Ancient Science of Life

: 2015  |  Volume : 34  |  Issue : 3  |  Page : 156--161

Effect of Calendula officinalis hydroalcoholic extract on passive avoidance learning and memory in streptozotocin-induced diabetic rats

Shirin Moradkhani1, Iraj Salehi2, Somayeh Abdolmaleki2, Alireza Komaki2,  
1 Neurophysiology Research Center, Hamadan University of Medical Sciences, Hamadan; Department of Pharmacognosy and Pharmaceutical Biotechnology, School of Pharmacy, Hamadan University of Medical Sciences, Hamadan, Iran
2 Neurophysiology Research Center, Hamadan University of Medical Sciences, Hamadan, Iran

Correspondence Address:
Dr. Alireza Komaki
Neurophysiology Research Center, Hamadan University of Medical Sciences, Shahid Fahmideh Street, Hamadan


Background: Medicinal plants, owing to their different mechanisms such as antioxidants effects, may improve learning and memory impairments in diabetic rats. Calendula officinalis (CO), has a significant antioxidant activity. Aims: To examine the effect of hydroalcoholic extract of CO on passive avoidance learning (PAL) and memory in streptozotocin (STZ)-induced diabetic male rats. Settings and Design: A total of 32 adult male Wistar rats were randomly allocated to four groups: Control, diabetic, control + extract of CO and diabetic control + extract of CO groups with free access to regular rat diet. Subjects and Methods: Diabetes in diabetic rats was induced by single intraperitoneal injection of 60 mg/kg STZ. After confirmation of diabetes, oral administration of 300 mg/kg CO extract to extract-treated groups have been done. PAL was tested 8 weeks after onset of treatment, and blood glucose and body weight were measured in all groups at the beginning and end of the experiment. Statistical Analysis Used: The statistical analysis of data was performed by ANOVA followed by least significant difference post-hoc analysis. Results: Diabetes decreased learning and memory. Effect of CO extract in retention test (after 24 and 48 h) has been shown a significant decrease in step-through latency and increase in time spent in the dark compartment part. Also the extract partially improved hyperglycemia and reduced body weight. Conclusion: Taken together, CO extract can improve PAL and memory impairments in STZ-diabetic rats. This improvement may be due to its antioxidant, anticholinergic activities or its power to reduce hyperglycemia.

How to cite this article:
Moradkhani S, Salehi I, Abdolmaleki S, Komaki A. Effect of Calendula officinalis hydroalcoholic extract on passive avoidance learning and memory in streptozotocin-induced diabetic rats.Ancient Sci Life 2015;34:156-161

How to cite this URL:
Moradkhani S, Salehi I, Abdolmaleki S, Komaki A. Effect of Calendula officinalis hydroalcoholic extract on passive avoidance learning and memory in streptozotocin-induced diabetic rats. Ancient Sci Life [serial online] 2015 [cited 2022 Jul 1 ];34:156-161
Available from:

Full Text


Cognitive impairment occurs in diabetes mellitus due to abnormalities in carbohydrate and lipid metabolism. [1],[2],[3],[4],[5] Recently, the focus has shifted to the use of plant extracts for the treatment of diabetes mellitus and its complications. The WHO has estimated that approximately 80% of the worldwide population relies on traditional medicine for their primary healthcare needs, and most of this therapy involves the use of plant extracts. [6]

