproof test room with various visual cues. A white platform was then placed in one of the pool quadrants. During the four subsequent days the rats were given four trials sessions per day with the platform in place. When a rat located the platform, it was permitted to remain on it for 10 s. The time interval between trial sessions was 30 min. During each trial session, the time taken to find the hidden platform (latency) was recorded using a video camera based (Ethovision System). One day after the final training trial sessions, the rats were individually subjected to a probe trial session in which the platform was removed from the pool and the rats were allowed to swim for 120 s to search for it. A record was kept of the swimming time in the pool quadrant where the platform had previously been placed.
2.3. Total phenolic compounds 2.8. Passive avoidance test The concentration of total phenolic compounds in M. chamomilla extract was determined using Folin–Ciocalteau method as described previously with minor modification. Briefly, to 0.5 mL of a 5.5 g/L diluted extract, 2.5 mL of Folin–Ciocalteau reagent (diluted 10 times with water) was added. The standard curve was plotted using 12.5, 25, 50, 62.5, 100, and 125 mg/L solutions of gallic acid in methanol and water (60:40, v/v). The absorbance was measured at 760 nm. The total phenolic contents of the extracts were expressed as gallic acid equivalent (mg/g extract) [12].
2.4. Total flavonoid and flavonol The amounts of total flavonoids and flavonols in the M. chamomilla extract were determined calorimetrically as described by Rabiei et al. Total flavonoids and flavonols were expressed in terms of rutin equivalent (mg/g), which is a common reference compound [13].
2.5. Animals Male Wistar rats, weighing 150–250 g, were obtained from Pasteur institution (Tehran, Iran). Rats were housed in groups of four in cage at 25 ◦ C with controlled 12 h light-dark cycle. Food and water were freely available. All experiments were executed in accordance with the Guide for the Care and Use at Laboratory Animals and were approved by Research and Ethics Committee at Shahrekord University of Medical Sciences.
Testing of passive avoidance performance was carried out in two identical light and dark square boxes as described in our previous reports. The rats were initially placed in the light chamber and 20 s later the door between compartments was opened. When the rat entered the dark compartment with all four paws, the guillotine door was closed, and the latency to enter was recorded (t1) (from the time the door is lifted), and an electrical foot shock (1 mA, lasting 1 s) was delivered through the stainless steel rods. Twentyfour hours after the training trial, the rats (individually) were again placed in the light compartment. The step-through latency (t2) to enter the dark compartment was measured. If the rat did not enter the dark compartment within 120 s, the experiment was stopped. 2.9. Ferric reducing/antioxidant power (FRAP) assay At the end of behavioral tests, the blood samples were obtained through cardiac puncture for biochemical estimations, and then the serum and plasma were immediately separated from the blood samples by centrifugation. Antioxidant capacity of the plasma was determined by measuring its ability to reduce Fe3+ to Fe2+ with ferric reducing ability of plasma (FRAP) test. FeSO4 (100–1000 M concentration range) was used as a standard in FRAP assay. The results are expressed in M [14]. 2.10. Measurement of plasma malondialdehyde (MDA) The plasma MDA level was measured as LPO by the thiobarbituric acid reactive substances (TBARS) method. Briefly, to 100 L plasma or standard, 100 L sodium dodecyl sulfate (8.1%) and
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2.11. Malondialdehyde (MDA) levels of brain Following the behavioral tests, rats were killed. The brains were quickly removed and were washed twice with cold saline solution, different parts of brains such as hippocampus, cortex and subcortex separated, placed into glass bottles, labeled, and stored in a deep freeze (−80 ◦ C) until processing. Tissues were homogenized in ice-cold Tris–HCl buffer (50 mM, pH7.4) for 2 min at 5000 rpm. The homogenized solution was then centrifuged for 60 min at 5000 g. Malondialdehyde (MDA) level was then measured. Lipid peroxidation was evaluated by measuring the TBARS content according to the TBA test method with slight modification. The MDA level was determined by a method based on the reaction with thiobarbituric acid (TBA) at 90–100 ◦ C. The reaction was performed at PH 2–3 at 90 ◦ C for 15 min. The sample was mixed with 2 volumes of cold 10% (w/v) trichloroacetic acid to precipitate protein. The precipitate was pelleted by centrifugation and an aliquot of the supernatant was reacted with an equal volume of 0.67% TBA (w/v) in a boiling water bath for 10 min. After cooling, the absorbance was read at 532 nm using spectrophotometer [16]. 2.12. Statistical analysis All the results were expressed as mean ± SEM and statistical analyses were performed using SPSS 11.0 statistical software. P < 0.05 was considered statistically significant. 3. Results 3.1. Standardization of M. chamomilla extract To standardize the plant extract, total phenolic, flavonoid and flavonol components in M. chamomilla extract were measured. Total amount of phenolic compounds in M. chamomilla extract was 78.4 mg galic acid equivalent per one gram dried extract. Total amount of flavonoid and flavonol compounds were 47.6 mg/g and 26.5 mg, respectively per one gram of dry matter. 3.2. Effect of M. chamomilla extract on DPPH free radical scavenging
Table 1 Free radical scavenging activity of different concentrations of Matricaria chamomilla extract. Sample
Concentration (g/mL)
DPPH radical scavenging activity Inhibition (%) IC50 (g/mL)
Matricaria chamomilla extract
80 70 60 50 40 30 25 20 15 10
74.8 65.3 56.9 49.2 (IC50 ) 41.29 32.4 27.5 22.15 18 5.8
IC50 values are highlighted in bold. DPPH: 2, 2-diphenyl-1-picrylhydrazyl.
