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

Active ingredients in Chinese medicines promoting blood circulation as Na+/K+-ATPase inhibitors

The positive inotropic effect of cardiac glycosides lies in their reversible inhibition on the membrane-bound Na+/K+-ATPase in human myocardium. Steroid-like compounds containing a core structure similar to cardiac glycosides are found in many Chinese medicines conventionally used for promoting blood circulation. Some of them are demonstrated to be Na+/K+-ATPase inhibitors and thus putatively responsible for their therapeutic effects via the same molecular mechanism as cardiac glycosides. On the other hand, magnesium lithospermate B of danshen is also proposed to exert its cardiac therapeutic effect by effectively inhibiting Na+/K+-ATPase. Theoretical modeling suggests that the number of hydrogen bonds and the strength of hydrophobic interaction between the effective ingredients of various medicines and residues around the binding pocket of Na+/K+-ATPase are crucial for the inhibitory potency of these active ingredients. Ginsenosides, the active ingredients in ginseng and sanqi, substantially inhibit Na+/K+-ATPase when sugar moieties are attached only to the C-3 position of their steroid-like structure, equivalent to the sugar position in cardiac glycosides. Their inhibitory potency is abolished, however, when sugar moieties are linked to C-6 or C-20 position of the steroid nucleus; presumably, these sugar attachments lead to steric hindrance for the entrance of ginsenosides into the binding pocket of Na+/K+-ATPase. Neuroprotective effects of cardiac glycosides, several steroid-like compounds, and magnesium lithospermate B against ischemic stroke have been accordingly observed in a cortical brain slice-based assay model, and cumulative data support that effective inhibitors of Na+/K+-ATPase in the brain could be potential drugs for the treatment of ischemic stroke

Steroid-like compounds in Chinese medicines promote blood circulation via inhibition of Na+/K+-ATPase

Aim: To examine if steroid-like compounds found in many Chinese medicinal products conventionally used for the promotion of blood circulation may act as active components via the same molecular mechanism triggered by cardiac glycosides, such as ouabain. Methods: The inhibitory potency of ouabain and the identified steroid-like compounds on Na+/K+-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of these compounds to Na+/K+-ATPase. Results: All the examined steroid-like compounds displayed more or less inhibition on Na+/K+-ATPase, with bufalin (structurally almost equivalent to ouabain) exhibiting significantly higher inhibitory potency than the others. In the pentacyclic triterpenoids examined, ursolic acid and oleanolic acid were moderate inhibitors of Na+/K+-ATPase, and their inhibitory potency was comparable to that of ginsenoside Rh2. The relatively high inhibitory potency of ursolic acid or oleanolic acid was due to the formation of a hydrogen bond between its carboxyl group and the Ile322 residue in the deep cavity close to two K+ binding sites of Na+/K+-ATPase. Moreover, the drastic difference observed in the inhibitory potency of ouabain, bufalin, ginsenoside Rh2, and pentacyclic triterpenoids is ascribed mainly to the number of hydrogen bonds and partially to the strength of hydrophobic interaction between the compounds and residues around the deep cavity of Na+/K+-ATPase. Conclusion: Steroid-like compounds seem to contribute to therapeutic effects of many cardioactive Chinese medicinal products. Chinese herbs, such as Prunella vulgaris L, rich in ursolic acid, oleanolic acid and their glycoside derivatives may be adequate sources for cardiac therapy via effective inhibition on Na+/K+-ATPase

Acid-sensing ion channel 3 mediates peripheral anti-hyperalgesia effects of acupuncture in mice inflammatory pain

Background: Peripheral tissue inflammation initiates hyperalgesia accompanied by tissue acidosis, nociceptor activation, and inflammation mediators. Recent studies have suggested a significantly increased expression of acid-sensing ion channel 3 (ASIC3) in both carrageenan- and complete Freund's adjuvant (CFA)-induced inflammation. This study tested the hypothesis that acupuncture is curative for mechanical hyperalgesia induced by peripheral inflammation. Methods: Here we used mechanical stimuli to assess behavioral responses in paw and muscle inflammation induced by carrageenan or CFA. We also used immunohistochemistry staining and western blot methodology to evaluate the expression of ASIC3 in dorsal root ganglion (DRG) neurons. Results: In comparison with the control, the inflammation group showed significant mechanical hyperalgesia with both intraplantar carrageenan and CFA-induced inflammation. Interestingly, both carrageenan- and CFA-induced hyperalgesia were accompanied by ASIC3 up-regulation in DRG neurons. Furthermore, electroacupuncture (EA) at the ST36 rescued mechanical hyperalgesia through down-regulation of ASIC3 overexpression in both carrageenan- and CFA-induced inflammation. Conclusions: In addition, electrical stimulation at the ST36 acupoint can relieve mechanical hyperalgesia by attenuating ASIC3 overexpression

Effect of aristolochic acid on intracellular calcium concentration and its links with apoptosis in renal tubular cells

Aristolochic acid (AA) has been demonstrated to play a causal role in Chinese herbs nephropathy. However, the detailed mechanism for AA to induce apoptosis of renal tubular cells remains obscure. In this study, we show that AA evokes a rapid rise in the intracellular Ca2+ concentration of renal tubular cells through release of intracellular endoplasmic reticulum Ca2+ stores and influx of extracellular Ca2+, which in turn causes endoplasmic reticulum stress and mitochondria stress, resulting in activation of caspases and finally apoptosis. Ca2+ antagonists, including calbindin-D-28k (an intracellular Ca2+ buffering protein) and BAPTA-AM (a cell-permeable Ca2+ chelator), are capable of ameliorating endoplasmic reticulum stress and mitochondria stress, and thereby enhance the resistance of the cells to AA. Moreover, we show that overexpression of the anti-apoptotic protein Bcl-2 in combination with BAPTA-AM treatment can provide renal tubular cells with almost full protection against AA-induced cytotoxicity. In conclusion, our results demonstrate an impact of AA to intracellular Ca2+ concentration and its link with AA-induced cytotoxicity

Ubiquitination of oleosin-H and caleosin in sesame oil bodies after seed germination

Sesame (Sesamum indicum L) seed oil bodies are composed of triacylglycerols encapsulated by a monolayer of phospholipids embedded with three classes of proteins, oleosin, caleosin and steroleosin. Among proteins extracted from sesame oil bodies after germination, laddering bands higher than the original antigens were recognized by antibodies against oleosin-H (17 kDa) and caleosin (27 kDa), but not those against oleosin-L (15 kDa), steroleosin-A (39 kDa) and steroleosin-B (41 kDa). Regardless the original antigens, the lowest but relatively abundant laddering band (32 kDa) detected by antibodies against oleosin-H and that (42 kDa) detected by antibodies against caleosin were eluted from SOS-PAGE gels, and then subjected to mass spectrometric analyses. The results showed that the 32 kDa and 42 kDa bands were ubiquitinated oleosin-H and caleosin, respectively. The ubiquitination was further confirmed by immunological detection using antibodies against ubiquitin. Ubiquitination sites were found at three lysine residues (130, 143 and 145) of oleosin-H and two lysine residues (165 and 235) of caleosin. Two ubiquitination sites of oleosin-H, Lys(143) and Lys(145), were located in the extra 18-residue segment found only in oleosin-H, but not oleosin-L isoforms. (C) 2010 Elsevier Masson SAS. All rights reserved

