Prunellae Spica Research Articles

English

JiMaiTong (JMT), a Chinese herb compound prescription consisted of Flos chrysanthemi Indici, Spica prunellae and Semen cassiae for anti-hypertension. Luteolin is one of the major bioactivity compositions in F. chrysanthemi Indici in JMT. There are some reports about pharmacokinetics of luteolin in extract of F. chrysanthemi and husks of peanut in normal rats, but it lacked pharmacokinetic information of luteolin residing in a Chinese herb compound prescription in hypertensive animal models. The present study aimed to develop a high-performance liquid chromatography with photodiode array detection (HPLC-DAD) method for determination of luteolin in rat plasma and for pharmacokinetic study after oral administration of JMT to spontaneous hypertensive rats (SHR) and normal Sprague Dawley (SD) rats. After oral administration of JMT to SHR and SD rats, respectively the content of luteolin in blood samples at different time points were determined by a reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with liquid-liquid phase extraction. This revealed a difference in disposition of luteolin in SHR and SD rats and a relationship between plasma concentration and hypertension of pathological state within the organism. This was the first report on the pharmacokinetic compare of luteolin between normal and hypertension pathological state rats. The hypertension pathological state would effects the disposition of luteolin, and it may provide a meaningful basis for the clinical application of JMT. Key words: Luteolin, pharmacokinetic, Flos chrysanthemi Indici, hypertension, Chinese herb.

Preliminary analysis of bitter substances in spica of Prunella vulgaris

Volatile oil components and the contents and types of amino acid in spica of Prunella vulgaris were analysed by GC-MS and amino acid analyzer. Esters, fatty acids, aromatic hydrocarbon, ketone and several alcohol compounds were identified by mass spectrum comparison. In these ingredients, beta-ionone smelled aroma of cedar, raspberry, nerolidol showed weak sweet soft orange blossom flavor, neroli tasted sweet and fresh, nerolidol tasted sweet with light aroma of wood, hexadecanal showed a weak aroma of flowers and wax, alpha-sinensal had rich and fresh sweet orange flavor. To some extent, these types of aromatic substances can affect the taste of herbal tea or decoction made of Spica Prunellae. Among amino acids detected, natural amino acids accounted for a larger proportion, and those natural amino acids showed bitterness, slight bitterness, sourness (freshness), sweetness, slight sweetness, sourness (slight freshness). The results indicated that bitter and slightly bitter amino acids have the greatest impacts on the sense of Spica Prunellae.

Study on inhibitory effect of <i>Spica Prunellae</i> extract on T Lymphoma cell EL-4 tumor

The objective of the study was to investigate the in-vivo anti-tumour activity of Spica prunellae extract, and to preliminarily explore the possible mechanism of in-vivo anti-tumour effect of Spica prunellae extract. Tumour inhibition rate and tumour apoptosis-related protein status were determined using the mice model of transplanted T lymphoma cell EL-4 tumour, and by immunohistochemical method. The results revealed that Spica prunellae extract showed certain tumour inhibitory effect, and compared with the model group. Tumour weight in Spica prunellae high-dose group was highly significantly different (P<0.01). Tumour weight in Spica prunellae medium-dose group was also significantly different (P<0.05) compared with the model group. Spica prunellae high-dose group enabled the high expression of Bcl-2 protein (47.54%) and low expression of Bax protein (13.14%). The study concluded that Spica prunellae extract has certain in vivo anti-tumour effect, which may be achieved through regulation of apoptosis related proteins.

Spica Prunellae extract inhibits the proliferation of human colon carcinoma cells via the regulation of the cell cycle

Spica Prunellae has long been used as a significant component in numerous traditional Chinese medicine (TCM) formulas to clinically treat cancers. Previously, Spica Prunellae was shown to promote cancer cell apoptosis and inhibit angiogenesis in vivo and in vitro. To further elucidate the precise mechanism of its tumoricidal activity, the effect of the ethanol extract of Spica Prunellae (EESP) on the proliferation of human colon carcinoma HT-29 cells was elucidated and the underlying molecular mechanisms were investigated. The proliferation of HT-29 cells was evaluated using 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation analyses. The cell cycle was determined using fluorescence-activated cell sorting (FACS) with propidium iodide (PI) staining. The mRNA and protein expression of cyclin-dependent kinase 4 (CDK4) and cyclin D1 was examined using RT-PCR and western blotting, respectively. EESP was observed to inhibit HT-29 viability and survival in a dose- and time-dependent manner. Furthermore, EESP treatment blocked G1/S cell cycle progression and reduced the expression of pro-proliferative cyclin D1 and CDK4 at the transcriptional and translational levels. Altogether, these data suggest that the inhibition of cell proliferation via G1/S cell cycle arrest may be one of the mechanisms through which Spica Prunellae treats cancer.

Spica prunellae promotes cancer cell apoptosis, inhibits cell proliferation and tumor angiogenesis in a mouse model of colorectal cancer via suppression of stat3 pathway

BackgroundConstitutive activation of STAT3 is one of the major oncogenic pathways involved in the development of various types of malignancies including colorectal cancer (CRC); and thus becomes a promising therapeutic target. Spica Prunellae has long been used as an important component in many traditional Chinese medicine formulas to clinically treat CRC. Previously, we found that Spica Prunellae inhibits CRC cell growth through mitochondrion-mediated apoptosis. Furthermore, we demonstrated its anti-angiogenic activities in vivo and in vitro. To further elucidate the precise mechanism of the potential tumoricidal activity of Spica Prunellae, using a CRC mouse xenograft model, in this study we evaluated its therapeutic efficacy against CRC and investigated the underlying molecular mechanisms.MethodsCRC mouse xenograft model was generated by subcutaneous injection of human colon carcinoma HT-29 cells into nude mice. Animals were given intra-gastric administration with 6 g/kg of the ethanol extract of Spica Prunellae (EESP) daily, 5 days a week for 16 days. Body weight and tumor growth were measured every two days. Tumor growth in vivo was determined by measuring the tumor volume and weight. HT-29 cell viability was examined by MTT assay. Cell apoptosis and proliferation in tumors from CRC xenograft mice was evaluated via immunohistochemical staining (IHS) for TUNEL and PCNA, and the intratumoral microvessel density (MVD) was examined by using IHS for the endothelial cell-specific marker CD31. The activation of STAT3 was evaluated by determining its phosphorylation level using IHS. The mRNA and protein expression of Bcl-2, Bax, Cyclin D1, VEGF-A and VEGFR2 was measured by RT-PCR and IHS, respectively.ResultsEESP treatment reduced tumor volume and tumor weight but had no effect on body weight change in CRC mice; decreased HT-29 cell viability in a dose-dependent manner, suggesting that EESP displays therapeutic efficacy against colon cancer growth in vivo and in vitro, without apparent toxicity. In addition, EESP significantly inhibited the phosphorylation of STAT3 in tumor tissues, indicating its suppressive action on the activation of STAT3 signaling. Consequently, the inhibitory effect of EESP on STAT3 activation resulted in an increase in the pro-apoptotic Bax/Bcl-2 ratio, decrease in the expression of the pro-proliferative Cyclin D1 and CDK4, as well as down-regulation of pro-angiogenic VEGF-A and VEGFR-2 expression. Finally, these molecular effects led to the induction of apoptosis, the inhibition of cell proliferation and tumor angiogenesis.ConclusionsSpica Prunellae possesses a broad range of anti-cancer activities due to its ability to affect STAT3 pathway, suggesting that Spica Prunellae could be a novel potent therapeutic agent for the treatment of CRC.

Prunellae Spica Extract Contains Antagonists for Human Endothelin Receptors

Endothelin-1 (ET-1) is a powerful vasoconstrictor that contributes to blood pressure elevation. The biological effects of ETs are mediated by two receptors, namely, endothelin type A receptor (ET(A)R) and endothelin type B receptor (ET(B)R). Chinese herbal medicines (CHM) with antagonist activity for these two receptors were screened by establishing stable clones of CHO-K1 cells expressing high levels of human ET(A)R and ET(B)R, namely CHO-ET(A)R and CHO-ET(B)R.The aqueous extract of Prunellae Spica (P1) inhibited the binding of (125)I-ET-1 to ET(A)R and ET(B)R in CHO-ET(A)R and CHO-ET(B)R cells, respectively. P1 suppressed the ET-1-induced mobilization of intracellular Ca(2+) . Through the alcohol fractionation of P1, the antagonists of human ET(A)R and ET(B)R were found to belong to different, separable ingredients and the antagonist of ET(A)R is more soluble in alcohol. The two antagonists were also effective in the test on human primary cells, HASMC and HUVEC. P1 successfully prevented the development of ET-1-associated hypertension in rats without further purification. These results indicate the presence of anti-hypertensive ingredients in P. Spica extract, at least through the inactivation of ET(A)R and/or ET(B)R.

Anti-angiogenic effect of Spica prunellae extract in vivo and in vitro

Angiogenesis is a fundamental component of cancer growth and metastasis; therefore, inhibition of angiogenesis has become a promising strategy for the discovery and development of cancer chemotherapeutics. Recently, natural products have attracted significant interest as therapeutic agents for cancer since they have relatively fewer side effects as compared to modern chemotherapeutics and have long been used clinically to treat a variety of diseases, including cancer. Spica prunellae is an important component in several traditional Chinese medicine (TCM) formulations for cancer treatment. Recently, we reported that S. prunellaeinhibits cancer cell growth through the induction of mitochondrion-dependent apoptosis. However, the precise mechanisms of its overall anti-cancer activity remain largely unknown. In the present study, we investigated the anti-angiogenic effects of the ethanol extract of S. prunellae (EESP) in vitro on human umbilical vein endothelial cells (HUVECs) and in vivo using the chick embryo chorioallantoic membrane (CAM) assay. We found that EESP inhibited the proliferation of HUVECs via blockade of cell cycle G1 to S progression. In addition, EESP inhibited the migration and tube formation of HUVECs. Moreover, EESP treatment decreased VEGF-A expression in HT-29 human colon carcinoma cells as well as the expression of VEGF-A and VEGFR-2 in HUVECs. Furthermore, EESP exposure reduced the formation of blood vessels in chick embryos. These results suggest that the inhibition of angiogenesis is one of the mechanisms by which S. prunellae can mediate an anti-cancer effect. Key words: Spica prunellae, angiogenesis, cancer treatment, herbal medicine.

A Potential In Vitro and In Vivo anti‐HIV Drug Screening System for Chinese Herbal Medicines

Chinese herbal medicines are often applied as an alternative therapy for viral diseases. However, the development of anti-HIV herbal drugs has proceeded slowly, partly because of the lack of a high-throughput system for screening these drugs. The present study evaluated 16 herbal medicines for anti-HIV activities in vitro and in vivo. Herbal medicines were first screened for the ability to regulate C-X-C receptor 4 (CXCR4) and C-C receptor 5 (CCR5) promoter activities. A single-round pseudotyped HIV-luciferase reporter virus system (HIV-Luc) was used to identify potential anti-HIV mechanisms. CD4+ T cells from healthy volunteers were examined for changes in CXCR4 and CCR5 levels. HIV-1 replication was evaluated by ELISA. Spica Prunellae and Herba Andrographitis were found to down-regulate the activities of both the CXCR4 and CCR5 promoters. Also, Spica Prunellae and Herba Andrographitis (>1000 μM) inhibited HIV-1 in a dose-dependent manner. CXCR4 and CCR5 levels were reduced in CD4+ T cells from healthy volunteers (p<0.05). Spica Prunellae and Herba Andrographitis (EC₅₀: 3.18 and 5.49 μg/mL, respectively) could suppress cell fusion and decrease p24 antigen. In conclusion, the data demonstrated that Spica Prunellae and Herba Andrographitis possessed anti-HIV-1 capabilities, perhaps through the inhibition of the CXCR4 and CCR5 promoters and HIV-1 replication.

English

Spica Prunellae&nbsp;(Prunella vulgaris&nbsp;fruiting spikes) has long been used as an important component in formulated prescriptions of Chinese traditional medicine to treat various kinds of cancer. However, the precise mechanism of the anti-cancer activity of Spica Prunellae remains to be elucidated. In this report, we investigated the cellular effects of the ethanol extract of Spica Prunellae&nbsp;(EESP) in the HT-29 human colon carcinoma cell line. We found that EESP inhibited the growth of HT-29 cells as evidenced by EESP-induced cell morphological changes and reduced cell viability in dose- and time-dependent manners. Furthermore, we demonstrated that the HT-29 cell growth inhibitory activity of EESP was due to apoptosis, as EESP treatment resulted in the loss of plasma membrane asymmetry (externalization of phosphatidylserine), collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and increase in the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Taken together, these results suggest that Spica Prunellae inhibits the growth of HT-29 colon cancer cells through mitochondrion-mediated apoptosis, which may, in part, explain its anti-cancer activity. &nbsp; Key words:&nbsp;Apoptosis, anti-tumor, HT-29 cells, phytotherapy, mitochondria, Spica Prunellae.

