Dragon's blood dropping pills have protective effects on focal cerebral ischemia rats model.
Received 7 May 2013
Received in revised form 28 June 2013
Accepted 8 August 2013
Dragon's blood dropping pills
Middle Cerebral Artery Occlusion (MCAO)
Focal cerebral ischemia
Neurological deficit scores
Cerebral water content
Dragon's blood is a bright red resin obtained from Dracaena cochinchinensis (Lour.) S.C.Chen (Yunnan, China). As a traditional Chinese medicinal herb, it has great traditional medicinal value and is used for wound healing and to stop bleeding. Its main biological activity comes from phenolic compounds. In this study, phenolic compounds were made into dropping pills and their protective effects were examined by establishing focal cerebral ischemia rats model used method of Middle Cerebral Artery Occlusion (MCAO), and by investigating indexes of neurological scores, infarct volume, cerebral index, cerebral water content and oxidation stress. Compared to model group, high, middle and low groups of Dragon's blood dropping pills could improve the neurological function significantly (p <0.01) and reduce cerebral infarct volume of focal cerebral ischemia rats remarkably (p < 0.05-0.01). Meanwhile, each group could alleviate cerebral water col.tent and cerebral index (p <0.05-0.01) and regulate oxidative stress of focal cerebral ischemia rats obviously (p <0.05-0.01). Activities of middle group corresponded with that treated with positive control drug. The results obtained here showed that Dragon's blood dropping pills had protective effects on focal cerebral ischemia rats.
[c] 2013 Elsevier GmbH. All rights reserved.
Ischemic cerebrovascular disease (ICVD) is harm to human health seriously. It is one of the three major diseases which have the characteristics of high incidence, disability, fatality and recurrence, next only to heart disease and cancer (Diener et at. 2008). In clinic, most cerebral infarction is due to cerebral arterial thrombosis. It can make cell apoptosis of cerebral ischemic and hypoxic regions with "ischemic waterfall-like reaction" immediately. Ischemic stroke accounts for 70-80% of the stroke incidence and its prevention and treatment are necessary without delay. Therefore, it is a very important theoretical research and practical applications on complicated mechanisms and effective drugs study of ICVD.
Chinese herbal medicine has a long history in China, and is still in wide use up to now because of its definite therapeutic effect, wide indications, safety and convenient application. Dragon's blood is a kind of red resin obtained from Dracaena cochinchinensis (Lour.) S.C.Chen (Yunnan, China). And it is a recently discovered native dracaena species that has become the main source of dragon's blood in China from then on (Cai and Xu 1979; Fan et al. 2008; Wang et al. 2010, 2011; Zheng et al. 2012). As a "panacea of blood activating" resin, it has great medicinal value and its main biological activity comes from phenolic compounds (Zhou et al., 2001; Zhu etal. 2002). Pharmacological studies have showed that it has positive effects on treatment of blood stasis syndrome, trauma, tumors, inflammation, gynecopathy, allergic dermatitis and so on. It can promote blood circulation and serve as an antithrombotic, antioxidant, antiseptic, and antiinflammatory compound (Choy et al. 2008; Gurgel et al. 2001; Jones 2003; Peres et al. 1997; Rao et al. 2007; Zheng et al. 2005). It can improve blood circulation, metabolism, immune function and so on. Our previous study showed that Dragon's blood also had raclioprotective effects in radiation-induced neuro-injury in rat (Xin et al. 2012). All these indicate that Dragon's blood has enormous potential for further study.
In this study, phenolic compounds from Dragon's blood were made into dropping pills and their effects on focal cerebral ischemia rats model were examined by establishing focal cerebral ischemia rats model used method of Middle Cerebral Artery Occlusion (MCAO). The protective effects were further determined to validate the new dosage forms of dropping pills and to obtain information for further drug development.
Materials and methods
Reagents Sodium carboxymethyl cellulose (CMC-Na, No. 050803), sodium chloride (No. 20081205), 2,3,5-triphenytetrazolium chloride (ITC, No. 20070601) and chloral hydrate (No. 30037517) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Xue-saitong tablets (No. 20071201), as a positive control drug (Wei 2004), were purchased from Weihe Pharmaceutical Co. Ltd. (Yuxi, China). Superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-PX), nitric oxide synthase (NOS) and catalase (CAT) kits (No. 20070329) were supplied from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Else: MCAO monofilaments (No. 2634-100, AAAA grade, diameter of headend: 0.34 [+ or -] 0.02 mm; diameter of body: 0.26 mm) were purchased from Beijing Sunbio Biotech Co., Ltd. (Beijing, China).
The multi-phenolic fraction of crude Dragon's blood was prepared for this study. Crude Dragon's blood extract was dissolved in 40% ethanol and kept for 24h at room temperature to separate a clear supernatant. The clear supernatant was eluted with 95% ethanol on macroporous adsorption resin and prepared by concentration and desiccation. Some of phenolic compounds were identified by Beijing BIT&GY Pharmaceutical R&D Co., Ltd to be the active components of Dragon's blood (Xin et al. 2011) (Fig.1).
Chromatographic conditions were as follows (Teng et al. 2011). Column: Ultimate XB-[C.sub.18] (250 mm x 4.6 mm, 5 p.m) column (Welch Materials, MD, USA); eluent: A: 1% glacial acetic acid water solution, B: acetonitrile; linear gradient elution was adopted starting from 25% to 55% B in 80 min; flow rate was 1.0 mL [min.sup.-1] and the column temperature was 25 [degrees]C. The monitoring wavelength was set at 280 nm. The main phenolic compositions of Dragon's blood extracts were Resveratrol (No. 1, 3.6 mg [g.sup.-1]), Loureirin A (No. 2, 6.9 mg [g.sup.-1]), Loureirin B (No. 3, 6.2 mg [g.sup.-1]) and Pterostilbene (No. 4, 5.0 mg [g.sup.-1]) of the dry extracts. The retention time of 1-4 compounds were 18.11, 59.93, 61.56 and 71.51 min, respectively (Fig. 2).
Dragon's blood dropping pills (No. 20071201) contained 30% multi-phenolic fraction of crude Dragon's blood and was provided by Beijing BIT&GY Pharmaceutical R&D Co., Ltd.
Animals The study complied with the "Guide for the Care and use of Laboratory Animals" published by the US National Institutes of Health (NIH Publication No. 85-23, revised in 1985) and all animals was approved by the institutional animal experiments committee. SD rats (230-250g body weight, 6-8 weeks old) and their special granular food were provided by Experimental Animal Center, West China Medical Center of Sichuan University (Chengdu, China). Male and female were half different. Animals were maintained under 12h light/dark cycle at a temperature approximately (24 [+ or -] 1) [degrees]C with free access to food and water ad libitum.
Animal groups and drug administrations
A total of 70 SD rats were divided into 7 groups (n=10 for each group). Group 1 (sham operation group) and groups 2 (model group) received the same volume of solvent. Group 3 (positive group), group 4 (crude Dragon's blood group) and groups 5-7 (Dragon's blood dropping pills high, middle and low groups) received oral administrations of 0.18 g [kg.sup.-1][d.sup.-1] of Xue-saitong tablets, 0.24g [kg.sup.-1][d.sup.-1] of crude Dragon's blood and 0.80, 0.40, 0.20g [kg.sup.-1][d.sup.-1] of Dragon's blood dropping pills, respectively. All groups were orally administered once a day for 5 days.
The MCAO rats model was performed according to the intraluminal filament technique modified by Zea-Longa (Bederson et al. 1986; Koizumi et al. 1986; Mohagheghi et al. 2011; Xiao and Liu 2006).
On the 5th day after 12-h fasting and administration, the focal cerebral ischemia rats model was established by method of MCAO. All rats were anaesthetized by 10% chloral hydrate with dose of 350 mg [kg.sup.-1] after the last administration. All groups were received common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA) of right side isolation. CCA and ECA of all groups except sham operation group were ligatured and MCAO monofilament was inserted into ICA about 18.5 [+ or -] 0.5 mm from the intersection of CCA to block the origin of middle cerebral artery. Then, ICA and MCAO monofilament were ligatured together. All rats were kept their rectal temperature at 37 [+ or -] 0.5 [degrees]C during the operative procedure. MCAO monofilament was removed to the intersection of ECA and ICA after cerebral ischemia 2 h: The rats of sham operation group were only received CCA, ICA and ECA of right side isolation without ligation. Then, all rats were sutured and took normal diet after the operations.
Neurological scores. All rats of each group were assessed with neurological scores 24 h after reperfusion according to some protocols (De et al. 1989; Li et at. 2012; Longa et at. 1989). All rats were placed on the floor and examined: 0 point = normal activities without neurologic deficiency; 1 point = without extension of con tralateral forelimb completely; 2 points = ipsilateral Horner syndrome, contra lateral tumble and/or draw outward circles; 3 points = fall down to the contralateral hemiplegic side; 4 points = inability to walk freely and unconsciousness; 5 points = death. Among them, the effective models were from 1 point to 4 points.
Detection of cerebral infarction volume. All rats were sacrificed 24 h after reperfusion with anaesthetization by 10% chloral hydrate (350 mg [kg.sup.-1]). The rats were decapitated and the brains rapidly removed and cooled in saline at 4 [degrees]C for 15 min. Six 2-mm thick coronal sections were then cut. The slices were immersed and stained by 2% 2,3,5-triphenyltetrazolium chloride (ITC) solution and kept at 37 [degrees]C in a water bath for 15 min. Then, the slices were digitally photographed. Unstained areas were defined as infarct and were measured using image analysis software. The cerebral infarction volume was calculated according to the following formula:
Cerebral infarction volume (V/[mm.sup.3]) = ([A.sub.1]+ ... +[A.sub.n]) xt/2
(t = section thickness (mm); A = infarction area ([mm.sup.2)])
Detection of cerebral water content and cerebral index. The brains of all rats were weighed and cerebral index was calculated according to the following formula:
Cerebral index = brain weight (g) / body weight (g) x 100
The brain wet and dry weights were measured before and after drying for 24 h at 70[degrees]C and the cerebral water content was calculated according to the following formula:
Cerebral water content (%)
= brain wet weight (g) - brain dry weight (g) / brain wet weight (g) X 100
Detection of oxidative stress in serum. The serum of all rats was obtained for further analysis. SOD activity was measured using the xanthine oxidase test. MDA level was detected using the thiobar-bituric acid test. GSH-PX activity was measured using a modified glutathione exhaustion assay. NOS activity was assayed by nitrate reductase method. CAT activity was determined by molybdate col-ori metric method.
SPSS13.0 (SPSS, Chicago, IL USA) was used for statistical analysis. Values were expressed as mean [+ or -]Standard Deviation (S.D.). Data were analyzed using one-way ANOVA, followed by Student's two-tailed paired t-test for comparison between two groups. p<0.05 was considered to be significant.
Effect of Dragon's blood dropping pills on neurological scores in MCAO rats model
We established focal cerebral ischemia rats model used method of MCAO to investigate the protective effects of Dragon's blood dropping pills. The results showed that, compared with sham operation group, the neurological scores of model group (3.25 [+ or -] 0.71) were increased significantly (p <0.01). There were serious abnormal behaviors in model group because of cerebral ischemia, according to flexion of left limbs and tumble or circle to the left with rear collision. It was proved that MCAO rats model was made successfully. Compared with model group, each group of Dragon's blood dropping pills could decrease neurological scores remarkably (p <0.01). Among them, protective effect of high group (0.56 [+ or -] 0.20) on MCAO rats was higher than that of positive group (2.00 [+ or -] 0.76) and crude Dragon's blood group (1.63 [+ or -] 0.74) (p < 0.05). But the middle (1.75 [+ or -]0.71) and the low (2.00 [+ or -]0.76) groups were the same as that of positive group and crude Dragon's blood group (p >0.05). Therefore, protective effects of Dragon's blood dropping pills on MCAO rats were enhanced gradually with a tendency of increase in a dose-dependent manner (Fig. 3).
Effect of Dragon's blood dropping pills on cerebral infarction volume in MCAO rats model Staining with Trc showed that, compared with model group, the high and the middle groups of Dragon's blood dropping pills could reduce the cerebral infarction size in MCAO rats model. Thus, protective effects of Dragon's blood dropping pills on MCAO rats were enhanced gradually with a tendency of increase in a dose-dependent manner (Fig. 4).
The results showed that, compared with model group, each group of Dragon's blood dropping pills could reduce the cerebral infarction volume in MCAO rats model (p <0.05-0.01). The protective effects of high (53.33 [+ or -] 9.24) and middle (96.31 [+ or -] 8.39) groups were the same as that of positive group (96.85 [+ or -] 9.82) and crude Dragon's blood group (78.83 [+ or -] 22.44). Therefore, protective effects of Dragon's blood dropping pills on MCAO rats were enhanced gradually with a tendency of increase in a dose-dependent manner (Fig. 5).
Effect of Dragon's blood dropping pills on cerebral water content and cerebral index in MCAO rats model
The results showed that, compared with model group, each group of Dragon's blood dropping pills could decrease the cerebral index in MCAO rats model (p <0.05-0.01). The protective effects of high (0.75 [+ or -] 0.05) and middle (0.76 [+ or -] 0.07) groups were the same as that of positive group (0.77 [+ or -] 0.08) and crude Dragon's blood group (0.75 [+ or -] 0.07). Each group of Dragon's blood dropping pills could reduce the cerebral water content in MCAO rats model obviously (p <0.05-0.01). The protective effects of high (82.1 [+ or -] 0.9) group were the same as that of positive group (81.5 [+ or -] 0.7) and crude Dragon's blood group (81.7 0.8). Therefore, protective effects of Dragon's blood dropping pills on MCAO rats were enhanced gradually with a tendency of increase in a dose-dependent manner (Fig. 6a and b).
Effect of Dragon's blood dropping pills on oxidative stress in serum of MAO rats model
The results showed that, compared with model group, the high and the middle groups of Dragon's blood dropping pills could increase SOD activity (159.63[+ or -]2.20 and 157.64 [+ or -] 2.21) and reduce MDA level (5.93[+ or -] 1.06 and 6.27 [+ or -] 0.46) significantly (p < 0.05-0.01). Meanwhile, high group could improve GSH-PX activity (498.05 [+ or -] 41.37) remarkably (p<0.05). Each group of Dragon's blood dropping pills could decrease NOS activity (26.33 [+ or -] 3.04, 26.87 [+ or -] 2.70 and 26.63 [+ or -] 2.93) and increase CAT activity (7.70 [+ or -] 1.69, 7.62 [+ or -] 2.03 and 7.62 [+ or -] 2.03) evidently (p < 0.05-0.01). The protective effects of high and middle groups were almost equal to that of positive group and crude Dragon's blood group. Therefore, Dragon's blood dropping pills could improve the antioxidant activity on MCAO rats model (Fig. 7a-e).
Ischemic cerebrovascular disease is a common disease in clinical, which can cause neuronal damage and affect the patients' nervous function seriously (Qian et at. 2011). It is an irreversible local lesion caused by dysfunction of brain perfusion and successive ischemic-hypoxic necrosis because of vascular wall pathological changes or/and hemodynamics disturbance (Howells et al. 2012). It makes local or diffuse damage and results in a series of neurological deficit syndrome.
In this study, focal cerebral ischemia rats model was established by method of MCAO, which made cerebral cortex and basal ganglia ischemia and dysfunction. Then, corresponding neurological symptoms and pathological changes appeared. Modern extraction process was used in our laboratory to obtain the active ingredients - phenolic compounds from Dragon's blood and they were made into dropping pills and their protective effects were examined to validate the efficacy of this new dosage forms.
Ischemia and hypoxia of nerve cells could cause brain tissue metabolism disorders and infarction volume increases in cerebral ischemia, which led to a series of clinical manifestations and affect neurological scores (Shimazu et al. 2005). In this study, MCAO rats model was established successfully. Neurological scores of model group were decreased and cerebral infarction volume was increased because of serious cerebral injury. Each group of Dragon's blood dropping pills could decrease neurological scores significantly (p <0.01). At the same time, Dragon's blood dropping pills could reduce the cerebral infarction volume in MCAO rats model (p < 0.05-0.01). Therefore, protective effects of Dragon's blood dropping pills on MCAO rats were enhanced gradually with a tendency of increase in a dose-dependent manner.
Cerebral edema is one of the basic pathological changes of ischemic cerebrovascular disease, which is an important indicator to reflect cerebral ischemia. The cerebral index is able to respond to the case of the brain edema to a certain extent (Li et al. 2007). In this experiment. MCAO rats model was used to investigate the effect of Dragon's blood dropping pills on cerebral edema. Cerebral edema was occurred because of severe hypoxia and microcirculatory blood flow obstacles, which made cerebral water content and cerebral index increased. After administration of Dragon's blood dropping pills, each group could decrease cerebral index and alleviate cerebral edema significantly (p < 0.05-0.01). The results suggested that Dragon's blood dropping pills could improve symptoms of cerebral edema evidently.
At present, lots of researches have indicated that cerebral vascular occlusion, cerebral ischemia and hypoxia, degeneration and edema are occurred after cerebral infarction. Large amounts of free radicals are produced in the process of cerebral ischemic injury. Lipid peroxidation caused by free radicals is the important pathogenesis of ischemic brain damage (Cuzzocrea et al. 2001; Lakhan et al. 2009). MDA is one of the radical metabolites and its serum levels can reflect the oxidative degree of tissue and cells in vivo. However, SOD is an important free radical scavenger. When the balance between generation and scavenging of free radicals is broken, lots of pathological changes appear. Therefore, the determination of SOD activity and MDA content can reflect the extent of lipid peroxidation induced by free radicals. GSH-PX has the activity of free radical scavenging caused by reactive oxygen species and 'OH and protect the integrity of the cell membrane in structure and function. Nitric oxide (NO) is a new kind of free radicals and neurotransmitter. It is catalyzed and synthesized by the rate-limiting enzyme, NOS from its substrate 1-arginine in vivo. It has various and extensive biological effects (Asano 1999). After cerebral ischemia injury, dephosphorylation and activation of NOS are occurred because of energy metabolism disturbance and adenosine triphosphate (ATP) deficiency, which lead to generation of large amount of NO. Excessive NO can produce toxicity to nerve cells with the form of free radicals (Zhao etal. 2005). CAT, as a catalase, is a kind of enzymes scavenger and conjugase with prosthetic group of iron porphyrin. It can scavenge H202 in vivo and make it decompose into molecular oxygen and water so as to prevent cells from being poisoned. Meanwhile, it is one of the key enzymes in biological defence system.
In this study, compared with sham operation group, SOD activity, GSH-PX activity and CAT activity of model group in serum were decreased significantly (p <0.01). at the same time, MDA level and NOS activity were increased remarkably (p <0.01). Compared with model group, the high and the middle groups of Dragon's blood dropping pills could increase SOD activity and reduce MDA level significantly (p <0.05-0.01). Meanwhile, high group could improve GSH-PX activity remarkably (p <0.05). Each group of Dragon's blood dropping pills could decrease NOS activity and increase CAT activity evidently (p < 0.05-0.01). So, Dragon's blood dropping pills had protective effects and improved the antioxidant activity of MCAO rats on scavenging excessive free radicals and decreasing lipid peroxidation.
In summary, Dragon's blood dropping pills had protective effects on focal cerebral Ischemia rats model through improving neurological scores, infarct volume, cerebral index, cerebral water content and oxidation stress. The validity and the possible mechanisms of Dragon's blood dropping pills against focal cerebral ischemia were determined preliminarily by these experiments.
Abbreviations: ATP, adenosine triphosphate; CAT, catalase; CCA, common carotid artery; ECA, external carotid artery; GSH-PX, glutathione peroxidase; ICA, internal carotid artery; ICVD, ischemic cerebrovascular disease; MCAO, Middle Cerebral Artery Occlusion; MDA, malondialdehyde; NO, nitric oxide; NOS, nitric oxide synthase; SOD, superoxide dismutase; TrC, 2,3,5-triphenyltetrazolium chloride; V. cerebral infarction volume.
The authors wish to express their thanks for financial support from the Ministry of Science and Technology of the People's Republic of China (Nos. 2012YQ040140; 2009BAK59B01, 02 and 03), material or facilitative support of Beijing BIT&GY Pharmaceutical R&D Co., Ltd. and West China School of Preclinical and Forensic Medicine, Sichuan University.
* Financially supported by the Ministry of Science and Technology of the People's Republic of China (Nos. 2012YQ040140; 2009BAK591301, 02 and 03).
* Corresponding author at: School of Life Science, Beijing Institute of Technology (BIT), 5 South Zhongguancun Street, Haidian District, Beijing 100081, PR China. Tel.: +86 1068915996: fax: +86 10 68915996.
E-mail address: email@example.com (Y.-L. Deng).
1 Both anthors contributed equally to this work.
0944-7113/s - see front matter 2013 Elsevier GmbH. All rights reserved.
Nian Xin (a), (1), Fang-Ju Yang (b), (1), Yan Li (a), Yu-Juan Li (a), Rong-Ji Dai (a), Wei-Wei Meng (a), Yan Chen (c), Yu-Lin Deng (a), *
(a) School of Life Science, Beijing Institute of Technology, Beijing 100081, PR China
(b) West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, PR China
(c) Beijing BIT & GY Pharmaceutical R&D Co., Ltd., Beijing 100081, PR China
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|Author:||Xin, Nian; Yang, Fang-Ju; Li, Yan; Li, Yu-Juan; Dai, Rong-Ji; Meng, Wei-Wei; Chen, Yan; Deng, Yu-Lin|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Dec 15, 2013|
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