(T2) time of intact rats treated with 200 and 500 mg/kg of M. chamomilla extract were significantly increased compared to SCOP rats (P < 0.05) but did not increase compared with the control group (P > 0.05) (Fig. 1). 3.4. Morris water maze swimming test The time spent by the animal, searching for the missing platform in target quadrant (zone 1) with respect to other quadrant (zones 2–4) on day 5 was noted as an index of retrieval memory. In the probe trial following the last training session M. chamomilla with doses of 200 and 500 mg/kg increased the swimming time in the target quadrant (zone 1) after the platform was removed in SCOP + 200 MC, SCOP + 500 MC and intact + 500 MC groups when compared with scopolamine-treated group (P = 0.049, P = 0.002, P = 0.008, respectively) (Fig. 2). Animals in the control group rapidly learned the location of the platform. This was demonstrated by exhibiting a reduction in swimming times from the first to the second trial on day 1 and by reaching stable latencies at day 2. By contrast, rats treated with scopolamine (1 mg/kg body weight) failed to find the platform until given the maximum time. The escape latency did not decrease in the SCOP, SCOP + 200 MC and SCOP + 500 MC groups compared to the control group (P > 0.05). The latency before reaching platform on day 3 significantly decreased in control, intact + 200 MC and intact + 500 MC groups respect to SCOP group (P = 0.022, P = 0.002, P = 0.015, respectively). Escape latency significantly reduced in
80
***
60
Time(sec)
2.5 mL TBA/buffer (prepared by dissolving of 0.53% thiobarbituric acid in 20% acetic acid as adjusted to pH 3.5 with NaOH) were added. The tubes were covered with caps and incubated at 95 ◦ C for 60 min. The reaction was stopped by placing tubes on ice followed by centrifugation at 4000 rpm for 10 min to separate two phases. The supernatant (20 L) was injected into the HPLC system. Chromatographic determinations were performed on a high-performance liquid chromatograph equipped with an 1100 series pump and a UV absorbance detector. An HP 3395 integrator was employed to record retention times, chromatograms, and evaluate peak heights. A technopak 10u C18 reversed-phase column (emission 553 and excitation 515) was used [15].
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t1 t2
****** ***
***
40
20
C t5
In ta c
20 ct
00
0M
M
C
C ta In
0M
00 SC O P+ 5
20 P+ O
M
C
l co nt ro SC
3.3. Passive avoidance test
0 SC O P
The antioxidant activity of M. chamomilla extract was assessed on the basis of radical scavenging of the stable DPPH free radical. M. chamomilla extract showed strong free radical scavenging activity against DPPH radicals, with an IC50 value of 50 g/mL (Table 1).
Groups
The step-through latency of the scopolamine-treated group was significantly shorter than that of the control group (P < 0.05). The shorter step-through latency induced by scopolamine was significantly reversed by M. chamomilla extract with doses of 200 and 500 mg/kg (P < 0.05). Furthermore, the step-through latency
Fig. 1. The initial latency and step-through latency in the passive avoidance response. T1: initial latency; T2: step-through latency. The data are expressed as mean ± SD; n = 7 in each group. *** P < 0.01 SCOP vs. control, SCOP + 200 MC, SCOP + 500 MC, intact 200 MC, intact 500 MC groups (n = 7). MC: Matricaria chamomilla; SCOP: scopolamine.
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800
*** ***
FRAP(μ μ M)
600
400
200
C M 00
M
t5
00
ac
t2
In t
ac In t
SC
SC
O
O P+
P+
50
20
0M
C
C
C 0M
ol co nt r
SC
O P
0
Groups Fig. 2. Spent time in zone 1 during the probe trial in experimental groups. The data are expressed as mean ± SD; n = 7 in each group. * P < 0.05; *** P < 0.0.
Fig. 4. Plasma antioxidant capacity (ferric reducing/antioxidant power) in experimental groups. The data are expressed as mean ± SD; n = 7 in each group. *** P < 0.001.
control, intact + 200 MC and intact + 500 MC groups when compared with scopolamine-treated group in day 4 (P = 0.021, P = 0.006, P = 0.042, respectively) (Fig. 3).
