The neuroprotective effects of an extract of Gastrodia elata

Ethnopharmacological relevance

Gastrodia elata (GE) Blume (family Orchidaceae) is a traditional Chinese herbal medicine for treating headaches, dizziness, tetanus, and epilepsy, indicating neuronal protective functions.

Aim of the study

To evaluate the neuroprotection of GE and its molecular mechanism in preventing serum deprivation-induced PC12 cell apoptosis.

Materials and methods

An MTT assay and Hoechst staining were used to respectively validate serum deprivation-induced cell death and apoptosis. Cyclic (c)AMP formation and protein kinase (PK)A activity were also measured after GE treatment. Western blotting was used to detect the phosphorylation of the cAMP response element-binding (CREB) protein. Transient transfection of a dominant negative CREB was used to validate the importance of CREB.

Results

GE targeted the adenosine A_2A receptor (A_2A-R). GE increased cAMP formation, PKA activity, and phosphorylation of the CREB protein. GE-induced CREB protein phosphorylation and protection was blocked by a PKA inhibitor and overexpression of the dominant negative CREB, respectively.

Conclusions

These results support the neuroprotective effects of GE. The protective mechanism might be mediated through an A_2A-R/cAMP/PKA/CREB-dependent pathway.     

HPLC同时测定西藏栽培天麻中天麻素和8种核苷及碱基类成分

本实验建立了同时测定天麻药材中天麻素及8种核苷和碱基类成分的高效液相色谱法:Agilent Zorbax BonusRP(4.6 mm×250 mm,5μm)色谱柱,甲醇-(0.04%冰乙酸)水溶液为流动相,梯度洗脱,柱温36℃,流速1.0 m L·min-1,检测波长254 nm,进样量20μL。该方法分离度良好,天麻素、胞嘧啶、胞苷、尿嘧啶、腺嘌呤、尿苷、胸腺嘧啶、鸟苷和腺苷等的质量浓度在2.04~262.00,0.20~24.67,0.18~23.75,0.20~25.83,0.20~26.67,0.16~20.00,0.22~27.71,0.20~24.29,0.24~30.58 mg·L-1内,相关系数r在0.998 9~0.999 9,具有良好的线性。天麻素和8种核苷及碱基平均加标回收率在96.4%~99.6%,RSD均小于2.7%(n=6)。测定了7个西藏栽培天麻药材中上述9种成分的含量。结果表明,7个天麻样品中天麻素的含量均高于2.0 mg·g-1,均含有较高的腺苷、鸟苷、尿苷和胞嘧啶,而胞苷、尿嘧啶、腺嘌呤和胸腺嘧啶含量较低。该方法简便、准确,重复性好,适用于天麻药材中天麻素和腺苷等8种核苷及碱基类成分的含量测定。     

贵州仿野生栽培红天麻的生活史及物候期研究

为掌握仿野生栽培红天麻的生活史及物候期,在贵州大方县采用林下仿野生模式栽培红天麻,观察并记录其生长发育各阶段特征。整理的历时24个月的贵州仿野生栽培红天麻生活史,将贵州仿野生栽培红天麻无性繁殖与有性繁殖中箭麻繁育物候期各自划分出5个时期,并详述各期特点。其结果可明确贵州红天麻仿野生栽培流程,为仿野生栽培天麻技术标准的研究与制订提供理论支撑。     

Anti-inflammatory effects of methanol extracts of the root of Lilium lancifolium on LPS-stimulated Raw264.7 cells

Aim of the study

Lilium lancifolium is commonly used to treat bronchitis, pneumonia, etc. In this study, we investigated the anti-inflammatory effects of methanol extracts of the root of Lilium lancifolium (LL extracts) in LPS-stimulated Raw264.7 cells.

Material and methods

Levels of NO, PGE₂ and pro-inflammatory cytokines (IL-6 and TNF-α) in the supernatant fraction were determined using sandwich ELISA. Expression of COX-2 and iNOS, phosphorylation of MAPK subgroups (ERK and JNK), and NF-κB activation in extracts were detected via Western blot and immunocytochemistry assays.