Calendula officinalis (CO) Linn (Asteraceae), known as "gole- hamishe- bahar" in Persian, is used in traditional Iranian medicine. It has various biological activities such as: Analgesic, antiseptic, bactericide. It also finds use in skin problems and as an antifungal agent. [7],[8],[9],[10],[11],[12],[13] It is known as Pot Marigold and is an important medicinal plant used in Ayurveda and Italian folk medicine for treating various conditions such as fever, cancer, and inflammation. [14],[15] It has Ayurvedic actions such as granthi (reduction of tumors), gulmaghna (reduction of swollen glands and lumps), jvaraghna (antipyretic), stanya (wound healing). [16] CO has been widely used in homeopathic medicine for the treatment of many diseases.[7] It has been reported to possess effects such as antioxidant,[8] anti-inflammatory, [7],[17] antidiabetic, [18] anti-pyretic, cytotoxic as well as tumor reducing the potential. [19] Homeopathic calendula ointments are used in the healing stages of second and third degree burns to stimulate regrowth of skin and to diminish scar formation. [15] It is also used in gastrointestinal, gynecological and eye diseases. The plant is rich in many pharmaceutical active ingredients such as auroxanthin, carotenoids, flavonoids, flavoxanthin, glycosides, triterpenoid esters, sterols, and steroids. [7],[8],[9],[10],[11],[12],[13] Although there are reports on the antidiabetic effect of CO, [20],[21] to our best of knowledge, there is no report on its effect in cognitive impairment in diabetes. Thus, we seek to investigate the effect of CO hydroalcoholic extract on passive avoidance learning and memory (PAL).

 Subjects and Methods

Plant extraction

Calendula officinalis aerial parts were collected in spring and identified at the Botanic Institute of the Hamadan University of Medical Sciences. A voucher specimen was deposited in the Department of Pharmacognosy and Biotechnology, School of Pharmacy, Hamadan University of Medical Sciences. The dried aerial parts of the plant were ground to a fine powder. 200 g of the fine powder was macerated in ethanol 80% for 3 days. This process was repeated thrice. The resulting extract was filtered, and the filtrate was concentrated to dryness in a rotary evaporator under reduced pressure at a constant temperature of 40°C. The resulting extract was stored in a refrigerator.


Thirty-two male Wistar rats (250-280 g) were purchased from animal house, Hamadan University of Medical Sciences. All animals were maintained at a constant temperature (22 ± 0.5°C) with 12 h alternating light and dark cycles. They had free access to laboratory chow and tap water. Each experimental group consisted of 8 animals that were chosen randomly from different cages, and each rat was used only once. All research and animal care procedures were approved by the Veterinary Ethics Committee of the Hamadan University of Medical Sciences and were performed in accordance with the National Institutes of Health Guide for Care and use of Laboratory Animals (Publication number 85-23, revised 1985).

Experimental design

The animals were divided into two diabetic and two control groups (n = 8 each). Diabetes was induced by a single intraperitoneal (i.p.) injection of streptozotocin (Sigma-Aldrich, Germany) (60 mg/kg). Three days later, fasting blood glucose levels were determined. Blood samples were collected from the tail vein, and plasma glucose was measured using a glucometer. Animals were considered diabetic if plasma glucose levels exceeded 250 mg/dl. As soon as diabetes was confirmed, both diabetic and normal groups received saline or 300 mg/kg of the extract by oral gavage for 60 days. At the end of the experiment, all rats were weighed, and blood was collected for plasma glucose measurement.

Passive avoidance learning

The passive avoidance apparatus (shuttle box) consisted of illuminated and dark enclosures. The rat was placed in the illuminated enclosure facing away from the guillotine door, and 5 s later the door was raised. After the rat had entered the dark part, the door was closed, and a 50 Hz square wave, 1.2 mA constant current shock was applied for 1.5 s. [22],[23],[24],[25],[26],[27] The rat was retained in the apparatus and received a foot shock each time it reentered the dark part. Training was terminated when the rat remained in the illuminated enclosure for 120 consecutive s. On the retention test that given 24 and 48 h after the acquisition trial, the rat was again placed into the illuminated part and step-through latency (STLr) latency and time spent in the dark compartment (TDC) were recorded as a measure of retention performance. The ceiling score was 600 s and behavioral tests were performed at 8:00-11:00 h.

Measurement of plasma glucose levels

At the end of the experiment, after weighing, all rats were decapitated under ketamine HCl anesthesia (50 mg/kg, i.p.), and blood samples were drawn. Plasma glucose levels were measured using a glucometer.

Statistical analysis

The data were analyzed using SPSS 20 (IBM ® SPSS ® Statistics), and were expressed as mean ± standard error of the mean and compared by one-way ANOVA, post-hoc test least significant difference. P < 0.05 were considered to be significant.


