Therapeutic Effects of Lithospermate B Complexed with Mg2+ or Zn2+ on Metabolic Syndrome Induced in Rats Fed with High-Fat Diet.

Excessive food consumption and insufficient exercise lead to the prevalence of metabolic syndrome in modern life, which consequently increases the risk of many chronic diseases. Magnesium lithospermate B (MLB) from Danshen has been demonstrated to improve metabolic changes in high-fat diet-fed rats with metabolic syndrome. In this study, Mg2+ in MLB was successfully replaced with Zn2+ to form zinc lithospermate B (ZLB) complex. MLB (10 mg/kg /day) and ZLB of various concentrations (1, 2.5, 5, and 10 mg/kg/day) were prepared and examined for their therapeutic effects on metabolic syndrome induced in rats fed with a high-fat diet. The results showed that both MLB and ZLB were able to recover or alleviate the abnormal physiological states of high-fat diet-fed rats including weight gain, epididymal fat accumulation, fatty liver, retarded blood lipid and glucose metabolism putatively caused by insulin resistance, and elevated levels of proinflammatory cytokine, leptin, and oxidative stress. In an overall view of the animal study, the effectiveness of ZLB supplementation seemed to be better than that of MLB supplementation for the recovery of high-fat-fed rats from metabolic syndrome.

Development of Indirect Competitive ELISA for Lithospermic Acid B of Salvia miltiorrhiza with Its Specific Antibodies Generated via Artificial Oil Bodies.

Lithospermic acid B (LSB), the major water-soluble ingredient of Salvia miltiorrhiza (Danshen), has been shown to be an active ingredient responsible for the therapeutic effects of this traditional Chinese herb used to treat cardiac disorders. This study aimed to develop an indirect competitive enzyme linked immunosorbent assay (ELISA) for the detection of LSB. Firstly, LSB was chemically conjugated to a modified oil-body protein, lysine-enriched caleosin, recombinantly expressed in Escherichia coli. Antibodies against LSB (Ab-LSB) were successfully generated by immunizing hens with artificial oil bodies constituted with the LSB-conjugated caleosin. Western blotting showed that Ab-LSB specifically recognized LSB, but not the carrier protein, lysine-enriched caleosin. To detect LSB via indirect competitive ELISA, LSB was conjugated with bovine serum albumin (LSB-BSA) and coated on a microplate. The binding between Ab-LSB and LSB-BSA on the microplate was competed dose-dependently in the presence of free LSB with a concentration ranging from 5 to 5 × 104 ng/mL. The IC50 value was approximately determined to be 120 ng/mL for LSB regardless of its complex with a metal ion of Na+, K+ or Mg2+.

Echinacoside Isolated from Cistanche tubulosa Putatively Stimulates Growth Hormone Secretion via Activation of the Ghrelin Receptor.

Cistanche species, the ginseng of the desert, has been recorded to possess many biological activities in traditional Chinese pharmacopoeia and has been used as an anti-aging medicine. Three phenylethanoid glycosides-echinacoside, tubuloside A, and acteoside-were detected in the water extract of Cistanche tubulosa (Schenk) R. Wight and the major constituent, echinacoside, was further purified. Echinacoside of a concentration higher than 10-6 M displayed significant activity to stimulate growth hormone secretion of rat pituitary cells. Similar to growth hormone-releasing hormone-6, a synthetic analog of ghrelin, the stimulation of growth hormone secretion by echinacoside was inhibited by [D-Arg¹, D-Phe5, D-Trp7,9, Leu11]-substance P, an inverse agonist of the ghrelin receptor. Molecular modeling showed that all the three phenylethanoid glycosides adequately interacted with the binding pocket of the ghrelin receptor, and echinacoside displayed a slightly better interaction with the receptor than tubuloside A and acteoside. The results suggest that phenylethanoid glycosides, particularly echinacoside, are active constituents putatively responsible for the anti-aging effects of C. tubulosa and may be considered to develop as non-peptidyl analogues of ghrelin.

Detection of lithospermate B in rat plasma at the nanogram level by LC/MS in multi reaction monitoring mode.