Results

The LL extract significantly inhibited NO, PGE₂, IL-6 and TNF-α production in LPS-stimulated cells, and suppressed iNOS and COX-2 expression. A mechanism-based study showed that phosphorylation of ERK1/2 and JNK and translocation of the NF-κB p65 subunit into nuclei were inhibited by the LL extract. Furthermore, interleukin-4 and interleukin-13 production in Con A-induced splenocytes was suppressed.

Conclusion

These results indicate that anti-inflammatory effects of methanol extracts from Lilium lancifolium are due to downregulation of iNOS and COX-2 via suppression of NF-κB activation and nuclear translocation as well as blocking of ERK and JNK signaling in LPS-stimulated Raw264.7 cells.     

Acorus gramineus inhibits microglia mediated neuroinflammation and prevents neurotoxicity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson's disease

Ethnopharmacological relevance

Acorus gramineus Solander (Acoraceae, AG), is a widely distributed plant in Asian countries. Rhizome part of this plant has long been used as a traditional medicine for treating various symptoms including central nervous system (CNS) disorders.

Aim of study

The anti-neuroinflammatory effect of AG aqueous extract was investigated using in vitro cellular and in vivo Parkinson's disease (PD) mouse model.

Materials and methods

Lipopolysaccharide (LPS) is used to stimulate BV-2 microglial cells in vitro and the changes in neuroinflammatory expressional levels were measured using ELISA, Western blotting, RT-PCR and immunofluorescence techniques. In in vivo experiments, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mouse model of PD was developed followed by immunohistochemical analysis of specific brain tissues.

Results

LPS-stimulation to BV-2 cells increased the production of nitric oxide (NO) and proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β. Pretreatment with AG extract inhibited the increased levels of NO and pro-inflammatory cytokines in LPS-stimulated BV-2 cells. Mechanistic study revealed that AG acts via the regulation of nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPKs) and TRIF-dependent signaling pathways. Further, AG protected MPTP-induced neuronal cell death and inhibited neuroinflammation in vivo.

Conclusion

Our results indicated that AG extract exerted anti-neuroinflammatory effects against activated microglia mediated insults through multiple signaling pathways and prevented in vivo neuronal cell death in mouse model of PD substantiating the traditional claims for its use in CNS disorders.     

Gleditsia sinensis thorns inhibit the production of NO through NF-kappaB suppression in LPS-stimulated macrophages

The thorns of Gleditsia sinensis LAM. (Leguminosae) have been used in traditional medicine for the treatment of inflammatory diseases including swelling, suppuration, carbuncle and skin diseases in China and Korea. In this study, we investigated the mechanism responsible for anti-inflammatory effects of Gleditsia sinensis thorns in RAW 264.7 macrophages. The aqueous extract of Gleditsia sinensis thorns (AEGS) inhibited LPS-induced NO secretion as well as inducible nitric oxide synthase (iNOS) expression, without affecting cell viability. Furthermore, AEGS suppressed LPS-induced NF-kappaB activation, phosphorylation and degradation of IkappaB-alpha, phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). These results suggest that AEGS has the inhibitory effects on LPS-induced NO production and iNOS expression in macrophages through blockade in the phosphorylation of MAPKs, following IkappaB-alpha degradation and NF-kappaB activation.     

Mechanisms of anti-inflammatory property of Anacardium occidentale stem bark: Inhibition of NF-κB and MAPK signalling in the microglia

Ethnopharmacological relevance

Anacardium occidentale is used in traditional African medicine for the treatment of arthritis, fever, aches, pains, and inflammation of the extremities.

Aim of the study

In this study, we investigated the molecular mechanisms responsible for anti-inflammatory effects of a stem bark extract of A. occidentale (ANE) in LPS-stimulated microglia.

