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1.  Subacute toxicity and stability of Soshiho-tang, a traditional herbal formula, in Sprague–Dawley rats 
Backgroud
Soshiho-tang (SST, Xiao-chai-hu-tang in Chinese and Sho-saiko-to in Japanese), an oriental herbal formula, is used for treatment of chronic liver diseases. Although many researchers have studied the pharmacological properties of SST, information about its safety and toxicity is limited. Therefore, we evaluated the potential safety of SST in Sprague–Dawley rats over a period of 4-weeks.
Methods
The SST was administered once daily by gavage to male and female rats at doses of 0, 500, 1000 and 2000 mg/kg/day for 4 weeks. We measured the body weight, mortality, food consumption, ophthalmoscopy, urinalysis, hematology, serum biochemistry, gross pathological findings, absolute/relative organ weights and histopathology. In addition, we analyzed the component of SST and measured the stability of its component in SST according to study periods using high performance liquid chromatography.
Results
The SST treatment did not result in any toxicologically significant changes in mortality, food consumption, ophthalmoscopy, urinalysis, hematology, serum biochemistry, gross pathological findings, absolute/relative organ weights and histopathology, except for salivation and reduction in body weight in the 2000 mg/kg/day male group. These findings in the 2000 mg/kg/day male group are considered toxicologically insignificant because they are not accompanied by other pathological findings, including in hematology, serum biochemistry and histopatholgy, and they do not exhibit a dose–response relationship. SST is detected three components including liquiritin, baicalin, and glycyrrhizin. In addition, there were not observed the significant differences among the contents of three components in SST according to storage periods.
Conclusion
These results indicate that SST may be a safe material. Based on these results, the no-observed-adverse-effect level was more than 2000 mg/kg for both genders.
doi:10.1186/1472-6882-12-266
PMCID: PMC3553034  PMID: 23268740
Herbal formula; Soshiho-tang; Toxicity; Stability; Rat
2.  Hypoglycemic and hypolipidemic effects of Bersama engleriana leaves in nicotinamide/streptozotocin-induced type 2 diabetic rats 
Background
The present investigation was aimed at evaluating the hypoglycemic and hypolipidemic properties of the aqueous and methanolic extracts from Bersama engleriana leaves in streptozotocin/nicotinamide (STZ-NA)-induced type 2 diabetic rats.
Methods
Animals were orally treated for 4 consecutive weeks with Bersama engleriana extracts at doses of 300 or 600 mg/kg. The anti-diabetic effect was examined by measuring blood glucose (BG) at 0, 1, 14 and 28 days after STZ-NA treatment and, total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and triglycerides (TG) levels at sacrifice (day 29). Glibenclamide (0.25 mg/kg) was used for comparison.
Results
STZ-NA-induced diabetic rats showed moderate to significant increases in the levels of BG, TG, TC, LDL-C while body weight, HDL-C levels and relative weights of liver and pancreas were decreased compared to controls (non diabetic rats). Administration of the plant extracts to STZ-NA diabetic rats resulted in a significant decrease in BG, TG, TC and LDL-C and the dose 600 mg/kg of the methanolic extract was the most effective; HDL-C level was markedly increased after four weeks compared to untreated diabetic rats. A dose-dependent increase in the relative weights of the diabetogenic organs was observed in the Bersama engleriana groups. It can be also noticed that the methanolic extract, especially the dose 600 mg/kg (p<0.001), produced more effects than glibenclamide and aqueous extract. Rats treated with glibenclamide (0.25 mg/kg) generally gave lower results compared to groups treated with plant extracts.
Conclusion
Results of the present study showed that Bersama engleriana extracts and especially its methanolic extract possess antidiabetogenic properties and beneficial effects on diabetic hyperlipidemia. All these effects could be due to the bioactive components revealed in the Bersama engleriana extracts such as triterpenes and phenols and which could justify its ethnomedical use.
doi:10.1186/1472-6882-12-264
PMCID: PMC3546073  PMID: 23267560
Bersama engleriana; Diabetes mellitus; Hypoglycemic; Hypolipidemic; Rat
3.  Crude aqueous extracts of Pluchea indica (L.) Less. inhibit proliferation and migration of cancer cells through induction of p53-dependent cell death 
Background
Pluchea indica (L.) Less. (Asteraceae) is a perennial shrub plant with anti-inflammatory and antioxidant medicinal properties. However, the anti-cancer properties of its aqueous extracts have not been studied. The aim of this study was to investigate the anti-proliferation, anti-migration, and pro-apoptotic properties of crude aqueous extracts of P. indica leaf and root on human malignant glioma cancer cells and human cervical cancer cells, and the underlying molecular mechanism.
Methods
GBM8401 human glioma cells and HeLa cervical carcinoma cells were treated with various concentrations of crude aqueous extracts of P. indica leaf and root and cancer cell proliferation and viability were measured by cell growth curves, trypan blue exclusions, and the tetrazolium reduction assay. Effects of the crude aqueous extracts on focus formation, migration, and apoptosis of cancer cells were studied as well. The molecular mechanism that contributed to the anti-cancer activities of crude aqueous extracts of P. indica root was also examined using Western blotting analysis.
Results
Crude aqueous extracts of P. indica leaf and root suppressed proliferation, viability, and migration of GBM8401 and HeLa cells. Treatment with crude aqueous extracts of P. indica leaf and root for 48 hours resulted in a significant 75% and 70% inhibition on proliferation and viability of GBM8401 and HeLa cancer cells, respectively. Crude aqueous extracts of P. indica root inhibited focus formation and promoted apoptosis of HeLa cells. It was found that phosphorylated-p53 and p21 were induced in GBM8401 and HeLa cells treated with crude aqueous extracts of P. indica root. Expression of phosphorylated-AKT was decreased in HeLa cells treated with crude aqueous extracts of P. indica root.
Conclusion
The in vitro anti-cancer effects of crude aqueous extracts of P. indica leaf and root indicate that it has sufficient potential to warrant further examination and development as a new anti-cancer agent.
doi:10.1186/1472-6882-12-265
PMCID: PMC3575299  PMID: 23268709
Pluchea indica (L.) Less; Crude aqueous extracts; Cancer; p53; p21; Cell growth arrest; Apoptosis
4.  Biochemical composition and antioxidant capacity of extracts from Podophyllum hexandrum rhizome 
Background
Podophyllum hexandrum Royle (P. hexandrum) is a perennial herb and widely used in clinic. The present study was designed to separate and identify the biochemical composition and antioxidant capacity of extracts from P. hexandrum rhizome.