Low bioavailability and high binding affinity to plasma proteins led to the difficulty for the quantitative detection of lithospermate B (LSB) in plasma. This study aimed to develop a protocol for detecting LSB in plasma. A method was employed to quantitatively detect LSB of 5-500 ng/mL by LC/MS spectrometry in multi reaction monitoring mode via monitoring two major fragments with m/z values of 519 and 321 in the MS2 spectrum. To set up an adequate extraction solution to release LSB captured by plasma proteins, recovery yields of LSB extracted from rat plasma acidified by formic acid or HCl in the presence or absence of EDTA and caffeic acid were detected and compared using the above quantitative method. High recovery yield (∼90%) was achieved when LSB (5-500 ng/mL) mixed in rat plasma was acidified by HCl (5 M) in the presence of EDTA (0.5 M) and caffeic acid (400 µg/mL). The lower limit of detection and the lower limit of quantification for LSB in the spiked plasma were calculated to be 1.8 and 5.4 ng/mL, respectively. Good accuracy (within ±10%) and precision (less than 10%) of intra- and inter-day quality controlled samples were observed. Oral bioavailability of LSB in rat model was detected via this optimized extraction method, and the maximum plasma concentration (Cmax) was found to be 1034.3 ± 510.5 µg/L at tmax around 10 min, and the area under the plasma concentration-time curve (AUC) was 1414.1 ± 851.2 µg·h/L.

Characterization of Vasorelaxant Principles from the Needles of Pinus morrisonicola Hayata.

Pinus morrisonicola Hayata, usually called Taiwan five-leaf pine (5LP), is an endemic species in Taiwan and is traditionally used to relieve hypertension symptoms and improve cardiovascular function. In this study, the needle extract of 5LP was fractionated and analyzed by LC/MS/MS to search for possible antihypertensive candidates. In addition, bioassay-guided purification of the bioactive components was performed by Ca2+ fluorescent signal (Fluo 4-AM) assays. Two dihydrobenzofuran lignans, pinumorrisonide A (1) and icariside E4 (2), and one acylated flavonoid glycoside, kaempferol 3-O-α-(6‴-p-coumaroylglucosyl-ß-1,4-rhamnoside) (3) were characterized from the active fractions. The structure of a new compound 1 was established on the basis of 2D NMR spectroscopic and mass spectrometric analyses, and the known compounds 2 and 3 were identified by comparison of their physical and spectroscopic data with those reported in the literature. The purified compounds 1-3 exhibited significant inhibition of Ca2+ fluorescence with IC50 values of 0.71, 0.36, and 0.20 mM, respectively. A mechanism study showed that these compounds showed vasorelaxant effects by blocking the voltage-operated Ca2+ channel (VOCC) and inhibiting Ca2+ influx to the cytoplasmic. These results suggested that 5LP and the three characterized components could be promising antihypertensive candidates for the use as VOCC blockers.

Ginkgoghrelins, unique acylated flavonoid diglycosides in Folium Ginkgo, stimulate growth hormone secretion via activation of the ghrelin receptor.

ETHNOPHARMACOLOGICAL RELEVANCE: Folium Ginkgo, the dried leaf of Ginkgo biloba L. is a traditional Chinese medicine listed in the Pharmacopoeia of the People's Republic of China with several therapeutic effects, including prevention of aging. It is used as herbal medicine for the treatment of several aging-related diseases. The therapeutic effects of Folium Ginkgo on aging-related diseases are suspected to be similar to the anti-aging effects of growth hormone release induced by ghrelin. MATERIALS AND METHODS: Candidate components responsible for the anti-aging effects via the ghrelin receptor-activated pathway were searched from the known compounds found in Folium Ginkgo. Two acylated flavonoid diglycosides, tentatively named ginkgoghrelins in this study, were selected and isolated from the methanol extract of Folium Ginkgo, and their chemical structures were confirmed by spectroscopic analysis. These two compounds were examined for their capability of stimulating growth hormone release of rat primary anterior pituitary cells via activation of the ghrelin receptor. The major metabolites of ginkgoghrelins in rat bile were detected after intravenous injection and structurally analyzed by mass spectroscopy. Molecular modeling of ginkgoghrelins docking to the ghrelin receptor was exhibited to explore the possible interaction within the binding pocket. RESULTS: Similar to growth hormone-releasing hormone-6 (GHRP-6), a synthetic analog of ghrelin, ginkgoghrelins were demonstrated to stimulate growth hormone secretion of rat primary anterior pituitary cells in a dose dependent manner, and the stimulation was inhibited by [d-Arg1, d-Phe5, d-Trp7,9, Leu11]-substance P, an inverse agonist of the ghrelin receptor. Putative metabolites of ginkgoghrelins via glucuronidation and methylation were detected in bile of rats after intravenous injection. Molecular modeling and docking showed that ginkgoghrelins as well as GHRP-6 could fit in and adequately interact with the binding pocket of the ghrelin receptor. CONCLUSION: The results suggest that ginkgoghrelins are putative components partly accounting for the anti-aging effects of Folium Ginkgo possibly via activation of the ghrelin receptor, and possess great potential to be developed as non-peptidyl analogs of ghrelin.