30
20
***
*** 10
***
***
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C 0M
C ct ta In
In
ta
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20
50
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0M
ol
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0 co nt r
Administration of scopolamine with dose of 1 mg/kg in scopolamine-treated rats caused a slightly reduction (not significant, P > 0.05) of plasma antioxidant levels compared with control group. M. chamomilla extract treatment significantly increased the plasma antioxidant level in intact 200 MC and intact 500 MC groups when compared with scopolamine-treated group (P = 0.000, P = 0.000, respectively) and when compared with control group (P = 0.028, P = 0.004, respectively) (Fig. 4).
MDA (ng/ul)
SC O P
3.5. Plasma antioxidant level
Groups
Fig. 5. Plasma malondialdehyde level (MDA) in experimental groups. The data are expressed as mean ± SD; n = 7 in each group. *** P < 0.001.
3.6. Plasma level of malondialdehyde Scopolamine administration with dose of 1 mg/kg significantly increased the plasma MDA levels in SCOP group respect to control group (P = 0.000). M. chamomilla extract with doses of 200 and 500 mg/kg significantly reduced the plasma MDA levels in SCOP + 200 MC, SCOP + 500 MC, intact 200 MC and intact 500 MC groups (P = 0.000, P = 0.000, P = 0.000, P = 0.000, respectively) when compared with SCOP group (Fig. 5).
3.7. Brain MDA levels Administration with scopolamine (1 mg/kg) produced a significant increase in MDA content of hippocampus and cortex in SCOP group, an index of lipid peroxidation, when compared with control group (P = 0.031, P = 0.000, respectively). M. chamomilla extract
Latrency time (s)
60
40
20
* * ***
* * ***
SCOP control SCOP+200MC SCOP+500MC Intact200MC Intact500MC
4
3
2
1
0
Traning days Fig. 3. Spatial learning in a hidden platform model in experimental groups during four training days. The data are expressed as mean ± SD; n = 7 in each group. * P < 0.05; *** P < 0.001.
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Scopolamine treatment with the dose of 1 mg/kg significantly increased the brain and plasma MDA levels when compared with control group. Chamomile extract administration dramatically decreased the brain and plasma MDA levels in experimental groups. These results may suggest that Chamomile extract antioxidant property protected the brain by prevention of oxidative status due to administration of scopolamine. MDA is one of the major aldehydes formed after breakdown of lipid hydroperoxides. Therefore, it is considered a good biomarker of the involvement of free radical damage in pathologies associated with oxidative stress [20]. In this study, we proved Chamomile extract have antioxidant activity through the DPPH method and increased significantly the serum antioxidants levels in groups that received extract and might be beneficial in patients with memory deficit. Fig. 6. Brain malondialdehyde level (MDA) in experimental groups. The data are expressed as mean ± SD; n = 7 in each group. * P < 0.05; *** P < 0.001.
significantly decreased the MDA content in hippocampus, cortex and subcortex in SCOP + 200 MC group (P = 0.000, P = 0.001, P = 0.000, respectively) and SCOP + 500 MC group (P = 0.006, P = 0.000, P = 0.000, respectively) when compared with SCOP group. M. chamomilla extract treatment significantly reduced MDA content of hippocampus, cortex and subcortex in intact groups with doses of 200 (P = 0.000, P = 0.000, P = 0.000, respectively) and 500 (P = 0.000, P = 0.000, P = 0.000, respectively) when compared with SCOP groups (Fig. 6). 4. Discussion The present study aimed at evaluating the ethanolic extract of M. chamomilla for memory enhancing activity in scopolamineinduced amnesic rats using Morris water maze and passive avoidance paradigm and to carry out the biochemical estimation like MDA in brain tissue homogenate and in plasma at end of the study. The brain is considered to be more susceptible to peroxidative damage than other tissues due to the high content of their polyunsaturated lipid-rich neural parenchyma, high oxygen utilization and low antioxidative enzymes. Furthermore, previous studies indicate that oxidative stress is one of the earliest events in pathogenesis of memory impairment [17]. Previous studies showed that the cerebral cortex and hippocampus, which are thought to control cognitive and motor functions, seem to be sensitive to oxidative stress, and require antioxidants [18]. Medicinal plants having antioxidant properties, have been shown to improve memory and learning in rat model of Alzheimer’s [13]. There is notable evidence that scopolamine causes oxidative stress through the interference with acetylcholine in brain and leading to cognitive impairment [8,19]. This study aimed at investigating whether such impaired cognition due to scopolamine administration is associated with altered oxidative stress indices. However, brain oxidative status in this experimental animal model of amnesia that induced by scopolamine has yet to be evaluated. Scopolamine administration dramatically decreased the stepthrough latency compared with control group. The shorter stepthrough latency induced by scopolamine was significantly reversed by M. chamomilla extract. In Morris water maze test, in probe trial, M. chamomilla extract significantly increased the swimming time in the target quadrant.