KALIMATAR (VICIA FABA L.) COOKING CHARACTERISTICS AS AFFECTED BY SOAKING AND ROASTING

Kalimatar seeds were evaluated for their physical characteristics and cooking qualities. Physical characteristics of Kalimatar seeds measured in millimeters per seed were as follows: length 6.65 +/- 0.11; breadth 4.76 +/- 0.09; and thickness 4.49 +/- 0.07. Cooking qualities of the seeds were evaluated as follows: 1,000-seed weight 199.95 +/- 0.34 g; 1,000-seed volume 152.0 +/- 0.58 mL; seed density 1.27 +/- 0.02 g/mL; hydration capacity 1386.1 +/- 2.63 g/kg; and swelling capacity 1493.4 +/- 3.82 mL/L. The cooking time of untreated Kalimatar seed was 187 +/- 3.15 min; soaking the seeds in different media for 12 h reduced the cooking time considerably. Sodium carbonate solution (2%) was found as the most suitable soaking medium, particularly followed by roasting the seeds. Soaking and/or roasting did not cause pronounced reduction in nutrient content of the sample. PRACTICAL APPLICATIONS Long cooking time severely restricts the utilization of Kalimatar, a protein-rich source. This study aims to evaluate the effect of soaking and/or roasting on the cooking time of Kalimatar seed to find out a feasible way out of the problem. We found that soaking and quick cooking system considerably reduces the cooking time of Kalimatar without pronounced nutrient loss. Both methods can be adopted very successfully on a household scale as well as at industrial level. However, quick cooking method is much better than soaking system. Again, quick cooking seeds are suitable for preservation. Moreover, this method improves the nutritional quality and palatability of the seeds

Analysis of the three essential constituents of oil bodies in developing sesame seeds

Oil bodies of plant seeds contain a matrix of triacylglycerols surrounded by a monolayer of phospholipids embedded with alkaline proteins termed oleosins. Triacylglycerols and two oleosin isoforms of 17 and 15 kDa were exclusively accumulated in oil bodies of developing sesame seeds, During seed development, 17 kDa oleosin emerged later than 15 kDa oleosin, but it was subsequently found to be the most abundant protein in mature oil bodies. Phosphotidylcholine, the major phospholipid in oil bodies, was amassed in microsomes during the formation of oil bodies, Prior to the formation of these oil bodies, a few oil droplets of smaller size were observed both in vivo and in vitro. These oil droplets were unstable, presumably due to the lack of steric hindrance shielded by the oleosins. The temporary maintenance of these droplets as small entities seemed to be achieved by phospholipids, presumably wrapped in ER, Oil bodies assembled in late developing stages possessed a higher ratio of oleosin 17 kDa over oleosin 15 kDa and were utilized earlier during germination. It seems that the proportion of oleosin 17 kDa on the surface of oil bodies is related to the priority of their utilization

Two distinct steroleosins are present in seed oil bodies

In addition to oleosin isoforms, three minor proteins, Sop1, 2 and 3 are present in sesame oil bodies. Genes encoding Sop1 and Sop2, named caleosin and steroleosin for their calcium and sterol-binding capacity, respectively, have been cloned recently. Blast sequence analysis of the first 32 N-terminal residues revealed that Sop3 was presumably a steroleosin-like protein homologous to Sop2. A putative cDNA clone of Sop3 was obtained by PCR, and Subsequently confirmed by immunological recognition with antibodies against its over-expressed protein in Escherichia coli. Although Sop2 and Sop3, tentatively named steroleosin-A and -B, were found homologous, they could not be cross-recognized immunologically. Sequence comparison showed that these two steroleosins possessed a conserved NADP(+) binding subdomain but a diverse sterol-binding subdomain of different size. Both steroleosins were progressively accumulated in maturing seeds but with different cumulating patterns. Dehydrogenase activity detected in their expressed proteins indicated that steroleosin-B might comparably possess a broader sterol selectivity and higher NADP(+) specificity than steroleosin-A. Immunological cross-recognition implies that steroleosin-B is present in seed oil bodies of diverse species. A structural model of an oil-body was drawn with all its known essential constituents, and secondary structure organizations of the three classes of oil-body proteins were compared. (C) 2004 Elsevier SAS. All rights reserved