Antibacterial and laxative activities of strictinin isolated from Pu'er tea (Camellia sinensis).

Strictinin, the major phenolic compound in Pu'er teas produced from young leaves and buds of wild trees, was isolated to evaluate its antibacterial and laxative activities. The minimum inhibitory concentrations of strictinin against Propionibacterium acnes and Staphylococcus epidermidis were determined as 250 µM and 2000 µM, respectively, apparently higher than those of several antibiotics commonly used for bacterial infections. The additive and synergistic effects on the inhibitory activities of strictinin combined with other commercial antibiotics were observed in two bacteria tested in this study via the analysis of fractional inhibitory concentrations. Laxative activity was observed on defecation of the rats fed with strictinin. Further analysis showed that the laxative effect of strictinin was presumably caused by accelerating small intestinal transit, instead of enhancing gastric emptying, increasing food intake, or inducing diarrhea in the rats. Taken together with the antiviral activities demonstrated previously, it is suggested that strictinin is one of the active ingredients responsible for the antiviral, antibacterial, and laxative effects of wild Pu'er tea, and has the potential to be developed as a mild natural substitute for antibiotics and laxatives.

Emoghrelin, a unique emodin derivative in Heshouwu, stimulates growth hormone secretion via activation of the ghrelin receptor.

ETHNOPHARMACOLOGICAL RELEVANCE: Heshouwu, the root of Polygonum multiflorum, is an anti-aging Chinese traditional medicine. Fresh (raw) Heshouwu is commonly converted to processed Heshouwu by specialized heating to alleviate its side effects of diarrhea presumably caused by anthraquinones. However, raw Heshouwu has been noted to be better than processed Heshouwu regarding anti-aging effects. The therapeutic effects of raw Heshouwu on aging-related diseases were somehow similar to the anti-aging effects of growth hormone release induced by ghrelin MATERIALS AND METHODS: Major ingredients in the methanol extract from raw Heshouwu were separated and identified. Emodin-8-O-(6'-O-malonyl)-glucoside, a unique anthraquinone glycoside known to be completely eliminated in the conversion process of Heshouwu was isolated. This emodin derivative, tentatively named emoghrelin, was examined for its cytotoxicity and capability of stimulating growth hormone release of rat primary anterior pituitary cells via activation of the ghrelin receptor. Moreover, molecular modeling of emoghrelin docking to the ghrelin receptor was exhibited to explore the possible interaction within the binding pocket. RESULTS: No apparent cytotoxicity was observed for emoghrelin of 10(-7)-10(-4)M. Similar to growth hormone-releasing hormone-6 (GHRP-6), a synthetic analog of ghrelin, emoghrelin was demonstrated to stimulate growth hormone secretion of rat primary anterior pituitary cells in a dose dependent manner, and the stimulation was inhibited by [d-Arg(1), d-Phe(5), d-Trp(7,9), Leu(11)]-substance P, an antagonist of the ghrelin receptor. Molecular modeling and docking showed that emoghrelin as well as GHRP-6 could fit in and adequately interact with the binding pocket of the ghrelin receptor. CONCLUSION: The results suggest that emoghrelin is a key ingredient accounting for the anti-aging effects of Heshouwu, and possesses great potential to be a promising non-peptidyl analog of ghrelin.

Enhancing the potency of lithospermate B for inhibiting Na+/K+-ATPase activity by forming transition metal ion complexes.