5. Conclusion We herein aimed to test antioxidant, and antiamnesic activities of the M. chamomilla ethanolic extract in rat. Our results showed that M. chamomilla extract could be beneficial in the treatment of cognitive impairment on account of their various activities related to AD. References [1] Francis PT, Palmer AM, Snape M, Wilcock GK. The cholinergic hypothesis of Alzheimer’s disease: a review of progress. J Neurol Neurosurg Psychiatry 1999;66(2):137–47. [2] Rabiei Z, Rafieian-Kopaei M, Mokhtari S, Alibabaei Z, Shahrani M. The effect of pretreatment with different doses of Lavandula officinalis ethanolic extract on memory, learning and nociception. Biomed Aging Pathol 2014 [in press]. [3] Rabiei Z, Hojjati M, Rafieian-Kopaeia M, Alibabaei Z. Effect of Cyperus rotundus tubers ethanolic extract on learning and memory in animal model of Alzheimer. Biomed Aging Pathol 2013;3(4):185–91. [4] Rabiei Z, Bigdeli MR, Rasoulian B, Ghassempour A, Mirzajani F. The neuroprotection effect of pretreatment with olive leaf extract on brain lipidomics in rat stroke model. Phytomedicine 2012;19(10):940–6. [5] Rabiei Z, Bigdeli MR, Rasoulian B. Neuroprotection of dietary virgin olive oil on brain lipidomics during stroke. Curr Neurovasc Res 2013;10(3): 231–7. [6] Misane I, Ogren SO. Selective 5-HT1A antagonists WAY 100635 and NAD-299 attenuate the impairment of passive avoidance caused by scopolamine in the rat. Neuropsychopharmacology 2003;28(2):253–64. [7] Jeong EJ, Lee KY, Kim SH, Sung SH, Kim YC. Cognitive-enhancing and antioxidant activities of iridoid glycosides from Scrophularia buergeriana in scopolaminetreated mice. Eur J Pharmacol 2008;588(1):78–84. [8] El-Sherbiny DA, Khalifa AE, Attia AS, Eldenshary EEDS. Hypericum perforatum extract demonstrates antioxidant properties against elevated rat brain oxidative status induced by amnestic dose of scopolamine. Pharmacol Biochem 2003;76(3–4):525–33. [9] Viola H, Wasowski C, Levi de Stein M, Wolfman C, Silveira R, Dajas F, et al. Apigenin, a component of Matricaria recutita flowers, is a central benzodiazepine receptors-ligand with anxiolytic effects. Planta Med 1995;61: 213–6. [10] Cemek M, Kaga S, Simsek N, Buyukokuroglu ME, Konuk M. Antihyperglycemic and antioxidative potential of Matricaria chamomilla L. in streptozotocininduced diabetic rats. J Nat Med 2008;62(3):284–93. [11] Moon J-H, Terao J. Antioxidant activity of caffeic acid and dihydrocaffeic acid in lard and human low-density lipoprotein. J Agric Food Chem 1998;46(12):5062–5. [12] Folin O, Ciocalteau V. On tyrosine and tryptophan determination in protein. J Biol Chem 1927;73:627–50. [13] Rabiei Z, Rafieian-kopaei M, Heidarian E, Saghaei E, Mokhtari S. Effects of Zizyphus jujube extract on memory and learning impairment induced by bilateral electric lesions of the nucleus basalis of Meynert in rat. Neurochem Res 2014;39(2):353–60. [14] Benzie IFF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP Assay. Anal Biochem 1996;239(1): 70–6. [15] Karatas F, Karatepe M, Baysar A. Determination of free malondialdehyde in human serum by high-performance liquid chromatography. Anal Biochem 2002;311(1):76–9. [16] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95(2):351–8.
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[17] Fukui K, Onodera K, Shinkai T, Suzuki S, Urano S. Impairment of learning and memory in rats caused by oxidative stress and aging, and changes in antioxidative defense systems. Ann N Y Acad Sci 2001;928(1): 168–75. [18] Richardson JS. Free radicals in the genesis of Alzheimer’s diseasea. Ann N Y Acad Sci 1993;695(1):73–6.
[19] Shi J, Liu Q, Wang Y, Luo G. Coadministration of huperzine A and ligustrazine phosphate effectively reverses scopolamine-induced amnesia in rats. Pharmacol Biochem Behav 2010;96(4):449–53. [20] Pilz J, Meineke I, Gleiter CH. Measurement of free and bound malondialdehyde in plasma by high-performance liquid chromatography as the 2,4-dinitrophenylhydrazine derivative. J Chromatogr B 2000;742(2):315–25.
Available online at
ScienceDirect www.sciencedirect.com
Original article
Matricaria Chamomilla extract demonstrates antioxidant properties against elevated rat brain oxidative status induced by amnestic dose of scopolamine Zahra Alibabaei , Zahra Rabiei , Samira Rahnama , Shiva Mokhtari , Mahmoud Rafieian-kopaei ∗ Medical Plants Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran
a r t i c l e
i n f o
Article history: Received 12 April 2014 Accepted 10 July 2014 Available online 15 August 2014 Keywords: Matricaria Chamomilla Alzheimer’s disease Scopolamine
a b s t r a c t Alzheimer’s disease (AD) is clinically characterized by progressive loss of cognitive abilities and usually is accompany with elevated oxidative stress. Chamomile is a plant with antioxidant activity with is currently used in Iranian folk medicine as sedative, analgesic, antipyretic and antispasmodic agent. The present study was investigated the effect of Matricaria chamomilla (MC) on learning and memory functions in scopolamine-induced memory deficit in rats. Memory enhancing activity in scopolamine-induced amnesic rats was investigated by assessing the Morris water maze and passive avoidance paradigm. Forty-two male Wistar rats were divided into 6 equal groups as bellow: 1 – control (received water), 2 – SCOP (received scopolamine 1 mg/kg for 15 days), 3 and 4 – SCOP + MC (received scopolamine and MC extract 200 and 500 mg/kg b.w. per day for 15 days), 5 and 6 – intact groups (received MC extract 200 and 500 mg/kg b.w. per day for 15 days). M. Chamomilla ethanolic extract produced significant memory enhancing activity when evaluated by Morris water maze and passive avoidance paradigm models. Our results suggest that M. chamomilla ethanolic extract has repairing effects on memory deficit and might be beneficial in patients with Alzheimer’s disease and behavioral disorders. The memory enhancing activity of the extract may be attributed to the free radical scavenging activity, which would have been afforded by the active constituents present in the extract. © 2014 Published by Elsevier Masson SAS.
1. Introduction Alzheimer disease (AD) is the most common form of dementia. There is no cure for the disease and it worsens as it progresses. Most often, AD is diagnosed in people over 65 years of age although the less-prevalent early-onset Alzheimer’s can occur much earlier [1]. Because of this problem, alternative and complementary therapies are needed. Several studies have shown the neuroprotective and cognition-enhancing properties of herb extracts and other natural products using different animal models [2–5]. Scopolamine, a blocker of muscarinic acetylcholine receptor that has been used to induce experimental models of Alzheimer’s disease [6]. Scopolamine significantly increases AChE and malondialdehyde (MDA) levels in the cortex and hippocampus [7]. The
∗ Corresponding author. Tel.: +983813346692. E-mail address: rafi[email protected] (M. Rafieian-kopaei). http://dx.doi.org/10.1016/j.biomag.2014.07.003 2210-5220/© 2014 Published by Elsevier Masson SAS.
elevation of brain oxidative stress after administration of scopolamine further substantiates the value of scopolamine-induced amnesia as an animal model [8]. Chamomile is perennial flowering herb that grows in widespread regions, including Europe, Africa, and Asia. Chamomile is widely distributed in Iran and is used in Iranian traditional medicine for various conditions. One of the most important flavonoid of Chamomile is apigenin, which has mild relaxing property [9]. Previous studies showed that streptozotocin resulted in oxidative stress and affected the antioxidant status. Treatment with ethanolic extract of Chamomile significantly reduced postprandial hyperglycemia and oxidative stress, and augmented the antioxidant capacity. Treatment with ethanolic extract of Chamomile significantly increased the blood glutathione level [10]. In the present study, we investigated the behavioral recovery following chronic exposure to ethanolic extract of Matricaria chamomilla on scopolamine-induced dementia in rat model.
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2. Materials and methods
2.6. Experimental protocol
2.1. Preparation of the extract
• Group 1: received vehicle (distilled water) for 15 days consecutively. • Group 2: received scopolamine 1 mg/kg intraperitoneally (i.p) for 15 days consecutively (SCOP group). • Groups 3 and 4: received simultaneously scopolamine 1 mg/kg and M. Chamomilla ethanolic extract with doses of 200 and 500 mg/kg for 15 days (SCOP + 200, 500 MC). • Groups 5 and 6: intact groups that received only M. Chamomilla ethanolic extract with doses of 200 and 500 mg/kg for 15 days (intact 200, 500 MC).
Air-dried M. chamomilla was pulverized with a blender and the extract was prepared by adding ethanol (70%) on the plant powder. The pooled extract was filtered after 48 hours. The filtrate was concentrated using a rotary vacuum evaporator under reduced pressure until dryness. The extract was stored at 4 ◦ C, and reconstructed with distilled water to obtain different doses when needed to treat the animals.