Materials and methods

Nitric oxide (NO), prostaglandin E₂ and cytokine (TNFα and IL-6) production were evaluated in supernatants from LPS-stimulated BV-2 cells. Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and microsomal prostaglandin E2 synthase (mPGES-1) protein expressions in rat primary microglia were measured using western blot. The effects of ANE on NF-κB activation and nuclear translocation were evaluated in the luciferase reporter gene assay and ELISA, while ability of ANE to influence IκB phosphorylation was determined using ELISA specific for phospho-IκB. The involvement of MAPK phosphorylation in the anti-inflammatory actions of ANE was evaluated using specific ELISA for phospho-p38, phospho-p42/44 and phospho-JNK. The MTT assay was used to determine the effect of ANE on BV-2 microglia viability.

Results

ANE (25–100 μg/ml) produced significant (p<0.05) reduction in the production of NO, PGE₂, TNFα and IL-6 in BV-2 microglia stimulated with LPS for 24 h. Pre-treatment with ANE caused a significant (p<0.05) inhibition of COX-2, iNOS and mPGES-1 protein expressions in the rat primary microglia. Further experiments showed that ANE inhibited COX-2 and iNOS protein expression via IκB-mediated nuclear translocation and transactivation of NF-κB. Our studies also revealed that ANE produced significant (p<0.05) and dose-dependent inhibition of p38, p42/44 and JNK MAPK phosphorylation in LPS-activated BV-2 microglia.

Conclusions

We conclude that ANE has an anti-inflammatory property related to inhibition of inflammation-associated cytokine production as well as iNOS and COX-2 gene expression by blocking NF-κB and MAPK pathways in the microglia. It is also suggested that mPGES-1 inhibition contributes to the effect of ANE on PGE₂ production in the microglia.     

The production of nitric oxide and prostaglandin E2 in peritoneal macrophages is inhibited by Andrographis paniculata, Angelica sinensis and Morus alba ethyl acetate fractions

Aim of the study

Traditional Chinese medicine herbs (TCMHs) are used in medicines as well as in daily dietary supplements in Asia. In this study, we employed pNF-κB-Luc or pIFN-γ-Luc and BALB/c mice peritoneal macrophages or splenocytes to investigate both the immune and inflammatory effects of six selected plant species.

Materials and Methods

Specifically, we used ethyl acetate fractions of Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) Hsiao (Fabaceae) (AM), Andrographis paniculata (Burm. f.) Nees (Acanthaceae) (AP), Angelica sinensis (Oliv.) Diels (Apiaceae) (AS), Eucommia ulmodes Oliv. (Eucommiaceae) leaves (EU leaves), Isatis indigotica Fort. (Brassicaceae) (II) and Morus alba L. (Moraceae) (MA).

Results

We found that ethyl acetate fractions of AP, AS and MA significantly decreased NF-κB luciferase activity and also the secretion of NO and PGE₂ in LPS/IFN-γ stimulated mouse peritoneal macrophages (p < 0.05). In contrast, they did not affect IFN-γ luciferase activity or IFN-γ production in concanavalin A (Con A)-activated mouse splenocytes. Our results indicated that the anti-inflammatory properties of these plant extracts might be resulted from the inhibition of pro-inflammatory mediators (e.g., NO and PGE₂), at least in part via suppression of a signaling pathway such as NF-κB.

Conclusions

Collectively, we have found that three potent bioactive TCMH species exerted significant NF-κB inhibitory activity and acted in a cell type dependent fashion.     

Chrysanthemum indicum Linné extract inhibits the inflammatory response by suppressing NF-κB and MAPKs activation in lipopolysaccharide-induced RAW 264.7 macrophages

Aims of study

Although the flowers of Chrysanthemum indicum Linné (Asteraceae) have long been used in traditional Korean and Chinese medicine to treat inflammatory diseases, the underlying mechanism(s) by which these effects are induced remains to be defined. We investigated the effects of a 70% ethanolic extract of C. indicum (CIE) on the activities of cellular signaling molecules that mediate inflammatory responses.