Methods
The ethyl acetate and ethanol extracts from P. hexandrum rhizome were analyzed by GC-MS (gas chromatography–mass spectrometry), and the antioxidant capacity of the extracts and the components was tested by using the DPPH (2, 2-diphenylpicrylhydrazyl) and FRAP (Ferric reducing/antioxidant power) assays.
Results
The rhizome extracts had greater antioxidant capacity than the petiole extracts in DPPH and FRAP assays. About 16 kinds of main reactive oxygen components were identified in the extracts. Components of PADE (Phthalic acid, diisobutyl ester), BADE (1,2-Benzenedicarboxylic acid, diisooctyl ester), Polyneuridine, PODD (Podophyllotoxin, deoxy), β-Sitosterol and POD (Podophyllotoxin) showed the antioxidant capacity in some degree. PODD, POD, and Polyneuridine showed stronger antioxidant capacity with the IC50 and FRAP values of 9.61 ± 0.81 and 2923.98 ± 21.89 μM, 9.98 ± 0.24 and 2847.27 ± 14.82 μM, and 13.37 ± 0.35 and 2404.32 ± 36.88 μM, respectively, than the positive control ASA (Ascorbic acid) with the values of 60.78 ± 1.22 and 1267.5 ± 30.24 μM (P < 0.01).
Conclusions
PODD, POD, and Polyneuridine are very critical for the antioxidant capacity in the extract of P. hexandrum rhizome. These results provide useful biochemical basis and information for the potential use of this plant.
doi:10.1186/1472-6882-12-263
PMCID: PMC3542176  PMID: 23259745
Podophyllum hexandrum; Biochemical composition; Antioxidant capacity; GC-MS
5.  Solenostemon monostachyus, Ipomoea involucrata and Carica papaya seed oil versus Glutathione, or Vernonia amygdalina: Methanolic extracts of novel plants for the management of sickle cell anemia disease 
Background
Sickle cell disease (SCD) is a genetic disease caused by an individual inheriting an allele for sickle cell hemoglobin from both parents and is associated with unusually large numbers of immature blood cells, containing many long, thin, crescent-shaped erythrocytes. It is a disease prevalent throughout many populations. The use of medicinal plants and nutrition in managing SCD is gaining increasing attention.
Methods
The antisickling effects of Solenostemon monostachyus (SolMon), Carica papaya seed oil (Cari-oil) and Ipomoea involucrata (Ipocrata) in male (HbSSM) and female (HbSSF) human sickle cell blood was examined in vitro and compared with controls, or cells treated with glutathione or an antisickling plant (Vernonia amygdalina; VerMyg).
Results
Levels of sickle blood cells were significantly reduced (P < 0.05) in all the plant-extract treated SCD patients’ blood compared with that of untreated SCD patients. RBCs in SolMon, Ipocrata, and Cari-oil treated samples were significantly higher (P < 0.05) compared with VerMyg-treated samples. The Fe2+/Fe3+ ratio was significantly reduced (P < 0.05) in all plant extract-treated HbSSM samples compared with controls. Hemoglobin concentration was significantly increased (P < 0.05) by SolMon treatment in HbSSF compared with VerMyg. Sickle cell polymerization inhibition exhibited by SolMon was significantly higher (P < 0.05) compared with that of VerMyg in HbSSF blood. Sickle cell polymerization inhibition in SolMon and Ipocrata were significantly higher (P < 0.05) compared with VerMyg in HbSSM blood. All plant extracts significantly reduced (P < 0.05) lactate dehydrogenase activity in both HbSSM and HbSSF-treated blood. Catalase activity was significantly increased (P < 0.05) in HbSSF blood treated with Ipocrata compared with glutathione. Cari-oil treated HbSSM and HbSSF blood had significantly increased (P < 0.05) peroxidase activity compared with controls.
Conclusions
Methanolic extracts from S. monostachyus, C. papaya seed oil and I. involucrata exhibited particular antisickling properties coupled with the potential to reduce stress in sickle cell patients. Each plant individually or in combination may be useful for the management of sickle cell disease.
doi:10.1186/1472-6882-12-262
PMCID: PMC3553046  PMID: 23259718
Sickle cell disease; Health; Management; Antisickling; Underutilized; Plants
6.  Glucan-rich polysaccharides from Pleurotus sajor-caju (Fr.) Singer prevents glucose intolerance, insulin resistance and inflammation in C57BL/6J mice fed a high-fat diet 
Background
Pleurotus sajor-caju (P. sajor-caju) has been extremely useful in the prevention of diabetes mellitus due to its low fat and high soluble fiber content for thousands of years. Insulin resistance is a key component in the development of diabetes mellitus which is caused by inflammation. In this study, we aimed to investigate the in vivo efficacy of glucan-rich polysaccharide of P. sajor-caju (GE) against diabetes mellitus and inflammation in C57BL/6J mice fed a high-fat diet.
Methods
Diabetes was induced in C57BL/6J mice by feeding a high-fat diet. The mice were randomly assigned to 7 groups (n=6 per group). The control groups in this study were ND (for normal diet) and HFD (for high-fat diet). The treated groups were ND240 (for normal diet) (240 mg/kg b.w) and HFD60, HFD120 and HFD240 (for high-fat), where the mice were administrated with three dosages of GE (60, 120, 240 mg GE/kg b.w respectively). Metformin (2 mg/kg b.w) served as positive control. The glucose tolerance test, glucose and insulin levels were measured at the end of 16 weeks. Expressions of genes for inflammatory markers, GLUT-4 and adiponectin in the adipose tissue of the mice were assessed. One-way ANOVA and Duncan’s multiple range tests (DMRT) were used to determine the significant differences between groups.
Results
GE treated groups improved the glucose tolerance, attenuated hyperglycemia and hyperinsulinemia in the mice by up-regulating the adiponectin and GLUT-4 gene expressions. The mice in GE treated groups did not develop insulin resistance. GE also down-regulated the expression of inflammatory markers (IL-6, TNF-α, SAA2, CRP and MCP-1) via attenuation of nuclear transcription factors (NF-κB).
Conclusion
Glucan-rich polysaccharide of P. sajor-caju can serve as a potential agent for prevention of glucose intolerance, insulin resistance and inflammation.
doi:10.1186/1472-6882-12-261
PMCID: PMC3553037  PMID: 23259700
Pleurotus sajor-caju; Diabetes; Polysaccharides; C57BL/6J mice; High-fat diet
7.  Estrogen-like activity of aqueous extract from Agrimonia pilosa Ledeb. in MCF-7 cells 
Background
Postmenopausal women experience estrogen deficiency-related menopausal symptoms (e.g., hot flashes and mood swings) and a dramatic increase in the incidence of chronic diseases. Although estrogen-replacement therapy (ERT) can reduce mortality from cardiovascular disease and improve osteoporosis and menopausal symptoms, its side effects have limited recent use. This study investigated the estrogen-like activity of aqueous extract from Agrimonia pilosa Ledeb.
Methods
The estrogenic activity of A. pilosa was investigated by using several in vitro assays. The binding activity of A. pilosa on estrogen receptors was examined using a fluorescence polarization-based competitive binding assay. The proliferative activity of A. pilosa was also examined using MCF-7 cells. Furthermore, the effect of A. pilosa on the expression of 3 estrogen-dependent genes was assessed.
Results
Using liquid chromatography-mass spectrometry, the 3 major peaks of A. pilosa aqueous extract were identified as apigenin-hexose, luteolin-glucuronide, and apigenin-glucuronide. The aqueous extract induced the proliferation of estrogen receptor-positive MCF-7 cells (p < 0.05). A. pilosa-stimulated proliferation was blocked on adding the estrogen antagonist ICI 182,780. Moreover, A. pilosa treatment increased the mRNA expression of the estrogen-responsive genes pS2 and PR (p < 0.05).
Conclusions
These results suggest A. pilosa can be used to improve estrogen deficiency-related menopausal symptoms or to treat diseases in postmenopausal women.
doi:10.1186/1472-6882-12-260
PMCID: PMC3575283  PMID: 23259680
MCF-7 proliferation assay; Estrogenic activity; Agrimonia pilosa; Metabolic syndrome
8.  Translation and adaptation of an international questionnaire to measure usage of complementary and alternative medicine (I-CAM-G) 
Background
The growing body of data on prevalence of complementary and alternative medicine (CAM) usage means there is a need to standardize measurement on an international level. An international team has published a questionnaire (I-CAM-Q), but no validation has yet been provided. The aim of the present study was to provide a German measurement instrument for CAM usage (I-CAM-G) which closely resembles the original English version, and to assess it’s performance in two potential samples for measuring CAM usage.
Methods
The English I-CAM-Q questionnaire was translated into German, and adapted slightly. The resulting I-CAM-G questionnaire was then pre-tested on 16 healthy volunteers, and 12 cognitive interviews were carried out. The questionnaire was employed in a sample of breast cancer patients (N = 92, paper and pencil), and a sample from the general population (N = 210, internet survey). Descriptive analyses of items and missing data, as well as results from the cognitive interviews, are presented in this paper.
Results
The translated questionnaire had to be adapted to be consistent with the German health care system. All items were comprehensible, whereby some items were unambiguous (e.g. CAM use yes/no, helpfulness), while others gave rise to ambiguous answers (e.g. reasons for CAM use), or high rates of missing data (e.g. number of times the CAM modality had been used during the last 3 months). 78% of the breast cancer patients and up to 85% of a sample of the general population had used some form of CAM.
Conclusions
Following methodologically sound and comprehensive translation, adaptation and assessment processes using recognized translation procedures, cognitive interviews, and studying the performance of the questionnaire in two samples, we arrived at a German questionnaire for measuring CAM use which is comparable with the international (English) version. The questionnaire appropriately measures CAM use, with some items being more appropriate than others. We recommend the development of a short version.
doi:10.1186/1472-6882-12-259
PMCID: PMC3562528  PMID: 23256756
9.  Synergetic downregulation of 67 kDa laminin receptor by the green tea (Camellia sinensis) secondary plant compound epigallocatechin gallate: a new gateway in metastasis prevention? 
Background
In traditional Chinese medicine, green tea is considered to have a life-prolonging effect, possibly as a result of its rich content of antioxidant tea polyphenols, and hence has the potential to prevent cancer. This study investigated the role of the major tea secondary plant compound epigallocatechin gallate (EGCG) for its inhibitory effects on the metastasis-associated 67 kDa laminin receptor (67LR).
Methods
To clarify the impact of EGCG on siRNA-silenced expression of 67LR, we applied an adenoviral-based intestinal in vitro knockdown model, porcine IPEC-J2 cells. Quantitative real-time polymerase chain reaction was performed to analyze 67LR gene expression following treatment with physiological and pharmacological concentrations of EGCG (1.0 g/l, 0.1 g/l, 0.02 g/l and 0.002 g/l).
Results
We report co-regulation of EGCG and 67LR, which is known to be an EGCG receptor. siRNA selectively and highly significantly suppressed expression of 67LR under the impact of EGCG in a synergetic manner.
Conclusions
Our findings suggest that 67LR expression is regulated by EGCG via a negative feedback loop. The explicit occurrence of this effect in synergy with a small RNA pathway and a plant-derived drug reveals a new mode of action. Our findings may help to provide insights into the many unsolved health-promoting activities of other natural pharmaceuticals.
doi:10.1186/1472-6882-12-258
PMCID: PMC3536572  PMID: 23249430
Cancer; EGCG; IPEC-J2; miRNA; RNA interference
10.  A water extract of Samchulkunbi-tang attenuates airway inflammation by inhibiting inos and MMP-9 activities in an ovalbumin-induced murine asthma model 
Background
In this study, we investigated the effect of Samchulkunbi-tang water extract (SCTE) in an established mouse model of ovalbumin (OVA)-induced allergic asthma. The effects of SCTE on the production of Th1 and Th2 cytokines, eotaxin, and total and OVA-specific immunoglobulin E, inducible nitric oxide synthase expression, and matrix metalloproteinase-9 activity were measured.
Methods
Mice were sensitized on days 0 and 14 with an intraperitoneal injection of 20 μg ovalbumin (OVA) emulsified in 2 mg aluminum hydroxide in 200 μL PBS buffer. On days 21, 22, and 23, mice received an airway exposure to OVA (1%, w/v, in PBS) for 1 h. SCTE was administered orally to mice at doses of 200 and 400 mg/kg per day from days 18 to 23.
Results
SCTE reduced the number of inflammatory cells, cytokines, and chemokines in bronchoalveolar lavage fluids and iNOS expression and MMP-9 activity in mouse lung tissue. Histological studies using hematoxylin & eosin and periodic acid-schiff staining showed that SCTE substantially inhibited OVA-induced inflammatory cell infiltration in lung tissue and goblet cell hyperplasia in the airway. SCTE also reduced IL-4 and IL-13 expression in concanavalin-A-stimulated splenocytes. These results were similar to those obtained with montelukast as a positive control.
Conclusions
Collectively, these results suggest that SCTE may be an effective oral treatment for allergic airway inflammation by virtue of its anti-inflammatory activity.
doi:10.1186/1472-6882-12-257
PMCID: PMC3557135  PMID: 23244755
Samchulkunbi-tang; Asthma; Cytokines; iNOS; MMP-9; Inflammation
11.  Standardized butanol fraction of WIN-34B suppresses cartilage destruction via inhibited production of matrix metalloproteinase and inflammatory mediator in osteoarthritis human cartilage explants culture and chondrocytes 
Background
WIN-34B is a novel Oriental medicine, which represents the n-butanol fraction prepared from dried flowers of Lonicera japonica Thunb and dried roots of Anemarrhena asphodeloides BUNGE. The component herb of WIN-34B is used for arthritis treatment in East Asian countries. The aim of this study was to determine the cartilage-protective effects and mechanisms of WIN-34B and its major phenolic compounds, chlorogenic acid and mangiferin, in osteoarthritis (OA) human cartilage explants culture and chondrocytes.
Methods
The investigation focused on whether WIN-34B and its standard compounds protected cartilage in interleukin (IL)-1β-stimulated cartilage explants culture and chondrocytes derived from OA patients. Also, the mechanisms of WIN-34B on matrix metalloproteinases (MMPs), tissue inhibitor of matrix metalloproteinases (TIMPs), inflammatory mediators, and mitogen-activated protein kinases (MAPKs) pathways were assessed.
Results
WIN-34B was not cytotoxic to cultured cartilage explants or chondrocytes. WIN-34B dose-dependently inhibited the release of glycosaminoglycan and type II collagen, increased the mRNA expression of aggrecan and type II collagen, and recovered the intensity of proteoglycan and collagen by histological analysis in IL-1β-stimulated human cartilage explants culture. The cartilage protective effect of WIN-34B was similar to or better than that of chlorogenic acid and mangiferin. Compared to chlorogenic acid and mangiferin, WIN-34B displayed equal or greater decreases in the levels of MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, and markedly up-regulated TIMP-1 and TIMP-3. WIN-34B inhibited inflammatory mediators involved in cartilage destruction, such as prostaglandin E2, nitric oxide, tumor necrosis factor-alpha, and IL-1β. The phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38 was significantly reduced by WIN-34B treatment, while phosphorylation of JNK was only inhibited by chlorogenic acid or mangiferin in IL-1β-stimulated chondrocytes.
Conclusions
WIN-34B is potentially valuable as a treatment for OA by virtue of its suppression of MMPs, ADAMTSs, and inflammatory mediators, and it’s up-regulation of TIMP-1 and TIMP-3 involved in the MAPK pathway.
doi:10.1186/1472-6882-12-256
PMCID: PMC3559294  PMID: 23241445
WIN-34B; Standard compounds; Cartilage protection; Matrix proteinases; Inflammatory mediators
12.  Transcutaneous vagus nerve stimulation for the treatment of depression: a study protocol for a double blinded randomized clinical trial 
Background
Depressive disorders are the most common form of mental disorders in community and health care settings. Unfortunately, the treatment of Major Depressive Disorder (MDD) is far from satisfactory. Vagus nerve stimulation (VNS) is a relatively new and promising physical treatment for depressive disorders. One particularly appealing element of VNS is the long-term benefit in mood regulation. However, because this intervention involves surgery, perioperative risks, and potentially significant side effects, this treatment has been limited to those patients with treatment-resistant depression who have failed medication trials and exhausted established somatic treatments for major depression, due to intolerance or lack of response.
This double-blinded randomized clinical trial aims to overcome these limitations by introducing a novel method of stimulating superficial branches of the vagus nerve on the ear to treat MDD. The rationale is that direct stimulation of the afferent nerve fibers on the ear area with afferent vagus nerve distribution should produce a similar effect as classic VNS in reducing depressive symptoms without the burden of surgical intervention.
Design
One hundred twenty cases (60 males) of volunteer patients with mild and moderate depression will be randomly divided into transcutaneous vagus nerve stimulation group (tVNS) and sham tVNS group. The treatment period lasts 4 months and all clinical and physiological measurements are acquired at the beginning and the end of the treatment period.
Discussion
This study has the potential to significantly extend the application of VNS treatment for MDD and other disorders (including epilepsy, bipolar disorder, and morbid obesity), resulting in direct benefit to the patients suffering from these highly prevalent disorders. In addition, the results of this double-blinded clinical trial will shed new light on our understanding of acupuncture point specificity, and development of methodologies in clinical trials of acupuncture treatment.
Trials registration
Clinical Trials. ChiCTR-TRC-11001201 http://www.chictr.org/cn/
doi:10.1186/1472-6882-12-255
PMCID: PMC3537743  PMID: 23241431
Major depression disorder; Vagus nerve stimulation; Transcutanecous vagus nerve stimulation
13.  Frankincense essential oil prepared from hydrodistillation of Boswellia sacra gum resins induces human pancreatic cancer cell death in cultures and in a xenograft murine model 
Background
Regardless of the availability of therapeutic options, the overall 5-year survival for patients diagnosed with pancreatic cancer remains less than 5%. Gum resins from Boswellia species, also known as frankincense, have been used as a major ingredient in Ayurvedic and Chinese medicine to treat a variety of health-related conditions. Both frankincense chemical extracts and essential oil prepared from Boswellia species gum resins exhibit anti-neoplastic activity, and have been investigated as potential anti-cancer agents. The goals of this study are to identify optimal condition for preparing frankincense essential oil that possesses potent anti-tumor activity, and to evaluate the activity in both cultured human pancreatic cancer cells and a xenograft mouse cancer model.
Methods
Boswellia sacra gum resins were hydrodistilled at 78°C; and essential oil distillate fractions were collected at different durations (Fraction I at 0–2 h, Fraction II at 8–10 h, and Fraction III at 11–12 h). Hydrodistillation of the second half of gum resins was performed at 100°C; and distillate was collected at 11–12 h (Fraction IV). Chemical compositions were identified by gas chromatography–mass spectrometry (GC-MS); and total boswellic acids contents were quantified by high-performance liquid chromatography (HPLC). Frankincense essential oil-modulated pancreatic tumor cell viability and cytotoxicity were determined by colorimetric assays. Levels of apoptotic markers, signaling molecules, and cell cycle regulators expression were characterized by Western blot analysis. A heterotopic (subcutaneous) human pancreatic cancer xenograft nude mouse model was used to evaluate anti-tumor capability of Fraction IV frankincense essential oil in vivo. Frankincense essential oil-induced tumor cytostatic and cytotoxic activities in animals were assessed by immunohistochemistry.
Results
Longer duration and higher temperature hydrodistillation produced more abundant high molecular weight compounds, including boswellic acids, in frankincense essential oil fraactions. Human pancreatic cancer cells were sensitive to Fractions III and IV (containing higher molecular weight compounds) treatment with suppressed cell viability and increased cell death. Essential oil activated the caspase-dependent apoptotic pathway, induced a rapid and transient activation of Akt and Erk1/2, and suppressed levels of cyclin D1 cdk4 expression in cultured pancreatic cancer cells. In addition, Boswellia sacra essential oil Fraction IV exhibited anti-proliferative and pro-apoptotic activities against pancreatic tumors in the heterotopic xenograft mouse model.
Conclusion
All fractions of frankincense essential oil from Boswellia sacra are capable of suppressing viability and inducing apoptosis of a panel of human pancreatic cancer cell lines. Potency of essential oil-suppressed tumor cell viability may be associated with the greater abundance of high molecular weight compounds in Fractions III and IV. Although chemical component(s) responsible for tumor cell cytotoxicity remains undefined, crude essential oil prepared from hydrodistillation of Boswellia sacra gum resins might be a useful alternative therapeutic agent for treating patients with pancreatic adenocarcinoma, an aggressive cancer with poor prognosis.
doi:10.1186/1472-6882-12-253
PMCID: PMC3538159  PMID: 23237355
Apoptosis; Boswellia sacra, Boswellic acid; Essential oil; Frankincense; Hydrodistillation; Pancreatic cancer
14.  Paeoniflorin regulates macrophage activation in dimethylnitrosamine-induced liver fibrosis in rats 
Background
Macrophages in other organs (e.g. kidneys, lungs, and spleen, et. al) have rarely been reported in the development of liver fibrosis. Therefore, it is important to investigate macrophage activation in the main organs in liver fibrosis. We investigated the potential antifibrogenic effects of paeoniflorin (PF) in a dimethylnitrosamine (DMN)-induced rat model with special focus on inhibiting macrophage activation in the main organs.
Methods
Rat hepatic fibrosis was induced by treatment with DMN three times weekly over a 4-week period. DMN rats were treated with water, PF, or gadolinium chloride (GdCl3) from the beginning of the 3rd week. The expression of CD68, marker of macrophage, was investigated using immunohistochemical, real-time PCR, and western blot analysis.
Results
Hepatic hydroxyproline content markedly decreased and histopathology improved in the DMN-PF rats. Expression of desmin and collagen 1 decreased notably in DMN-PF liver. CD68 expression in the liver, spleen and kidney increased markedly after 2 weeks but decreased in DMN-water rats. PF and GdCl3 decreased CD68 expression in the liver and spleen and there was no effect on kidney. CD68 expression in the lung increased gradually during the course of DMN-induced liver fibrosis, and PF inhibited CD68 expression in the lung significantly while GdCl3 increased CD68 markedly. Expression of tumor necrosis factor (TNF-α) was decreased significantly by GdCl3 in the liver, as revealed by real-time PCR analysis. However, GdCl3 could not decrease TNF-α level in the serum by enzyme linked immunosorbent assay (ELISA).
Conclusions
Macrophage activation was disrupted in the liver, spleen, lung and kidney during development of DMN-induced liver fibrosis. PF administration attenuated DMN-induced liver fibrosis at least in part by regulating macrophage disruption in the main organs.
doi:10.1186/1472-6882-12-254
PMCID: PMC3575304  PMID: 23237422
Liver fibrosis; Macrophage; Paeoniflorin
15.  Young coconut juice can accelerate the healing process of cutaneous wounds 
Background
Estrogen has been reported to accelerate cutaneous wound healing. This research studies the effect of young coconut juice (YCJ), presumably containing estrogen-like substances, on cutaneous wound healing in ovairectomized rats.
Methods
Four groups of female rats (6 in each group) were included in this study. These included sham-operated, ovariectomized (ovx), ovx receiving estradiol benzoate (EB) injections intraperitoneally, and ovx receiving YCJ orally. Two equidistant 1-cm full-thickness skin incisional wounds were made two weeks after ovariectomy. The rats were sacrificed at the end of the third and the fourth week of the study, and their serum estradiol (E2) level was measured by chemiluminescent immunoassay. The skin was excised and examined in histological sections stained with H&E, and immunostained using anti-estrogen receptor (ER-α an ER-β) antibodies.
Results
Wound healing was accelerated in ovx rats receiving YCJ, as compared to controls. This was associated with significantly higher density of immunostaining for ER-α an ER-β in keratinocytes, fibroblasts, white blood cells, fat cells, sebaceous gland, skeletal muscles, and hair shafts and follicles. This was also associated with thicker epidermis and dermis, but with thinner hypodermis. In addition, the number and size of immunoreactive hair follicles for both ER-α and ER-β were the highest in the ovx+YCJ group, as compared to the ovx+EB group.
Conclusions
This study demonstrates that YCJ has estrogen-like characteristics, which in turn seem to have beneficial effects on cutaneous wound healing.
doi:10.1186/1472-6882-12-252
PMCID: PMC3538627  PMID: 23234369
Young coconut juice; Wound healing; Estrogen; ER-α; ER-β; Ovariectomy
16.  The cardioprotective effect of danshen and gegen decoction on rat hearts and cardiomyocytes with post-ischemia reperfusion injury 
Background
Danshen (Salviae Miltiorrhizae Radix) and Gegen (Puerariae Lobatae Radix) have been used for treating heart disease for several thousand years in China. It has been found that a Danshen and Gegen decoction (DG) exhibiting an anti-atherosclerosis effect, which improves the patients’ heart function recovery. Pre-treatment with DG was reported to have protective effects on myocardium against ischemia/reperfusion injury. In the present study, we aim to investigate the post-treatment effect of DG on ischemic-reperfusion injuries ex vivo or in vitro and the underlying mechanisms involved.
Methods
The rat heart function in an ischemia and reperfusion (I/R) model was explored by examining three parameters including contractile force, coronary flow rate and the release of heart specific enzymes within the heart perfusate. In vitro model of hypoxia and reoxygenation (H/R), the protective effect of DG on damaged cardiomyocytes was investigated by examining the cell structure integrity, the apoptosis and the functionality of mitochondria.
Results
Our results showed that DG significantly improved rat heart function after I/R challenge and suppressed the release of enzymes by damaged heart muscles in a dose-dependent manner. DG also significantly inhibited the death of cardiomyocytes, H9c2 cells, with a H/R challenge. It obviously decreased cell apoptosis, protected the mitochondrial function and cell membrane skeleton integrity on H9c2 cells. The cardio-protection was also found to be related to a decrease in intracellular calcium accumulation within H9c2 cells after I/R challenge.
Conclusion
The potential application of DG in treating rat hearts with an I/R injury has been implied in this study. Our results suggested that DG decoction could act as an anti-apoptotic and anti-ion stunning agent to protect hearts against an I/R injury.
doi:10.1186/1472-6882-12-249
PMCID: PMC3537695  PMID: 23228089
Danshen and Gegen decoction; Ischemia and reperfusion; Hypoxia and reoxygenation; Apoptosis; Calcium
17.  Prakriti (Ayurvedic concept of constitution) and variations in platelet aggregation 
Background
Ayurveda, the Indian traditional system of medicine describes a unique concept “prakriti”, genetically determined, categorising the population into several subgroups based on phenotypic characters like appearance, temperament and habits. The concept is claimed to be useful in predicting an individual’s susceptibility to a particular disease, prognosis of that illness and selection of therapy. The present study was carried out to study if the platelet aggregatory response and its inhibition by aspirin varied in the different prakriti subtypes.
Methods
After obtaining Institutional Ethics Committee permission, normal healthy individuals of either sex between the age group 18 to 30 years were recruited in the study. Their prakriti evaluation was done using a standardized validated questionnaire (TNMC Prakriti 2004). Their Platelet Rich Plasma was incubated with either aspirin [2.5micro-mole (μM) and 5μM] or distilled water as control for three minutes after which the aggregatory response to 5μM Adenosine Diphosphate (ADP) was measured over a period of 7 minutes.
Results
We observed that in the study population of normal healthy participants (n= 137), ADP-induced maximal platelet aggregation (MPA) was highest among the Vata-pitta prakriti individuals [Median (range), 83.33% (52.33-96)] as compared to the other prakriti types and these individuals responded better to lower dose of aspirin compared to other prakriti types.
Conclusions
Our results suggest that identifying the prakriti may help in individualising therapy or predicting proneness to a disease.
doi:10.1186/1472-6882-12-248
PMCID: PMC3562518  PMID: 23228069
Adenosine diphosphate; Aspirin; Pitta; Kapha; Vata
18.  Inhibition of lipopolysaccharide-induced nitric oxide and prostaglandin E2 production by chloroform fraction of Cudrania tricuspidata in RAW 264.7 macrophages 
Background
Cudrania tricuspidata extract is an important traditional herbal remedy for tumors, inflammation, gastritis, and liver damage and is predominantly used in Korea, China, and Japan. However, the anti-inflammatory effects of the extract have not yet been conclusively proved.
Methods
In this study, we investigated the effects of the CHCl3 fraction (CTC) of a methanol extract of C. tricuspidata on the lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW 264.7 macrophage cells and mouse peritoneal macrophages, and the levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in RAW 264.7 macrophage cells.
Results
We observed that the protein expression levels of inducible NO synthase and COX-2 enzymes were markedly inhibited by CTC in a concentration-dependent manner. In addition, CTC reduced the production of TNF-α, IL-1β, and IL-6 in the LPS-stimulated RAW 264.7 macrophage cells.
Conclusions
Our results show that the C. tricuspidata extract could modulate macrophage-mediated inflammatory functions such as the overproduction of cytokines, NO, and PGE2. The CTC was found to be the active fraction in this context.
doi:10.1186/1472-6882-12-250
PMCID: PMC3575384  PMID: 23228109
Cudrania tricuspidata; Nitric oxide; Prostaglandin E2; Lipopolysaccharide CHCl3 partitioned methanol extract of Cudrania tricuspidata; CTC, Lipopolysaccharide; LPS, prostaglandin E2; PGE2, nitric oxide; NO
19.  Anti-ulcerogenic activity of the root bark extract of the African laburnum “Cassia sieberiana” and its effect on the anti-oxidant defence system in rats 
Background
Despite the widespread use of roots of Cassia sieberiana in managing several health conditions including gastric ulcer disease, there is little scientific data to support the rational phytotherapeutics as an anti-ulcer agent. This paper reports an evaluation of the in vivo anti-oxidant properties of an aqueous root bark extract of C. sieberiana in experimental gastric ulcer rats in a bid to elucidate its mechanism of action.
Methods
Fisher 344 (F344) rats received pretreatment of C. sieberiana root bark extract (500, 750, and 1000 mg/kg body wt.) for 7 days after which there was induction of gastric injury with absolute ethanol. The mean ulcer index (MUI) was calculated and serum total anti-oxidant level determined. Gastric mucosal tissues were prepared and the activity level of the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and myeloperoxidase (MPO) were measured together with the level of lipid hydroperoxides (LPO). Statistical difference between treatment groups was analysed using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc t test. Statistical significance was calculated at P< 0.05.
Results
The administration of ethanol triggered severe acute gastric ulcer and pretreatment with C. sieberiana root bark extract significantly and dose dependently protected against this effect. The root bark extract also dose dependently and significantly inhibited the ethanol induced decrease in activity levels of the enzymes SOD, CAT and GPx. The extract also inhibited the ethanol-induced decrease in level of serum total anti-oxidant capacity. The increase in ethanol-induced LPO level and MPO activity were also significantly and dose-dependently inhibited by the root bark extract.
Conclusions
The gastro-cytoprotective effect, inhibition of decrease in activity of gastric anti-oxidant enzymes and MPO as well as the inhibition of gastric LPO level suggests that one of the anti-ulcer mechanisms of C. sieberiana is the anti-oxidant property.
doi:10.1186/1472-6882-12-247
PMCID: PMC3577646  PMID: 23228052
Gastric ulcer; Cassia sieberiana; Superoxide dismutase; Catalase; Glutathione peroxidase; Lipid hydroperoxide; Myeloperoxidase
20.  Inhibitory effects of crude extracts from some edible Thai plants against replication of hepatitis B virus and human liver cancer cells 
Background
Edible plants such as Cratoxylum formosum (Jack) Dyer, Curcumin longa Lin, Momordica charantia Lin and Moringa oleifera Lam have long been believed in Thai culture to relieve ulcers and the symptoms of liver disease. However, little is known about their anti-liver cancer properties and antiviral activity against hepatitis B virus (HBV). The aim of this study was to investigate the anti-liver cancer and anti-HBV activities of crude extracts from these edible plants on human liver cancer cells.
Methods
Plant samples were prepared and extracted using buffer and hydro-alcoholic solvents. The MTT assay was performed to investigate the effects of the plant extracts on the cell viability of HepG2 cells. The inhibitory effect on replication of HBV was analysed by determining the level of HBV covalently closed circular DNA (cccDNA) in transiently transfected HepG2 cells with the DNA expression plasmid of the HBV genome using a quantitative real-time PCR.
Results
Buffer and hydroalcoholic extracts from C. formosum (leaf) reduced cell viability of HepG2 cells and they also inhibited HBV cccDNA. Crude extracts from C. longa (bulb) in both solvents did not have any cytotoxic effects on the HepG2 cells, but they significantly decreased the level of HBV cccDNA. Buffer extracts from the leaves of M. charantia and the fruits of M. oleifera showed to have anti-HBV activity and also a mild cytotoxicity effect on the HepG2 cells. In addition, leaves of M. Oleifera extracted by hydroalcoholic solvent drastically decreased the level of cccDNA in transiently transfected HepG2 cells.
Conclusion
Some crude extracts of edible plants contain compounds that demonstrate anti-liver cancer and anti-HBV activities.
doi:10.1186/1472-6882-12-246
PMCID: PMC3553072  PMID: 23216691
Antiviral activity; Anti-liver cancer; HBV cccDNA; Hepatitis B virus; Edible Thai plants
21.  Analysis of chemical constituents and antinociceptive potential of essential oil of Teucrium Stocksianum bioss collected from the North West of Pakistan 
Background
Medicinal plants are used for the treatment of different diseases in almost all cultures. Teucrium species grow wildly at different geographical locations around the world. Teucrium stocksianum is used in folk medicine for the treatment of diarrhea, cough, jaundice and abdominal pain. Scientific study on Teucrium stocksianum shows that it possesses anthelmintic, cytotoxic and antispasmodic activity. The aim of our present study is to identify the chemical composition and antinociceptive potential of the essential oil extracted from Teucrium stocksianum bioss.
Method
Essential oil (EO) from the aerial parts of Teucrium stocksianum were extracted by hydrodistillation process. The qualitative and quantitative composition of essential oil was determined with Gas chromatography/Mass spectrometer. Antinociceptive activity was determined by acetic acid induced writhing method. Percent inhibition of writhes of the test concentration was determined by comparing it with that of control. Tween-80 emulsion 2.5% (5 ml/kg b.w) was used as a control while Diclofenic sodium 50 mg/kg (b.w) was used as a standard drug.
Results
The chromatogram of the essential oil of Teucrium stocksianum shows differences both qualitatively and quantatively from essential oil composition reported in other countries. Hydrodistillation of Teucrium stocksianum yielded 0.4% (v/w), pale yellowish oil on dry basis. A total of 26 chemicals were identified by GC-MS accounting for 90.28% of the oil. The major components of essential oil were δ-cadinene (12.92%), α-pinene (10.3%), myrcene (8.64%), β-caryophyllene (8.23%), germacrene D (5.18%) and limonene (2.36%). Essential oil of Teucrium stocksianum has shown outstanding antinociceptive activity. It has been observed that increase in percent writhe inhibition (PWI) occurred from 20-80 mg/kg (b.w) and maximum writhe inhibition has been noted at a concentration of 80 mg/kg (b.w), but PWI decreased at 160 mg/kg, which may be due to some toxic effect of higher dose. ED50 value for Teucrium stocksianum was calculated as 31.5 ± 1.72415 mg/kg (b.w).
Conclusion
Our results indicate that there is a lot of variation in the composition of essential oil of Teucrium stocksianum boiss, which may be due to different climatic and experimental conditions. Secondly, the essential oil possesses strong antinociceptive activity and could be used in analgesic preparations especially for topical use.
doi:10.1186/1472-6882-12-244
PMCID: PMC3534250  PMID: 23217213
Teucrium stocksianum; Antinociceptive; Essential oil
22.  Characterizing the cytoprotective activity of Sarracenia purpurea L., a medicinal plant that inhibits glucotoxicity in PC12 cells 
Background
The purple pitcher plant, Sarracenia purpurea L., is a widely distributed species in North America with a history of use as both a marketed pain therapy and a traditional medicine in many aboriginal communities. Among the Cree of Eeyou Istchee in northern Québec, the plant is employed to treat symptoms of diabetes and the leaf extract demonstrates multiple anti-diabetic activities including cytoprotection in an in vitro model of diabetic neuropathy. The current study aimed to further investigate this activity by identifying the plant parts and secondary metabolites that contribute to these cytoprotective effects.
Methods
Ethanolic extracts of S. purpurea leaves and roots were separately administered to PC12 cells exposed to glucose toxicity with subsequent assessment by two cell viability assays. Assay-guided fractionation of the active extract and fractions was then conducted to identify active principles. Using high pressure liquid chromatography together with mass spectrometry, the presence of identified actives in both leaf and root extracts were determined.
Results
The leaf extract, but not that of the root, prevented glucose-mediated cell loss in a concentration-dependent manner. Several fractions elicited protective effects, indicative of multiple active metabolites, and, following subfractionation of the polar fraction, hyperoside (quercetin-3-O-galactoside) and morroniside were isolated as active constituents. Phytochemical analysis confirmed the presence of hyperoside in the leaf but not root extract and, although morroniside was detected in both organs, its concentration was seven times higher in the leaf.
Conclusion
Our results not only support further study into the therapeutic potential and safety of S. purpurea as an alternative and complementary treatment for diabetic complications associated with glucose toxicity but also identify active principles that can be used for purposes of standardization and quality control.
doi:10.1186/1472-6882-12-245
PMCID: PMC3556504  PMID: 23216659
Diabetic neuropathy; Glucose toxicity; Traditional medicine; Quercetin-3-O-galactoside; Morroniside
23.  Antimicrobial, antimycobacterial and antibiofilm properties of Couroupita guianensis Aubl. fruit extract 
Background
Couroupita guianensis Aubl. (Lecythidaceae) is commonly called Ayahuma and the Cannonball tree. It is distributed in the tropical regions of northern South America and Southern Caribbean. It has several medicinal properties. It is used to treat hypertension, tumours, pain, inflammatory processes, cold, stomach ache, skin diseases, malaria, wounds and toothache.
Methods
The fruits of Couroupita guianensis were extracted with chloroform. Antimicrobial, antimycobacterial and antibiofilm forming activities of the chloroform extract were investigated. Quantitative estimation of Indirubin, one of the major constituent, was identified by HPLC.
Results
Chloroform extract showed good antimicrobial and antibiofilm forming activities; however it showed low antimycobacterial activity. The zones of inhibition by chloroform extract ranged from 0 to 26 mm. Chloroform extract showed effective antibiofilm activity against Pseudomonas aeruginosa starting from 2 mg/mL BIC, with 52% inhibition of biofilm formation. When the chloroform extract was subjected to HPLC-DAD analysis, along with Indirubin standard, in the same chromatographic conditions, it was found that Indirubin was one of the major compounds in this plant (0.0918% dry weight basis).
Conclusions
The chloroform extract showed good antimicrobial and antibiofilm properties. Chloroform extract can be evaluated further in drug development programmes.
doi:10.1186/1472-6882-12-242
PMCID: PMC3537531  PMID: 23206492
Antimicrobial; Antimycobacterial; Antibiofilm; Couroupita guianensis; Indirubin
24.  Ethanol extract of Gleditsia sinensis thorn suppresses angiogenesis in vitro and in vivo 
Background
Gleditsia sinensis thorns have been widely used in traditional Korean medicine for the treatment of several diseases, including obesity, thrombosis, and tumor-related diseases. The aim of the study is to determine the antiangiogenic effect of Gleditsia sinensis thorns in vitro and in vivo in a bid to evaluate its potential as an anticancer drug.
Methods
Ethanol extract of Gleditsia sinensis thorns (EEGS) were prepared and used for in vitro and in vivo assays. In vitro antiangiogenic effect of EEGS was determined in HUVEC primary cells by cell migration and tube formation assays. In vivo antiangiogenic effect of EEGS was determined by measuring vessel formation and vascular endothelial cells migrating into the implanted matrigels in nude mice. The angiogenesis-related proteins of which expression levels were altered by EEGS were identified by proteomic analysis.
Results
EEGS exerted a dose-dependent antiproliferative effect on HUVEC cells without significant cytotoxicity. Angiogenic properties, such as cell migration and tube formation, were significantly inhibited by EEGS in a dose-dependent manner. New vessel formation was also suppressed by EEGS, as determined by the directed in vivo angiogenesis assays in nude mice. EEGS reduced the expression of proangiogenic proteins, endothelin 1 and matrix metallopeptidase 2, in HUVEC cells.
Conclusions
Our findings suggest that EEGS can inhibit angiogenesis by down-regulating proangiogenic proteins, and therefore it should be considered as a potential anticancer drug targeting tumor-derived angiogenesis.
doi:10.1186/1472-6882-12-243
PMCID: PMC3556500  PMID: 23206527
Gleditsia sinensis thorn; Antiangiogenesis; Anticancer; Gene expression; Medicinal herb
25.  Differential proteomics analysis of the analgesic effect of electroacupuncture intervention in the hippocampus following neuropathic pain in rats 
Background
Evidence is building steadily on the effectiveness of acupuncture therapy in pain relief and repeated acupuncture-induced pain relief is accompanied by improvement of hippocampal neural synaptic plasticity. To further test the cellular and molecular changes underlying analgesic effect of acupuncture, the global change of acupuncture associated protein profiles in the hippocampus under neuropathic pain condition was profiled.
Methods
The chronic constrictive injury (CCI) model was established by ligature of the unilateral sciatic nerve in adult Wistar rats. Rats were randomized into normal control (NC) group, CCI group, and CCI with electroacupuncture (EA) stimulation group. EA was applied to bilateral Zusanli (ST36) and Yanglingquan (GB34) in the EA group. Differentially expressed proteins in the hippocampus in the three groups were identified by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The functional clustering of the identified proteins was analyzed by Mascot software.
Results
After CCI, the thermal pain threshold of the affected hind footpad was decreased and was reversed gradually by 12 sessions of acupuncture treatment. Following EA, there were 19 hippocampal proteins identified with significant changes in expression (>2-fold), which are involved in metabolic, physiological, and cellular processes. The top three canonical pathways identified were “cysteine metabolism”, “valine, leucine, and isoleucine degradation” and “mitogen-activated protein kinase (MAPK) signaling”.
Conclusions
These data suggest that the analgesic effect of EA is mediated by regulation of hippocampal proteins related to amino acid metabolism and activation of the MAPK signaling pathway.
doi:10.1186/1472-6882-12-241
PMCID: PMC3533837  PMID: 23198761
Acupuncture analgesia; Cumulative effect; Hippocampus; Chronic constrictive injury

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