2.2. DPPH free radical scavenging assay Antioxidant activity of the M. chamomilla extract was measured using the DPPH assay based on the scavenging ability to 2, 2diphenyl-1-picrylhydrazyl (DPPH) stable radical [11]. Butylated hydroxytoluene (BHT) was used as a positive control. The samples in different concentrations were mixed with DPPH solution and ethanol. After vortexing, the tubes were left in the dark at room temperature after which the absorbance was measured at 517 nm using a UV–vis spectrophotometer (Biochrom Ltd, England). Each measurement was performed in triplicate under identical conditions. Antioxidant activities were expressed as the IC50 values (the concentration of antioxidant required to cause 50% reduction in the original concentration of DPPH). Inhibition of free radical by DPPH (%) was calculated as follows:
I (%) = 100 × Acontrol − Asample /Acontrol
Animals were randomly divided into 6 groups with 7 in each group. 2.7. Water maze test A circular water pool with 183 cm in diameter and 60 cm in deep was used for a water maze test. The entire inside of the pool was painted black, and the pool was filled with muddy water containing ink at 22 ◦ C. The tank was placed in a dimly lit, soundproof test room with various visual cues. A white platform was then placed in one of the pool quadrants. During the four subsequent days the rats were given four trials sessions per day with the platform in place. When a rat located the platform, it was permitted to remain on it for 10 s. The time interval between trial sessions was 30 min. During each trial session, the time taken to find the hidden platform (latency) was recorded using a video camera based (Ethovision System). One day after the final training trial sessions, the rats were individually subjected to a probe trial session in which the platform was removed from the pool and the rats were allowed to swim for 120 s to search for it. A record was kept of the swimming time in the pool quadrant where the platform had previously been placed.
2.3. Total phenolic compounds 2.8. Passive avoidance test The concentration of total phenolic compounds in M. chamomilla extract was determined using Folin–Ciocalteau method as described previously with minor modification. Briefly, to 0.5 mL of a 5.5 g/L diluted extract, 2.5 mL of Folin–Ciocalteau reagent (diluted 10 times with water) was added. The standard curve was plotted using 12.5, 25, 50, 62.5, 100, and 125 mg/L solutions of gallic acid in methanol and water (60:40, v/v). The absorbance was measured at 760 nm. The total phenolic contents of the extracts were expressed as gallic acid equivalent (mg/g extract) [12].
2.4. Total flavonoid and flavonol The amounts of total flavonoids and flavonols in the M. chamomilla extract were determined calorimetrically as described by Rabiei et al. Total flavonoids and flavonols were expressed in terms of rutin equivalent (mg/g), which is a common reference compound [13].
2.5. Animals Male Wistar rats, weighing 150–250 g, were obtained from Pasteur institution (Tehran, Iran). Rats were housed in groups of four in cage at 25 ◦ C with controlled 12 h light-dark cycle. Food and water were freely available. All experiments were executed in accordance with the Guide for the Care and Use at Laboratory Animals and were approved by Research and Ethics Committee at Shahrekord University of Medical Sciences.
Testing of passive avoidance performance was carried out in two identical light and dark square boxes as described in our previous reports. The rats were initially placed in the light chamber and 20 s later the door between compartments was opened. When the rat entered the dark compartment with all four paws, the guillotine door was closed, and the latency to enter was recorded (t1) (from the time the door is lifted), and an electrical foot shock (1 mA, lasting 1 s) was delivered through the stainless steel rods. Twentyfour hours after the training trial, the rats (individually) were again placed in the light compartment. The step-through latency (t2) to enter the dark compartment was measured. If the rat did not enter the dark compartment within 120 s, the experiment was stopped. 2.9. Ferric reducing/antioxidant power (FRAP) assay At the end of behavioral tests, the blood samples were obtained through cardiac puncture for biochemical estimations, and then the serum and plasma were immediately separated from the blood samples by centrifugation. Antioxidant capacity of the plasma was determined by measuring its ability to reduce Fe3+ to Fe2+ with ferric reducing ability of plasma (FRAP) test. FeSO4 (100–1000 M concentration range) was used as a standard in FRAP assay. The results are expressed in M [14]. 2.10. Measurement of plasma malondialdehyde (MDA) The plasma MDA level was measured as LPO by the thiobarbituric acid reactive substances (TBARS) method. Briefly, to 100 L plasma or standard, 100 L sodium dodecyl sulfate (8.1%) and
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2.11. Malondialdehyde (MDA) levels of brain Following the behavioral tests, rats were killed. The brains were quickly removed and were washed twice with cold saline solution, different parts of brains such as hippocampus, cortex and subcortex separated, placed into glass bottles, labeled, and stored in a deep freeze (−80 ◦ C) until processing. Tissues were homogenized in ice-cold Tris–HCl buffer (50 mM, pH7.4) for 2 min at 5000 rpm. The homogenized solution was then centrifuged for 60 min at 5000 g. Malondialdehyde (MDA) level was then measured. Lipid peroxidation was evaluated by measuring the TBARS content according to the TBA test method with slight modification. The MDA level was determined by a method based on the reaction with thiobarbituric acid (TBA) at 90–100 ◦ C. The reaction was performed at PH 2–3 at 90 ◦ C for 15 min. The sample was mixed with 2 volumes of cold 10% (w/v) trichloroacetic acid to precipitate protein. The precipitate was pelleted by centrifugation and an aliquot of the supernatant was reacted with an equal volume of 0.67% TBA (w/v) in a boiling water bath for 10 min. After cooling, the absorbance was read at 532 nm using spectrophotometer [16]. 2.12. Statistical analysis All the results were expressed as mean ± SEM and statistical analyses were performed using SPSS 11.0 statistical software. P < 0.05 was considered statistically significant. 3. Results 3.1. Standardization of M. chamomilla extract To standardize the plant extract, total phenolic, flavonoid and flavonol components in M. chamomilla extract were measured. Total amount of phenolic compounds in M. chamomilla extract was 78.4 mg galic acid equivalent per one gram dried extract. Total amount of flavonoid and flavonol compounds were 47.6 mg/g and 26.5 mg, respectively per one gram of dry matter. 3.2. Effect of M. chamomilla extract on DPPH free radical scavenging
Table 1 Free radical scavenging activity of different concentrations of Matricaria chamomilla extract. Sample
Concentration (g/mL)
DPPH radical scavenging activity Inhibition (%) IC50 (g/mL)
Matricaria chamomilla extract
80 70 60 50 40 30 25 20 15 10
74.8 65.3 56.9 49.2 (IC50 ) 41.29 32.4 27.5 22.15 18 5.8
IC50 values are highlighted in bold. DPPH: 2, 2-diphenyl-1-picrylhydrazyl.