Materials and methods

Production of NO, PGE₂, TNF-α, and IL-1β by ELISA, mRNA and protein expression of iNOS and COX-2, phosphorylation of MAPKs, and activation of NF-κB by RT-PCR and Western blotting were examined in LPS-induced RAW 264.7 macrophages.

Results

The CIE strongly inhibited NO, PGE₂, TNF-α, and IL-1β production, and also significantly inhibited mRNA and protein expression of iNOS and COX-2 in LPS-induced RAW 264.7 macrophages, in a dose-dependent manner. Furthermore, the CIE clearly suppressed nuclear translocation of NF-κB p65 subunits, which correlated with an inhibitory effect on IκBα phosphorylation. The CIE also attenuated the activation of ERK1/2 and JNK in a dose-dependent manner.

Conclusion

Our results suggest that the anti-inflammatory properties of CIE might result from the inhibition of inflammatory mediators, such as NO, PGE₂, TNF-α, and IL-1β, via suppression of MAPKs and NF-κB-dependent pathways.     

RP-HPLC同时测定天麻中4种成分的含量

目的：建立RP-HPLC同时测定天麻药材中天麻素、腺苷、对羟基苯甲醇和对羟基苯甲醛的含量。方法：采用Kromasil C₁₈色谱柱(4.6 mm×250 mm,5 μm),以甲醇-0.1%醋酸水溶液为流动相,梯度洗脱,流速1.0 mL·min^-1,检测波长270 nm,柱温35 ℃。结果：天麻素、腺苷、对羟基苯甲醇和对羟基苯甲醛分别在19.1～383 μg·mL^-1(r=0.999 9),0.620～12.4 μg·mL^-1(r=0.999 9),2.45～49.0 μg·mL^-1(r=0.999 9),0.280～5.63 μg·mL^-1(r=0.999 6)与峰面积线性关系良好;平均加样回收率(n=9)均在96.7%～97.7%,RSD均小于1.6%。结论：本法准确可靠,分离度好,适用于天麻药材中天麻素、腺苷、对羟基苯甲醇和对羟基苯甲醛的含量测定。     

RP-HPLC同时测定天麻中4种成分的含量

目的:建立RP-HPLC同时测定天麻药材中天麻素、腺苷、对羟基苯甲醇和对羟基苯甲醛的含量.方法:采用Kromasil C_(18)色谱柱(4.6 mm×250 mm,5μm),以甲醇-0.1%醋酸水溶液为流动相,梯度洗脱,流速1.0 mL·min~(-1),检测波长270 nm,柱温35℃.结果:天麻素、腺苷、对羟基苯甲醇和对羟基苯甲醛分别在19.1～383(r=0.999 9),0.620～12.4(r=0.999 9),2.45～49.0(r=0.999 9),0.280～5.63 mg·L~(-1)(r=0.999 6)与峰面积线性关系良好;平均加样回收率(n=9)均在96.7%～97.7%,RSD均小于1.6%.结论:本法准确可靠,分离度好,适用于天麻药材中天麻素、腺苷、对羟基苯甲醇和对羟基苯甲醛的含量测定.     

大川芎方中天麻效应组分的体内移行研究

目的:借鉴血清药物化学的方法,探讨大川芎方中天麻的药效物质基础,为有效、全面地控制大川芎方及其制剂的质量奠定基础.方法:通过HPLC指纹图谱,比较空白血浆与含药血浆,空白脑脊液与含药脑脊液的成分异同,确定天麻效应组分的体内移行成分,并应用HPLC-DAD-MSn分析技术推测移行成分的化学结构.结果:灌胃大川芎方效应组分后,血浆中较明显来源于天麻效应组分的色谱峰有2个,经推断分别为天麻素和巴利森苷类成分;而脑脊液中未明显检测到来源于天麻效应组分的色谱峰.结论:天麻体内移行成分研究有助于阐明其效应物质基础.     

天麻对大鼠脑缺血再灌注神经细胞凋亡的影响

目的:研究天麻对大鼠脑缺血再灌注引起脑损伤的保护作用,探讨其作用机制。方法:线栓法制备大鼠脑缺血再灌注模型,144只Wistar大鼠随机分为6组,即假手术组、模型组、天麻提取物高剂量组(150 mg.kg-1)、天麻提取物中剂量组(100 mg.kg-1)、天麻提取物低剂量组(50 mg.kg-1)、尼莫地平组(25 mg.kg-1),各组再分为缺血再灌注6,12,24 h 3个亚组,分别于再灌注6,12,24 h处死,经海马CAI区连续冠状切片,分别用TUNEL法观察神经细胞凋亡细胞数,免疫组织化学法检测半胱氨酸蛋白酶8(caspase-8)蛋白阳性细胞数。结果:①再灌注6,12,24 h模型组与假手术组相比,神经元凋亡率明显升高(P<0.05),再灌注6,12,24 h天麻提取物低、中、高剂量组、尼莫地平组细胞凋亡率与模型组的比较,明显降低(P<0.05,P<0.01)。②再灌注6,12,24 h模型组与假手术组相比,caspase-8阳性细胞数明显升高(P<0.05,P<0.01),再灌注6,12,24 h天麻提取物低、中、高剂量组、尼莫地平组caspase-8阳性细胞数与模型对照组比较,明显降低(P<0.05,P<0.01)。结论:天麻能通过有效的抑制脑缺血再灌注后大鼠神经细胞的凋亡起到脑保护作用。     

Anti-inflammatory effects of Polygala tenuifolia root through inhibition of NF-κB activation in lipopolysaccharide-induced BV2 microglial cells

Ethnopharmacological relevance

The root of Polygala tenuifolia Willd is a well-known traditional Oriental medicine and has been prescribed for treatment of dysfunction in memorial systems and various brain inflammatory diseases. The present study was designed to validate the anti-inflammatory effects of the water extract of Polygala tenuifolia root (WEPT).

Materials and methods

The anti-inflammatory properties of WEPT were studied using lipopolysaccharide (LPS)-stimulated murine BV2 microglia model. As inflammatory parameters, the production of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin E₂ (PGE₂), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β were evaluated. We also examined the extract's effect on the activity of nuclear factor-kappaB (NF-κB), and toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (Myd-88) expression.

Results

WEPT suppressed LPS-induced production of NO, PGE₂, and expression of iNOS and COX-2 in a dose-dependent manner, without causing cytotoxicity. It also significantly reduced generation of proinflammatory cytokines, including IL-1β and TNF-α. In addition, WEPT suppressed NF-κB translocation by blockade of IkappaB-α (IκB-α) degradation and inhibited TLR4 and Myd-88 expression in LPS-stimulated BV2 cells.

Conclusions

These results indicate that the inhibitory effects of WEPT on LPS-stimulated inflammatory mediator production in BV2 microglia are associated with suppression of the NF-κB and toll-like receptor signaling pathways. Therefore, Polygala tenuifolia extracts may be useful in treatment of neurodegenerative diseases by inhibition of inflammatory mediator production in activated microglia.     

Suppression of lipopolysaccharide-induced inflammatory responses in RAW 264.7 murine macrophages by aqueous extract of Clinopodium vulgare L. (Lamiaceae)

Ethnopharmacological relevance

The wild basil Clinopodium vulgare L. is commonly used in Bulgarian folk medicine for treatment of irritated skin, mastitis- and prostatitis-related swelling, as well as for some disorders accompanied with significant degree of inflammation (e.g. gastric ulcers, diabetes, and cancer).

Aim of study

To determine the effect of aqueous extract of Clinopodium vulgare L. on LPS-induced inflammatory responses of murine RAW 264.7 macrophages.

Materials and methods

Cell cytotoxicity was evaluated by MTT assay. Protein expression levels were monitored by Western blot analysis. Production of NO and PGE₂ was measured by the Griess colorimetric method and enzyme immunoassay, respectively. Activation of MMP-9 was visualized by gelatin zymography. Cytokine levels were determined by BioPlex assay. Intracellular ROS and free radical scavenging potential were measured by DCFH-DA and DPPH method, respectively. Xanthine oxidase activity was evaluated spectrophotometrically.