Effects of diabetes on the passive avoidance learning and memory

Results of the one-way ANOVA indicated that there was no significant difference in the STLa and in the number of trials of the diabetic and control groups during the first acquisition trial [before the administration of the electrical shock; P > 0.05, [Figure 1]a and b. During the retention test, after 24 and 48 h the diabetic group had a decreased STLr, and increased TDC, compared to the control group respectively [both P < 0.01; [Figure 1]c-f].{Figure 1}

Effects of Calendula officinalis administration on passive avoidance learning and memory in nondiabetic rats

There was no significant difference in the STLa and in the number of trials of the control rats treated with extract and untreated control rats during the first acquisition trial [before the administration of the electrical shock; P > 0.05, [Figure 1]a and b]. During the retention test, after 24 and 48 h the control rats treated with extract had an increased STLr and decreased TDC compared to the untreated control rats respectively [both P < 0.001; [Figure 1]c-f].

Effects of Calendula officinalis administration to diabetic rats on passive avoidance learning and memory

There was no significant difference in the STLa and in the number of trials of the extract-treated diabetic rats and untreated diabetic rats during the first acquisition trial [before the administration of the electrical shock; P > 0.05, [Figure 1]a and b]. During the retention test, after 24 and 48 h the extract-treated diabetic rats had an increased STLr and decreased TDC compared to the untreated diabetic rats respectively [both P < 0.001; [Figure 1]c-f].

Effects of Calendula officinalis administration on body weight and plasma glucose

There was a significant increase in blood glucose and decrease in body weight in diabetic rats as compared to the normal ones. Oral administration of an extract of CO significantly lowered the blood glucose and elevated body weight when compared with the untreated diabetic group.

The body weight and blood glucose levels of different animal groups at the beginning and at the end of the experiment are shown in [Table 1]. There was no significant difference in body weight and plasma glucose between any of the groups before the onset of diabetes. Body weight and plasma glucose levels were measured at the end of behavioral assays (60 days after the onset of hyperglycemia). At the end of assays, the body weight of the untreated (165.13 ± 1.63) and extract-treated diabetic rats (191.88 ± 1.39) were lower than control rats (226.63 ± 1.17). Furthermore, there was no significant difference in the body weight of extract-treated (224.25 ± 1.19) and untreated control (226.63 ± 1.17) animals. Considering plasma glucose levels, untreated diabetic animals had significantly (P < 0.001) elevated plasma glucose levels (474.63 ± 3.7) compared with control animals (167.25 ± 2.25). Oral administration of CO extract to diabetic rats decreased the plasma glucose levels of the treated groups (461.38 ± 3.14) compared with the untreated diabetic group (474.13 ± 3.7; P < 0.001).{Table 1}


This study reveals that oral administration of a hydro-alcoholic extract of CO improved PAL and memory of control rats and alleviated the negative influence of diabetes on learning and memory. Although there were no significant effects in the STLa and in the number of trials in the diabetic groups during the first acquisition trial, but in retention test (after 24 and 48 h) a significant decrease in STLr and increase in TDC, has shown promising results.

Literature survey revealed that CO had a profound effect on the antioxidant defense system, but there was no report on the effect of CO in PAL and memory in diabetic rats. Diabetes is associated with an increased production of reactive oxygen species (ROS), enhanced oxidative stress and changes in the antioxidant capacity. [28] Oxidative stress is involved in the pathogenesis of many central nervous system disorders (e.g., neurodegenerative diseases) or in the physiological process of aging. [29] According to the literature, the brain is very vulnerable to oxidative stress due to its high polyunsaturated fatty acids content, which are particularly susceptible to ROS damage. [30],[31] Increased thiobarbituric acid reactive substances (lipid peroxidation productions) of frontal cortex and hippocampus may lead to apoptosis of nerve cells, which eventually affects the function of learning and memory, because lipid peroxidation induces changes in the structure and function of biological membranes resulting in a change of the structural arrangement of membrane lipids. [32]