(T2) time of intact rats treated with 200 and 500 mg/kg of M. chamomilla extract were significantly increased compared to SCOP rats (P < 0.05) but did not increase compared with the control group (P > 0.05) (Fig. 1). 3.4. Morris water maze swimming test The time spent by the animal, searching for the missing platform in target quadrant (zone 1) with respect to other quadrant (zones 2–4) on day 5 was noted as an index of retrieval memory. In the probe trial following the last training session M. chamomilla with doses of 200 and 500 mg/kg increased the swimming time in the target quadrant (zone 1) after the platform was removed in SCOP + 200 MC, SCOP + 500 MC and intact + 500 MC groups when compared with scopolamine-treated group (P = 0.049, P = 0.002, P = 0.008, respectively) (Fig. 2). Animals in the control group rapidly learned the location of the platform. This was demonstrated by exhibiting a reduction in swimming times from the first to the second trial on day 1 and by reaching stable latencies at day 2. By contrast, rats treated with scopolamine (1 mg/kg body weight) failed to find the platform until given the maximum time. The escape latency did not decrease in the SCOP, SCOP + 200 MC and SCOP + 500 MC groups compared to the control group (P > 0.05). The latency before reaching platform on day 3 significantly decreased in control, intact + 200 MC and intact + 500 MC groups respect to SCOP group (P = 0.022, P = 0.002, P = 0.015, respectively). Escape latency significantly reduced in
80
***
60
Time(sec)
2.5 mL TBA/buffer (prepared by dissolving of 0.53% thiobarbituric acid in 20% acetic acid as adjusted to pH 3.5 with NaOH) were added. The tubes were covered with caps and incubated at 95 ◦ C for 60 min. The reaction was stopped by placing tubes on ice followed by centrifugation at 4000 rpm for 10 min to separate two phases. The supernatant (20 L) was injected into the HPLC system. Chromatographic determinations were performed on a high-performance liquid chromatograph equipped with an 1100 series pump and a UV absorbance detector. An HP 3395 integrator was employed to record retention times, chromatograms, and evaluate peak heights. A technopak 10u C18 reversed-phase column (emission 553 and excitation 515) was used [15].
357
t1 t2
****** ***
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40
20
C t5
In ta c
20 ct
00
0M
M
C
C ta In
0M
00 SC O P+ 5
20 P+ O
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3.3. Passive avoidance test
0 SC O P
The antioxidant activity of M. chamomilla extract was assessed on the basis of radical scavenging of the stable DPPH free radical. M. chamomilla extract showed strong free radical scavenging activity against DPPH radicals, with an IC50 value of 50 g/mL (Table 1).
Groups
The step-through latency of the scopolamine-treated group was significantly shorter than that of the control group (P < 0.05). The shorter step-through latency induced by scopolamine was significantly reversed by M. chamomilla extract with doses of 200 and 500 mg/kg (P < 0.05). Furthermore, the step-through latency
Fig. 1. The initial latency and step-through latency in the passive avoidance response. T1: initial latency; T2: step-through latency. The data are expressed as mean ± SD; n = 7 in each group. *** P < 0.01 SCOP vs. control, SCOP + 200 MC, SCOP + 500 MC, intact 200 MC, intact 500 MC groups (n = 7). MC: Matricaria chamomilla; SCOP: scopolamine.
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800
*** ***
FRAP(μ μ M)
600
400
200
C M 00
M
t5
00
ac
t2
In t
ac In t
SC
SC
O
O P+
P+
50
20
0M
C
C
C 0M
ol co nt r
SC
O P
0
Groups Fig. 2. Spent time in zone 1 during the probe trial in experimental groups. The data are expressed as mean ± SD; n = 7 in each group. * P < 0.05; *** P < 0.0.
Fig. 4. Plasma antioxidant capacity (ferric reducing/antioxidant power) in experimental groups. The data are expressed as mean ± SD; n = 7 in each group. *** P < 0.001.
control, intact + 200 MC and intact + 500 MC groups when compared with scopolamine-treated group in day 4 (P = 0.021, P = 0.006, P = 0.042, respectively) (Fig. 3).