English

The ITS sequences of the original plants of&nbsp;Spica prunellae&nbsp;and its confusable species including 16 individuals belonging to 6 species from 12 localities, were analyzed. The results showed that the length of ITS sequences was 584 bp after alignment and the overhanging terminals were cut. When alignment gaps were treated as missing data, there were 150 variable sites. The percent divergence between species was 10.9%, with a transitions/transversions ratio of 1.53. There were 11 variable and 9 informative sites among the ITS sequences of 4&nbsp;species in&nbsp;Prunella&nbsp;Linn. There were specific sites at 5, 474 and 525 nt of the ITS sequence of&nbsp;P. grandiflora. There were 122 specific sites between genus&nbsp;Ajugaand genus&nbsp;Prunella. Both phylogenetic trees constructed by Maximum parsimony method and Neighbor-Joining method indicated that&nbsp;Prunella&nbsp;vulgarisLinn.,&nbsp;P. asiatica&nbsp;Nakai. and&nbsp;P. hispida&nbsp;Benth. had a very close relationship.&nbsp;The character of ITS sequences can be used for providing the molecular markers for identifying Chinese&nbsp;Prunella&nbsp;(S. prunellae). &nbsp; Key words:&nbsp;Prunella&nbsp;vulgaris&nbsp;Linn,&nbsp;Prunella asiatica&nbsp;Nakai, ITS sequence, molecular identification.

Simultaneous determination of four active componentsin Spica Prunellae by HPLC

To develop a HPLC method for simultaneous determination of caffeic acid, rosmarinic acid, oleanolic acid and ursolic acid in Spica Prunellae. After ultrasonic extraction with 75% ethanol solution containing 1% formic acid, the ethanol-extract of Spica Prunellae was analyzed on an Elite SinoChrom ODS-AP column using gradient elution of 0.01% phosphoric acid (A) and acetonitrile (B) at a flow rate of 0.9-1.0 mL x min(-1). A wavelength switch program was used for detection at 330 nm (0-33 min) and 203 nm (33-40 min). The column temperature was set at 20 degrees C and injection volume was 50 microL. The calibration curves of all analytes were linear. The average recoveries were 93.7%-105.2% with RSDs not more than 4.5%. The contents of caffeic acid, rosmarinic acid, ursolic acid and oleanolic acid in Spica Prunellae were 0.0401-0.0968, 0.99-2.57, 0.243-0.556, 4.06-8.13 mg x g(-1), respectively. The described method is sensitive, convenient and accurate, and is suitable for the simultaneous determination of caffeic acid, rosmarinic acid, oleanolic acid and ursolic acid in Spica Prunellae.

Determination of Total Ash and Acid-insoluble Ash of Chinese Herbal Medicine Prunellae Spica by Near Infrared Spectroscopy

Rapid and accurate determination of total ash and acid-insoluble ash of Chinese herbal medicine would be valuable for the quality illustration because current approaches are laborious and time-consuming. This study investigated the feasibility of near infrared spectroscopy in predicting simultaneously the total ash and acid-insoluble ash contents of Prunellae Spica (one of the most widely used Chinese herbal medicine). Samples were collected from ten different geographic regions and scanned under the near-infrared spectroscopy region (4000-12000 cm(-1)). Models between the spectral data and the results of reference analysis were developed, and subsequently validated. Calibration models based on partial least squares were accurate for the prediction of total ash (R2=0.914; standard errors of prediction=0.373), and acid-insoluble ash (R2=0.905; standard errors of prediction=0.452).

Development of a validated liquid chromatographic method for the quality control of Prunellae Spica: Determination of triterpenic acids

A simple and rapid reversed-phase HPLC-UV method was developed for the determination of triterpenic acids in the crude extract of Prunellae Spica. Five triterpenic acids were extracted and isolated from P. Spica as marker compounds for use in the quality control of herbal medicines. Various solvent extraction techniques were evaluated, and the greatest efficiency was observed with sonication in 100% ethanol. Elemental compositions of the five marker compounds were determined by high-resolution mass spectroscopy. The dynamic range of the HPLC-UV method depended on the specific analyte, and acceptable quantitation was obtained between 10 and 250 μg mL −1 for oleanolic acid, between 10 and 300 μg mL −1 for ursolic acid, between 3 and 75 μg mL −1 for 2α,3α,24-trihydroxyolean-12en-28oic acid, between 5 and 100 μg mL −1 for euscaphic acid, and between 5 and 100 μg mL −1 for 2α,3α-dihydroxyurs-12en-28oic acid. The method was deemed satisfactory by inter- and intra-day validation and exhibited both high accuracy and precision (relative standard deviation <9.4%). Overall limits of quantitation and detection were approximately 0.5–2.5 μg mL −1 at a signal-to-noise ratio (S/N) of 3 and were about 3.0–10.0 μg mL −1 at a S/N of 10. In addition, principal component analysis (PCA) was performed on the analytical data of 15 different P. Spica samples in order to classify samples collected from different regions.