Impairment of learning and memory of diabetic groups may be related to the increased oxidative stress in diabetic animal's brain, but CO as an antioxidant may reduce the oxidative stress, and lead to the better behavioral activity of the animals in extract-treated diabetic groups. Clinical and experimental studies suggest that hyperglycemia and/or insulin-deficiency itself may be responsible for impaired cognitive function in type 1 diabetes. [33] In vivo and in vitro evaluation of CO flower extract revealed that it has a profound effect on the antioxidant defense system. [8] It has been claimed that the antioxidant activity of plants might be due to their phenolic compounds. [34] Flavonoids are a group of polyphenolic compounds with known properties which include free radical scavenging, inhibition of hydrolytic and oxidative enzymes and anti-inflammatory action. [35] Accordingly, it has been reported that CO has several ingredients with reported antioxidant and anticlastogenic activities. CO, with profound antioxidant potential and having the ability to trigger cellular antioxidants, can be exploited for its use against a number of disorders including cardiovascular diseases, inflammation, and cancer. [8] CO has been reported to contain flavonoids (including lutein, quercetin, protocatechuic acid, etc.), triterpenoids (including faradiol, oleanolic acid, beta-amyrin, calenduladiol, etc.), and the alkaloid narcissin. [36] Flavonoids and carotenoids present in CO are potent antioxidants at very low concentrations. The chemopreventive properties of flavonoids are generally believed to reflect their ability to scavenge endogenous ROS. By inhibiting or stimulating various signaling pathways, flavonoids at low concentration could affect cellular function. [37],[38] Flowers also are rich in carotenoids of which flavoxanthin has been reported to be present at 28.5% of total carotenoids, followed by luteoxanthin. [39] Flowers are also found to contain lycopene and beta-carotene. Coumarins are also active constituents in CO.[40] These constituents may contribute to the antioxidant potential of this extract. The administration of acetylcholinesterase (AChE) inhibitors effectively decreased hyperglycemia and incidence of diabetes, and restored plasma insulin levels and plasma creatinine clearance. Induction and accumulation of AChE in pancreatic islets and the protective effects of AChE inhibitors on the onset and development of type 1 diabetes indicate a close relationship between AChE and type 1 diabetes. [40] AChE inhibitors enhance cholinergic function in the brain when loss or decline in memory and cognitive impairment has occurred. [41],[42] Polyphenols have shown AChE inhibitory effect [43] and considering that CO contains flavonoids, [36] another possible mechanism of CO effect on PAL can be involved in AChE inhibitory potential of the plant.


This study indicates that CO can improve cognitive impairment in diabetic rats. Cellular and animal studies, as well as clinical trials, support its role as useful preparation in alternative medicine. Because of the easy access to the CO in various countries, this plant can be used to treat diseases associated with diabetes such as memory and learning impairment.


This research was supported by Hamadan University of Medical Sciences, Hamadan, Iran.