30
20
***
*** 10
***
***
***
C 0M
C ct ta In
In
ta
ct
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50
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O
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50
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ol
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Administration of scopolamine with dose of 1 mg/kg in scopolamine-treated rats caused a slightly reduction (not significant, P > 0.05) of plasma antioxidant levels compared with control group. M. chamomilla extract treatment significantly increased the plasma antioxidant level in intact 200 MC and intact 500 MC groups when compared with scopolamine-treated group (P = 0.000, P = 0.000, respectively) and when compared with control group (P = 0.028, P = 0.004, respectively) (Fig. 4).
MDA (ng/ul)
SC O P
3.5. Plasma antioxidant level
Groups
Fig. 5. Plasma malondialdehyde level (MDA) in experimental groups. The data are expressed as mean ± SD; n = 7 in each group. *** P < 0.001.
3.6. Plasma level of malondialdehyde Scopolamine administration with dose of 1 mg/kg significantly increased the plasma MDA levels in SCOP group respect to control group (P = 0.000). M. chamomilla extract with doses of 200 and 500 mg/kg significantly reduced the plasma MDA levels in SCOP + 200 MC, SCOP + 500 MC, intact 200 MC and intact 500 MC groups (P = 0.000, P = 0.000, P = 0.000, P = 0.000, respectively) when compared with SCOP group (Fig. 5).
3.7. Brain MDA levels Administration with scopolamine (1 mg/kg) produced a significant increase in MDA content of hippocampus and cortex in SCOP group, an index of lipid peroxidation, when compared with control group (P = 0.031, P = 0.000, respectively). M. chamomilla extract
Latrency time (s)
60
40
20
* * ***
* * ***
SCOP control SCOP+200MC SCOP+500MC Intact200MC Intact500MC
4
3
2
1
0
Traning days Fig. 3. Spatial learning in a hidden platform model in experimental groups during four training days. The data are expressed as mean ± SD; n = 7 in each group. * P < 0.05; *** P < 0.001.
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Scopolamine treatment with the dose of 1 mg/kg significantly increased the brain and plasma MDA levels when compared with control group. Chamomile extract administration dramatically decreased the brain and plasma MDA levels in experimental groups. These results may suggest that Chamomile extract antioxidant property protected the brain by prevention of oxidative status due to administration of scopolamine. MDA is one of the major aldehydes formed after breakdown of lipid hydroperoxides. Therefore, it is considered a good biomarker of the involvement of free radical damage in pathologies associated with oxidative stress [20]. In this study, we proved Chamomile extract have antioxidant activity through the DPPH method and increased significantly the serum antioxidants levels in groups that received extract and might be beneficial in patients with memory deficit. Fig. 6. Brain malondialdehyde level (MDA) in experimental groups. The data are expressed as mean ± SD; n = 7 in each group. * P < 0.05; *** P < 0.001.
significantly decreased the MDA content in hippocampus, cortex and subcortex in SCOP + 200 MC group (P = 0.000, P = 0.001, P = 0.000, respectively) and SCOP + 500 MC group (P = 0.006, P = 0.000, P = 0.000, respectively) when compared with SCOP group. M. chamomilla extract treatment significantly reduced MDA content of hippocampus, cortex and subcortex in intact groups with doses of 200 (P = 0.000, P = 0.000, P = 0.000, respectively) and 500 (P = 0.000, P = 0.000, P = 0.000, respectively) when compared with SCOP groups (Fig. 6). 4. Discussion The present study aimed at evaluating the ethanolic extract of M. chamomilla for memory enhancing activity in scopolamineinduced amnesic rats using Morris water maze and passive avoidance paradigm and to carry out the biochemical estimation like MDA in brain tissue homogenate and in plasma at end of the study. The brain is considered to be more susceptible to peroxidative damage than other tissues due to the high content of their polyunsaturated lipid-rich neural parenchyma, high oxygen utilization and low antioxidative enzymes. Furthermore, previous studies indicate that oxidative stress is one of the earliest events in pathogenesis of memory impairment [17]. Previous studies showed that the cerebral cortex and hippocampus, which are thought to control cognitive and motor functions, seem to be sensitive to oxidative stress, and require antioxidants [18]. Medicinal plants having antioxidant properties, have been shown to improve memory and learning in rat model of Alzheimer’s [13]. There is notable evidence that scopolamine causes oxidative stress through the interference with acetylcholine in brain and leading to cognitive impairment [8,19]. This study aimed at investigating whether such impaired cognition due to scopolamine administration is associated with altered oxidative stress indices. However, brain oxidative status in this experimental animal model of amnesia that induced by scopolamine has yet to be evaluated. Scopolamine administration dramatically decreased the stepthrough latency compared with control group. The shorter stepthrough latency induced by scopolamine was significantly reversed by M. chamomilla extract. In Morris water maze test, in probe trial, M. chamomilla extract significantly increased the swimming time in the target quadrant.
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