In vitro and in vivo immunosuppressive activity of Spica Prunellae ethanol extract on the immune responses in mice

The immunosuppressive activity of the ethanol extract of Spica Prunellae ( Prunella vulgaris fruiting spikes) (EESP) consisting of a mixture of triterpenoids, flavonoids, tannins and polysaccharide was studied on the immune responses in mice. The effects of EESP on mice splenocyte proliferation in vitro were measured. EESP significantly suppressed concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated splenocyte proliferation in vitro in a concentration-dependent manner. Furthermore, the effects of EESP at three dose levels (a single dose of 0.25, 0.5, and 1.0 mg) on the humoral and cellular immune responses of mice subcutaneously immunized with ovalbumin (OVA) were examined. ICR mice were immunized subcutaneously with OVA on day 0 and 14. Starting on the day of immunization, the mice were administered subcutaneously with EESP at 0.25, 0.5, and 1.0 mg at intervals of 7 days for a total of five doses. On day 28, antigen-specific antibodies in serum, mitogen- and OVA-induced splenocyte proliferation were investigated. EESP significantly suppressed Con A-, LPS- and OVA-induced splenocyte proliferation in the immunized mice in a dose-dependent manner. Total IgG, IgG1 and IgG2b levels in the immunized mice were significantly reduced by EESP. Moreover, the suppressing effects on the antibody responses to OVA in the immunized mice were dose-dependently enhanced according to the increase of EESP. The results suggest that EESP could suppress the cellular and humoral response in mice.

Application of capillary electrophoresis for organic acid analysis in herbal studies.

A capillary electrophoresis (CE) procedure has been developed for the separation of 25 inorganic and organic acid anions using a buffer system consisting of 15 mM tris(hydroxymethyl)aminomethane, 3 mM 1,2,4-benzenetricarboxylic acid, 1.5 mM tetraethylenepentaamine (TEPA) and 20% methanol with pH adjusted to 8.4. A good separation of organic acids extracted from a mixture of Chinese traditional medicine (TCM) containing three herbs, Flos chrysthemi, Spica prunellae, and Folium mori was obtained using the procedure developed with satisfactory working range (0.20-77 mg/g), low detection limit (90-190 microg/g), and good repeatability (relative standard deviation 4.47-6.99%, n = 4). A satisfactory extraction of organic acids was achieved within 20 min using 0.1 M NaOH. The addition of TEPA to provide a reduced electroosmotic flow (EOF) environment was shown to remove interfering organic compounds extracted from TCM. The applicability of using organic acids as markers for determining the mixing ratio of constituent herbs for a TCM mixture was investigated using a three-component mixture with a 1:1:1 mixing ratio. A satisfactory mixing ratio of 1.04:1.09:0.98 was obtained using the methodology developed based on organic acids as markers. The application of our method for determining more complicated TCM mixtures has been discussed.

Literature researches of Herba Lagopsis

The usage of Herba Lagopsis as a drug has a long history, and it was arranged under the item of Semen Leonuri in the herb books of past dynasties and used as a kind of Semen Leonuri. The alias of Semen Leonuri is Spica Prunellae in records, and the withered Semen Leonuri after the summer solstice is also Herba Lagopsis. Now, there are many Flora and local Flora and records of materia medica embodying it, and its function is similar with motherwort.

Effects of Shikunshito-Kamiho on fecal enzymes and formation of aberrant crypt foci induced by 1,2-dimethylhydrazine.

Shikunshito-Kamiho (SKTK) is a traditional Chinese medicine composed of eight crude drugs (Ginseng Radix, Hoelen, Atractylodis Rhizoma, Glycyrrhizae Radix, Prunellae Spica, Ostreae Testa, Laminaria Thallus, Sargassum). We investigated the effects of SKTK on pH, ammonia, fecal enzymes such as beta-glucuronidase, tryptophanase, urease, and formation aberrant crypt foci in the colon carcinogenesis model induced by 1,2-dimethylhydrazine (DMH). Water extract of SKTK was administered orally for 5 weeks to DMH-treated mice as 0.5% and 1.5% of the diet. Beta-glucuronidase, pH and tryptophanase were significantly inhibited after treatment of 0.5% and 1.5% SKTK, while urease was significantly reduced only during and after treatment of 1.5% SKTK as compared with control data. However, the ammonia concentration wasn't different in SKTK treated groups from control group. The incidence number of aberrant crypts foci (ACF) and aberrant crypts/focus in colon was significantly decreased by 0.5% and 1.5% SKTK mixed diets compared with that in rats treated with DMH alone. These results suggest that SKTK exterts anticarcinogenic activity on experimental murine colorectal cancer.