1Kodl CT, Seaquist ER. Cognitive dysfunction and diabetes mellitus. Endocr Rev 2008;29:494-511.
2Kumari M, Brunner E, Fuhrer R. Minireview: Mechanisms by which the metabolic syndrome and diabetes impair memory. J Gerontol A Biol Sci Med Sci 2000;55:B228-32.
3Biessels GJ. Cerebral complications of diabetes: Clinical findings and pathogenetic mechanisms. Neth J Med 1999;54:35-45.
4Stolk RP, Breteler MM, Ott A, Pols HA, Lamberts SW, Grobbee DE, et al. Insulin and cognitive function in an elderly population. The Rotterdam study. Diabetes Care 1997;20:792-5.
5Craft S, Peskind E, Schwartz MW, Schellenberg GD, Raskind M, Porte D Jr. Cerebrospinal fluid and plasma insulin levels in Alzheimer′s disease: Relationship to severity of dementia and apolipoprotein E genotype. Neurology 1998;50:164-8.
6Craig WJ. Health-promoting properties of common herbs. Am J Clin Nutr 1999;70 3 Suppl: 491S-9.
7Zitterl-Eglseer K, Sosa S, Jurenitsch J, Schubert-Zsilavecz M, Della Loggia R, Tubaro A, et al. Anti-oedematous activities of the main triterpendiol esters of marigold (Calendula officinalis L.). J Ethnopharmacol 1997;57:139-44.
8Preethi KC, Kuttan G, Kuttan R. Antioxidant potential of Calendula officinalis flowers in vitro and in vivo. Pharm Biol 2006;44:691-7.
9Della Loggia R, Tubaro A, Sosa S, Becker H, Saar S, Isaac O. The role of triterpenoids in the topical anti-inflammatory activity of Calendula officinalis flowers. Planta Med 1994;60:516-20.
10Efstratiou E, Hussain AI, Nigam PS, Moore JE, Ayub MA, Rao JR. Antimicrobial activity of Calendula officinalis petal extracts against fungi, as well as Gram-negative and Gram-positive clinical pathogens. Complement Ther Clin Pract 2012;18:173-6.
11Dumenil G, Chemli R, Balausard G. Evaluation of antibacterial properties of Calendula officinalis flowers and mother homeopathic tinctures of Calendula officinalis. Ann Pharma Fr 1980;38:493-9.
12Kasiram K, Sakharkar PR, Patil AT. Antifungal activity of Calendula officinalis. Indian J Pharm Sci 2000;6:464-6.
13Barbour EK, Sagherian V, Talhouk S, Talhouk R, Farran MT, Sleiman FT, et al. Evaluation of homeopathy in broiler chickens exposed to live viral vaccines and administered Calendula officinalis extract. Med Sci Monit 2004;10:BR281-5.
14Chakraborthy GS. Phytochemical screening of Calendula officinalis Linn leaf extract by TLC. Int J Res Ayurveda Pharm 2010;1:131-4.
15Mathur R, Mamta G. Antimicrobial and phytochemical estimation of Calendula officinalis against human pathogenic microorganism. Int J Innov Bio Sci 2011;1:1-10.
16McIntyre A. Calendula officinalis Zergul Marigold. Available from: [Last accessed on 2015 April].
17Di Lorenzo C, Dell′Agli M, Badea M, Dima L, Colombo E, Sangiovanni E, et al. Plant food supplements with anti-inflammatory properties: A systematic review (II). Crit Rev Food Sci Nutr 2013;53:507-16.
18Yoshikawa M, Murakami T, Kishi A, Kageura T, Matsuda H. Medicinal flowers. III. Marigold. (1): Hypoglycemic, gastric emptying inhibitory, and gastroprotective principles and new oleanane-type triterpene oligoglycosides, calendasaponins A, B, C, and D, from Egyptian Calendula officinalis. Chem Pharm Bull (Tokyo) 2001;49:863-70.
19Boucaud-Maitre Y, Algernon O, Raynaud J. Cytotoxic and antitumoral activity of Calendula officinalis extracts. Pharmazie 1988;43:220-1.
20Singh MK, Sahu P, Nagori K, Dewangan D, Kumar T, Alexander A, et al. Organoleptic properties in-vitro and in-vivo pharmacological activities of Calendula officinalis Linn: An over review. J Chem Pharm Res 2011;3:655-63.
21Chakraborthy GS, Arora R, Majee C. Antidiabetic and antihyperlipidemic effect of hydroalcoholic extract of Calendula officinalis. IRJP 2011;2:61-5.
22Lashgari R, Motamedi F, Zahedi Asl S, Shahidi S, Komaki A. Behavioral and electrophysiological studies of chronic oral administration of L-type calcium channel blocker verapamil on learning and memory in rats. Behav Brain Res 2006;171:324-8.
23Sarihi A, Yazdi M, Heshmatian B, Saleh I, Behzadi G, Naghdi N, et al. The effects of lidocaine reversible inactivation of the dorsal raphe nucleus on passive avoidance learning in rats. Basic Clin Neurosci 2011;2:27-35.
24Shahidi S, Komaki A, Mahmoodi M, Lashgari R. The role of GABAergic transmission in the dentate gyrus on acquisition, consolidation and retrieval of an inhibitory avoidance learning and memory task in the rat. Brain Res 2008;1204:87-93.
25Rasuli B, Rasuli S, Komaki A. Study the effects of endogenous cannabinoid breakdown inhibitor on learning and memory in rat. Cell J (Yakhteh) 2011;12:33-4.
26Khodamoradi N, Komaki A, Salehi I, Shahidi S, Sarihi A. Effect of vitamin E on lead exposure-induced learning and memory impairment in rats. Physiol Behav 2015;144:90-4.
27Komaki A, Karimi SA, Salehi I, Sarihi A, Shahidi S, Zarei M. The treatment combination of vitamins E and C and astaxanthin prevents high-fat diet induced memory deficits in rats. Pharmacol Biochem Behav 2015;131:98-103.
28Xie W, Du L. High-cholesterol diets impair short-term retention of memory in alloxan-induced diabetic mice, but not acquisition of memory nor retention of memory in prediabetic mice. Life Sci 2005;77:481-95.
29Halliwell B, Gutteridge JM. Oxygen radicals and the nervous system. Trends Neurosci 1985;8:22-6.
30Cui K, Luo X, Xu K, Ven Murthy MR. Role of oxidative stress in neurodegeneration: Recent developments in assay methods for oxidative stress and nutraceutical antioxidants. Prog Neuropsychopharmacol Biol Psychiatry 2004;28:771-99.
31Halliwell B. Role of free radicals in the neurodegenerative diseases: Therapeutic implications for antioxidant treatment. Drugs Aging 2001;18:685-716.
32Palmeira CM, Oliveira CR. Partitioning and membrane disordering effects of dopamine antagonists: Influence of lipid peroxidation, temperature, and drug concentration. Arch Biochem Biophys 1992;295:161-71.
33Schoenle EJ, Schoenle D, Molinari L, Largo RH. Impaired intellectual development in children with Type I diabetes: Association with HbA(1c), age at diagnosis and sex. Diabetologia 2002;45:108-14.
34Cook NC, Samman S. Flavonoids-chemistry, metabolism, cardioprotective effects, and dietary sources. Nutr Biochem 1996;7:66-76.
35Frankel E. Nutritional Benefits of Flavonoids. International Conference on Food Factors: Chemistry and Cancer Prevention, Hamamatsu, Japan; 1995. [Abstracts, C6-2].
36Matysik G, Wójciak-Kosior M, Paduch R. The influence of Calendulae officinalis flos extracts on cell cultures, and the chromatographic analysis of extracts. J Pharm Biomed Anal 2005;38:285-92.
37Kundu JK, Surh YJ. Breaking the relay in deregulated cellular signal transduction as a rationale for chemoprevention with anti-inflammatory phytochemicals. Mutat Res 2005;591:123-46.
38Chandran PK, Kuttan R. Effect of Calendula officinalis flower extract on acute phase proteins, antioxidant defence mechanism and granuloma formation during thermal burns. J Clin Biochem Nutr 2008;43:58-64.
39Kishimoto S, Maoka T, Sumitomo K, Ohmiya A. Analysis of carotenoid composition in petals of calendula (Calendula officinalis L.). Biosci Biotechnol Biochem 2005;69:2122-8.
40Zhang B, Yang L, Yu L, Lin B, Hou Y, Wu J, et al. Acetylcholinesterase is associated with apoptosis in β cells and contributes to insulin-dependent diabetes mellitus pathogenesis. Acta Biochim Biophys Sin (Shanghai) 2012;44:207-16.
41Mesulam M. The cholinergic lesion of Alzheimer′s disease: Pivotal factor or side show? Learn Mem 2004;11:43-9.
42Fodale V, Quattrone D, Trecroci C, Caminiti V, Santamaria LB. Alzheimer′s disease and anaesthesia: Implications for the central cholinergic system. Br J Anaesth 2006;97:445-52.
43Yan T, Wang T, Wei W, Jiang N, Qin YH, Tan RX, et al. Polyphenolic acetylcholinesterase inhibitors from Hopea chinensis. Planta Med 2012;78:1015-9.