Results

The extract suppresses NF-κB activation by preventing Iκ-B phosphorylation and inhibits the phosphorylation of p38 and SAPK/JNK MAPKs. It down-regulates iNOS expression which manifests as a drastic decrease of NO production, inhibits MMP-9 activation, but does not affect COX-2 protein levels and reduces only slightly the released PGE₂. Secretion of IL-1β and Il-10 is greatly reduced, whereas suppression of TNF-α and GM-CSF production is less dramatic. The extract has strong free radical scavenging properties and exerts inhibitory effect on xanthine oxidase activity, which lowers the levels of intracellular ROS.

Conclusion

The study provides evidence for the anti-inflammatory potential of Clinopodium vulgare L. aqueous extract.     

免疫亲和柱净化UPLC-MS-MS测定天麻药材中黄曲霉毒素的含量

目的：研究建立了免疫亲和柱净化UPLC-MS-MS测定天麻药材中黄曲霉毒素B₁,B₂,G₁,G₂的含量测定方法。方法：采用甲醇溶液超声提取、玻璃纤维滤纸滤过、黄曲霉毒素亲和免疫柱净化的方法对天麻药材样品进行处理,以超高液相色谱-三重四级杆串联质谱仪（UPLC-MS-MS）对黄曲霉毒素B₁,B₂,G₁,G₂含量进行研究并测定。结果：被测定的4种黄曲霉毒素分别在选定的范围内线性关系良好,精密度、重复性、准确度均良好,利用标准参考物质对本方法的验证结果良好,采用所建立的方法对30批不同产地天麻药材中黄曲霉毒素B₁,B₂,G₁,G₂含量进行了测定,结果均未检出。结论：该方法科学、可行、方便、快速,适用于对天麻药材中黄曲霉毒素B₁,B₂,G₁,G₂进行含量测定。     

Methylene chloride fraction of the leaves of Thuja orientalis inhibits in vitro inflammatory biomarkers by blocking NF-κB and p38 MAPK signaling and protects mice from lethal endotoxemia

Aim of the study

Thuja orientalis (TO) has been a recognized herbal medicine across Northeast Asian countries for thousands of years and used for the treatment of various inflammatory diseases through as yet undefined mechanisms. In this study, we set out to determine whether the anti-inflammatory effects of this plant are mediated to suppress mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) activation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells.

Materials and methods

RAW 264.7 cells were pretreated with the methylene chloride fraction of TO (MTO) and stimulated with LPS. Nitric oxide (NO) release was determined by the accumulation of nitrite in the culture supernatants and tumor necrosis factor-α (TNF-α) and IL-6 secretion were determined by immunoenzymatic assay. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression were evaluated via RT-PCR and Western blotting. NF-κB activation was also evaluated by reporter gene assay and electrophoretic mobility shift assay (EMSA). In addition, the protective effect of MTO was evaluated by use of the LPS-induced endotoxin shock model in mice.

Results

We found that MTO significantly suppressed LPS-stimulated NO and IL-6 production without affecting cell viability. MTO inhibited the expression of LPS-induced iNOS and COX-2 protein and their mRNA expression. Also, TNF-α and IL-6 secretion were decreased by MTO in both PMA and ionomycin-stimulated splenocytes. As a result, MTO inhibited pro-inflammatory cytokines such as TNF-α and IL-6, which is hypothesized as being due to the suppression of LPS-induced p38 MAPK and NF-κB activation. Moreover, MTO improved the survival rate during lethal endotoxemia by inhibiting the production of TNF-α in an animal model and our LC-MS analysis showed that a major component of MTO was pinusolide.

Conclusions

We demonstrate here the evidence that the methylene chloride fraction of Thuja orientalis (MTO) potentially inhibits the biomarkers related to inflammation in vitro and in vivo, and might be provided as a potential candidate for the treatment of inflammatory diseases.