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Tropical Journal of Pharmaceutical Research December 2016; 15 (12): 2571-2578 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. All rights reserved.

Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v15i12.6 Original Research Article

Neuroprotective effect of whole- extract of leptophylla in isoflurane-treated rats

Yong Fang1, Liang Lv2, Yong-Jian Chai3 and Fan Yu1* 1Department of Anesthesiology, Jinan Maternity and Child Care Hospital, Jinan, Shandong 250001, 2Department of Anesthesiology, Shandong Police Hospital, Jinan, Shandong 250002, 3Department of Anesthesiology, The First People's Hospital of Jinan City, Jinan, Shandong 250011, PR China

*For correspondence: Email: [email protected]; Tel/Fax: 0086-531-89029000

Received: 20 September 2016 Revised accepted: 25 November 2016

Abstract

Purpose: To investigate the neuroprotective effect of Torilis leptophylla (TL) extract on animals exposed to isoflurane-induced anesthesia. Methods: General anesthesia was induced in diabetic rats by administration of 2 % sevoflurane (v/v) in 100 % oxygen. The animals from the treatment group were orally administered TL extract. Behavioral parameters, the explicit objection recognition index, and performance in water maze experiments were observed. Additionally, the neuroprotection provided by the herbal extract was measured in terms of the reduction in oxidative stress in isolated animal brain tissue. Results: The study showed approximately 1.5-fold improvement in object recognition index of TL- extract-treated anesthetized rats compared with the control group of anesthetized animals. Additionally, the escape latency values of the anesthetized rats in Morris water maze experiments were reduced following TL extract treatment at all time points throughout the 5-day study protocol. Finally, biomarkers of oxidative stress in the brain indicate that the levels of superoxide dismutase and catalase enzymes were improved by approximately 1.43- and 1.36-fold in TL-extract-treated rats compared with those in the control group. Conclusion: The results of the present study show that cognitive dysfunction and brain injury associated with enhanced oxidative stress can be reversed by the antioxidant properties of the TL extract.

Keywords: Anesthesia, Torilis leptophylla, Neuroprotection, Brain injury, Cognitive impairment, Isoflurane

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INTRODUCTION Isoflurane is a transparent, stable substance with a feebly pungent musty odor. It is supplied as a General anesthesia in patients of different age 99.9 % pure liquid with no pharmaceutical groups, ranging from infants to geriatrics, with additives. Isoflurane is known to produce its varying pathological conditions and mechanical pharmacological action by agonistic binding to trauma has been a common medical practice for gamma aminobutyric acid (GABA), glutamate, several decades [1,2]. Among the various and glycine-type receptors. In addition, it anesthetic drugs, isoflurane has been the most antagonizes N-methyl-D-aspartate (NMDA) widely utilized inhalational anesthetic agent. receptor activity. Isoflurane is one of the most popular inhalational general anesthetics (Forane®) used to induce anesthesia,

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Fang et al particularly in pediatric patients, because of its Shadong, China) to obtain a coarse powder. quick onset of action and rapid recovery. Thereafter, 250 g of the coarse powder was Moreover, isoflurane is less irritating to the extracted twice with 1,500 mL of 95 % ethanol at airways than other anesthetic agents. However, room temperature for 24 h. The solvent was some recent studies have depicted setbacks concentrated in a rotary evaporator (Rotavapor® associated with the use of isoflurane in R215; Buchi Shanghai Ltd., Shanghai, China) experimental subjects, particularly regarding under reduced pressure at 40 °C to yield the TL younger age groups. The detrimental effects of extract. isoflurane-induced anesthesia comprise neurodegeneration and cognitive impairment Animals resulting mainly from increased oxidative stress and inflammatory response mediators. The experimental protocol was conducted after approval of the Institutional Animal Ethics This prompted us to investigate the prophylactic Committee of Jinan Maternity and Child Care potential of Torilis leptophylla (TL) extract on Hospital (approval ref no. 201208342) and was young rats exposed to isoflurane-induced performed in compliance with Directive anesthesia. TL is a plant known as bristle fruit 2010/63/EU on careful handling of animals for hedge parsley, which is mainly found in several scientific purposes [5]. Male neonatal Wistar rats regions of Asia, Europe, and North Africa [3]. (postnatal day 14) were randomly assigned to Historically, TL has been known for its anti- different study groups and were housed at 22 °C inflammatory and antimicrobial properties. It is in polypropylene rat cages (9 animals per cage) widely used in traditional medicine for the [6]. The animals were kept in a light-controlled treatment of gastrointestinal disorders and as a environment with a 12-h light-dark cycle and general tonic. Recent studies have suggested were allowed continuous access to a rat pellet the presence of multiple antioxidant components diet. in TL [3,4]. Despite the holistic therapeutic applications of the TL plant, its potential seems Treatment with the TL extract largely unexplored in modern medicine. Before starting the anesthesia, the TL extract Therefore, the current study aimed to investigate was administered to the rats in the treatment the prophylactic properties of a whole-plant group for 3 days. The extract was provided as extract of TL in reducing the extent of cognitive twice daily oral doses separated by 12 h (5 impairment and brain injury in neonatal subjects. mL/kg body weight) using an oral cannula feeder. Wistar rats were used as animal subjects for our The animals receiving the TL extract were studies. Various evaluations were performed referred to as the TL-extract-treated group. using behavioral and biochemical parameters. Isoflurane-induced anesthesia EXPERIMENTAL The rats (n = 18) were placed in plexiglass Materials sealed chambers maintained at a temperature of 37 ± 1°C. The chambers comprised a provision TL plant was obtained in August 2014 from for 1.5 % isoflurane in a gaseous mixture of 35 % Shanghai Botanical Gardens (Shanghai, China). O2/65 % N2 (1 L/min). The rats were exposed to Authentication of the plant material was isoflurane for 5 h. Following the anesthesia performed by Dr Feng Shucheng, Professor of phase, the animals were transferred to another Plant , Shanghai Botanical Gardens. A glass chamber with a provision of oxygen to voucher specimen (accession no. YF-014408) attain recovery for 1 h. The TL extract-treated was deposited at the Herbarium of Shanghai animals exposed to isoflurane anesthesia were Botanical Garden for future reference. Isoflurane termed the TL-ISF-treated group, whereas naïve was procured from a local pharmacy store in rats receiving anesthesia were called as the ISF- Shandong, China while streptozotocin was treated group. The rats in the ISF-control group obtained from Sigma-Aldrich (Shanghai, China). were only administered pure oxygen (without The other reagents and chemicals used were of anesthesia) and were termed the SVF-control analytical grade. group. Twenty-four hours after inducing anesthesia, a subgroup of animals (n = 6) from Preparation of extract each treatment group was sacrificed to collect blood samples for biochemical analysis. The All of the (5 kg) were dried under shade to remaining animals were subjected to behavioral obtain 1 kg of dry sample, which was milled in a determinations after 32 days. 7.5-kg ball-mill (JHMA®; Weifang Jinghua Ltd.,

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Object recognition test USA), and the homogenates were centrifuged at 4000 × g for 15 min. The supernatants were A plastic tank (60 cm × 60 cm × 25 cm) was used isolated and utilized for several estimations [8,9]. in the studies, and the animals were placed in the tanks for at least 10 sessions of 15 min for 2 Determination of oxidative stress biomarkers days before initiating the studies. The object recognition experiments were performed in two The protein content was quantified in the tissue phases. First, the rats were individually placed in homogenates using Gornall’s biuret technique, the arena, wherein two identical objects were with bovine serum albumin (BSA) as the already placed. The time taken to explore the reference protein [10]. Briefly, each tissue objects was recorded. The arena was thoroughly homogenate sample (0.1 mL) was mixed with cleaned in-between such sessions. Object sodium chloride (2.9 mL) and working biuret exploration refers to the incidence of sniffing (at reagent (3.0 mL). This mixture was allowed to less than 2 cm) and prolonged rearing on the rest at room temperature for 30 min, and its object (> 5 s). The second phase of the study absorbance was recorded at 540 nm in a double- comprised replacing of 1 of the 2 identical beam UV spectrophotometer (Shimadzu UV-200; objects with a different object (different color, Shimadzu, Osaka, Japan). Thereafter, oxidative odor, and texture). The rats were again placed in stress was estimated in terms of superoxide the arena, and the exploration time was dismutase (SOD), lipid peroxidation (expressed recorded. The test parameter was the recognition as malondialdehyde, MDA), and catalase (CAT). cognitive index [7], calculated as in Eq 1. Superoxide dismutase (SOD) Recognition index = [Td/(Td+Tf)] x 100 …… (1) Freshly prepared aqueous solutions of nitroblue- where Td and Tf represent the time taken to tetrazolium chloride (1.0 mL, 7.8 %) and explore different objects and the time taken to hydroxylamine hydrochloride (1.0 mL, 13.9 %) explore familiar objects, respectively. were added to 0.1 mL of the tissue homogenate. Thereafter, the change in absorbance was Morris water maze test recorded at 560 nm over 120-s intervals [11].

The Morris water maze test was performed using Malondialdehyde (MDA) a conventional Morris maze (Shanghai Jiliang Ltd., Shanghai, China). The swim pattern, path Acetic acid (1.5 mL, 20 %), aqueous sodium length, and time needed to reach the platform lauryl sulfate (0.2 mL, 8.1 %), aqueous were recorded by a video camera and analyzed thiobarbituric acid (1.5 mL, 0.8 %), and triple- by integrated computer software. Prior to distilled water (0.6 mL) were added to the tissue commencing the studies, the rats were trained homogenate (0.2 mL). The mixture was exposed for 1 day to swim and rest on a visible platform to heat (70 °C) and then allowed to cool destination. Similarly, the rats were subsequently gradually. Afterward, 1 mL of triple-distilled water trained to swim and rest on a submerged and an n-butanol:pyridine mixture (15:1 v/v; 5 (hidden) platform for the next 2 days. After mL) was added and agitated vigorously until the completion of the swim training, the Morris water organic layer separated. The organic layer was maze test was performed for 5 consecutive days. isolated using a phase separation funnel, and its The animals were transferred from four different absorbance was measured at 532 nm [12]. locations in the maze and were allowed to search for the platform for 2 min; if the animal failed to Catalase (CAT) activity find the platform, they were helped to the platform and kept there undisturbed for another The catalase activity in the tissue homogenates 30 s. The parameters observed were the time was measured using Thiele’s method [13]. The spent in the quadrants (Qtime) and the escape disappearance of hydrogen peroxide (H2O2) is an latency, which refers to the time taken by the indication of the consumption of CAT. Hydrogen animal to find the platform. peroxide (30 mmol/L) was mixed along with the tissue homogenate, and changes in the Collection of brain tissue samples absorbance values were monitored at 240 nm for 120-s intervals. CAT level was derived from Eq The rats were sacrificed by decapitation. Tissue 2. from the hippocampus was homogenized in radioimmuno-precipitation assay (RIPA) buffer CAT (K/min) = 2.3/∆t × logA1/A2 ……..………. (2) using a tissue homogenizer (PRO250® tissue homogenizer; Pro-Scientific Inc., Oxford, CT,

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where A1 and A2 represent the initial and final used as the target quadrant throughout the absorbance values, and the time interval ∆t is 60 studies. Additionally, the minimum values for the s. escape latency was on Day 1 in naïve and STN- control animals, values that were further reduced Statistical analysis with time. Post-anesthesia, the animals from the ISF-treated group exhibited noticeable cognitive All of the data were analyzed using one-way impairment as evidenced by the significant (p < analysis of variance (ANOVA) and expressed as 0.05) reduction in Qtime in the target quadrant. mean ± standard deviation (SD); p < 0.05 was Additionally, a reduced Qtime in the target considered to be statistically significant. quadrant was observed in animals in the TL- Statistical analysis was performed using Graph- control group. On the other hand, the animals Pad Prism® 4.0 (Graph-Pad Software Inc., San treated with TL extract showed a markedly Diego, CA, USA). improved Qtime in the target quadrant, indicating cognitive replenishment in the young rats. RESULTS The escape latency exhibited by SVF-treated Identical recognition index values were observed animals was also significantly higher (p < 0.05) for naïve rats and ISF-control rats (55.25 ± 4.05 than that of naïve and ISF-control animals. After and 51.29 ± 5.09, respectively) (Figure 1), which ISF-induced anesthesia, the cognitive loss in reflected the physiological memory function of neonatal rats (ISF-treated group) seemed to be healthy rats. Significantly lower (p < 0.05) more prominent than that in all other groups as recognition index values of ISF-treated rats the lowest value for Qtime in the fourth quadrant (36.21 ± 4.18) indicated that exposure to was observed. Furthermore, the escape latency anesthesia caused significant cognitive values were significantly higher (p < 0.05) during impairment in neonatal rats. The animals all of the study days. exposed to blank extract solvents (TL-control) showed no improvement in cognitive impairment, The levels of oxidative stress biomarkers in the whereas those treated with TL extract showed various groups are shown in Figure 3. It was tremendous improvement in cognitive function. observed that rats treated with anesthesia (ISF- The animals that received the phytochemical treated) showed a significant increase (p < 0.05) extract exhibited full replenished object in the MDA levels compared with naïve and ISF- recognition ability, as indicated by the high control animals. On the other hand, the levels of recognition index value (52.63 ± 3.88). SOD and CAT were significantly reduced (p < 0.05) after the administration of anesthesia The results of the water maze study are shown in compared with those in the naïve and ISF-control Figure 2A and B. Post-training, the naïve rats animals. and those from the ISF-control group spent the maximum Qtime in the fourth quadrant, which was

Figure 1: Recognition index values of different treatment groups

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Figure 2: Time spent in different quadrants (A) and escape latency (B) as shown by the Morris water maze test in naïve animals (X), the ISF-control group (◊), the ISF-treated group (O), the TL-control group (□), and the TL- treated group (∆)

DISCUSSION isoflurane-related cognitive impairment in neonatal animals. Isoflurane exposure has long been known to produce neurodegenerative effects rapidly in Additionally, surgical procedures in diabetic neonatal experimental rats [7,14]. These effects subjects are crucial. Very recently, the are especially critical because isoflurane is the antioxidant and neuroprotective functions of TL most widely used general anesthetic in neonatal extract have been reported [3,4]. The antioxidant and young patients. properties of the plant could provide an excellent prophylactic effect in neonatal patients, which are Isoflurane has been used as a general anesthetic required to be anesthetized. Therefore, the worldwide for several years. Recent studies have current study was aimed to evaluate the potential depicted the neurodegenerative effects of of TL extract in preventing cognitive and anesthesia induced using isoflurane. Bonin and neurophysiological dysfunction in neonatal Orser [14] reported severe tau-phosphorylation Wistar rats as animal subjects. of hippocampal tissue after 1 h of isoflurane administration in mice, leading to significant It was observed in the water maze test as well as memory loss. On the other hand, Yang et al [13] in the object recognition test that isoflurane- demonstrated short-term memory loss induced anesthesia in healthy animals (ISF- associated with isoflurane-induced anesthesia in treated group) as well as in placebo-treated rats [14], and Leopke et al [15] emphasized animals (TL-control group) produced significant memory loss with respect to naïve animals.

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Figure 3: SOD levels (A), MDA levels (B), and CAT activity (C) in the different treatment groups

This indicated that no pharmacological activity To determine the injury resulting from oxidative was shown by the placebo-treated animals. As stress due to isoflurane administration, the effect previously stated, isoflurane has been widely of TL extract on biomarkers of oxidative stress accepted to increase the symptoms of was studied. SOD is a defensive enzyme, which anesthesia-related cognitive dysfunction, which causes dismutation of toxic superoxide free can be attributed to several factors, such as radicals into oxygen and hydrogen peroxide. isoflurane-induced neuronal damage, the over- Similarly, CAT is also a defensive enzyme, which expression of mediators of inflammation, and catalyzes toxic hydrogen peroxide molecules into increased oxidative stress in the hippocampal nontoxic water and oxygen. Decreases in SOD region of the brain [15–17]. The observations of and CAT levels are an indicator of increased the current study indicate that the isoflurane- oxidative stress or brain cell injury. On the other induced neurodegeneration and cognitive loss in hand, the formation of MDA indicates a anesthetized neonatal animals was considerably detrimental effect of reactive oxygen on reduced upon treatment with TL extract. brain lipoproteins, and the end products are commonly termed lipoxidation end products.

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