AIM: To determine whether replacing Mg(2+) in magnesium lithospermate B (Mg-LSB) isolated from danshen (Salvia miltiorrhiza) with other metal ions could affect its potency in inhibition of Na(+)/K(+)-ATPase activity. METHODS: Eight metal ions (Na(+), K(+), Mg(2+), Cr(3+), Mn(2+), Co(2+), Ni(2+), and Zn(2+)) were used to form complexes with LSB. The activity of Na(+)/K(+)-ATPase was determined by measuring the amount of inorganic phosphate (Pi) liberated from ATP. Human adrenergic neuroblastoma cell line SH-SY5Y was used to assess the intracellular Ca(2+) level fluctuation and cell viability. The metal binding site on LSB and the binding mode of the metal-LSB complexes were detected by NMR and visible spectroscopy, respectively. RESULTS: The potencies of LSB complexed with Cr(3+), Mn(2+), Co(2+), or Ni(2+) increased by approximately 5 times compared to the naturally occurring LSB and Mg-LSB. The IC50 values of Cr-LSB, Mn-LSB, Co-LSB, Ni-LSB, LSB, and Mg-LSB in inhibition of Na(+)/K(+)-ATPase activity were 23, 17, 26, 25, 101, and 128 µmol/L, respectively. After treatment of SH-SY5Y cells with the transition metal-LSB complexes (25 µmol/L), the intracellular Ca(2+) level was substantially elevated, and the cells were viable for one day. The transition metals, as exemplified by Co(2+), appeared to be coordinated by two carboxylate groups and one carbonyl group of LSB. Titration of LSB against Co(2+) demonstrated that the Co-LSB complex was formed with a Co(2+):LSB molar ratio of 1:2 or 1:1, when [Co(2+)] was less than half of the [LSB] or higher than the [LSB], respectively. CONCLUSION: LSB complexed with Cr(3+), Mn(2+), Co(2+), or Ni(2+) are stable, non-toxic and more potent in inhibition of Na(+)/K(+)-ATPase. The transition metal-LSB complexes have the potential to be superior substitutes for cardiac glycosides in the treatment of congestive heart failure.

Active compounds in Chinese herbs and medicinal animal products which promote blood circulation via inhibition of Na+, K+-ATPase.

The therapeutic effect of cardiac glycosides for congestive heart failure lies in their reversible inhibition on Na+, K+-ATPase located in human myocardium. Several steroid-like compounds containing a core structure similar to cardiac glycosides have been found in many Chinese herbs and medicinal animal products conventionally used to promote blood circulation. They are putatively responsible for the therapeutic effect of those medicinal products via the same mechanism of inhibiting Na+, K+-ATPase. Inhibitory potency on Na+, K+-ATPase by ginsenosides, one of the identified steroid-like compounds, is significantly affected by sugar attachment that might cause steric hindrance of their binding to Na+, K+-ATPase. Ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure, equivalent to the sugar position in cardiac glycosides, substantially inhibit Na+, K+-ATPase. However, their inhibitory potency is abolished when sugar moieties are linked to the C-6 or C-20 position of the steroid-like structure. In contrast, no appreciable contents of steroid-like compounds are found in danshen, a well-known Chinese herb traditionally regarded as an effective medicine promoting blood circulation. Instead, magnesium lithospermate B (MLB), the major soluble ingredient in danshen, is assumed to be responsible for the therapeutic effect by inhibiting Na+, K+-ATPase in a manner comparable to cardiac glycosides. Neuroprotective effects of cardiac glycosides, ginsenosides and MLB against ischemic stroke were accordingly observed in a cortical brain slice-based assay model. Whether the neuroprotection is also triggered by inhibition of Na+, K+-ATPase remains to be investigated. Molecular modeling suggests that cardiac glycosides, ginsenosides and MLB presumably bind to the same extracellular pocket of the Na+, K+-ATPase alpha subunit.

Effect of sugar positions in ginsenosides and their inhibitory potency on Na+/K+-ATPase activity.

AIM: To determine whether ginsenosides with various sugar attachments may act as active components responsible for the cardiac therapeutic effects of ginseng and sanqi (the roots of Panax ginseng and Panax notoginseng) via the same molecular mechanism triggered by cardiac glycosides, such as ouabain and digoxin. METHODS: The structural similarity between ginsenosides and ouabain was analyzed. The inhibitory potency of ginsenosides and ouabain on Na+/K+-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of ginsenosides to Na+/K+-ATPase. RESULTS: Ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure, equivalent to the sugar position in cardiac glycosides, and possessed inhibitory potency on Na+/K+-ATPase activity. However, their inhibitory potency was significantly reduced or completely abolished when a monosaccharide was linked to the C-6 or C-20 position of the steroid-like structure; replacement of the monosaccharide with a disaccharide molecule at either of these positions caused the disappearance of the inhibitory potency. Molecular modeling and docking confirmed that the difference in Na+/K+-ATPase inhibitory potency among ginsenosides was due to the steric hindrance of sugar attachment at the C-6 and C-20 positions of the steroid-like structure. CONCLUSION: The cardiac therapeutic effects of ginseng and sanqi should be at least partly attributed to the effective inhibition of Na+/K+-ATPase by their metabolized ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure.