In this study, we aimed to identify the bioactive components in and to investigate their stability.


HPLC ngerprinting and stress test: storage at 40°C for 0, 1.5 and 3 months followed by the measurement of bioactive compounds.


Determination of total polyphenols as gallic acid by photo-spectrometry (optimised Folin-Ciocalteu method) and de- termination of 3,4,5-tri-O-galloylquinic acid by HPLC.


High content of polyphenols: polyphenols account for about 40 % of the dry mass. The abso- lute concentration was approximately 13,000 mg/l. The poly- phenol fraction was very stable over time: In a stress test (40°C), no more than 4 % and 10 % were degraded after 1.5 and 3 months, respectively (Figure 10).

3,4,5-tri-O-galloylquinic acid also was present in large quantities and extremely stable: it accounted for about 3 % of the dry mass. In a stress test at 40°C no degradation took place within 3 months (Figure 10).

We also found a high level of trehalose (approx. 5 % of dry weight), which is thought to act as a protein-shielding sub- stance, enabling enzymes and other proteins to stay intact and functional during long-term dehydration (not shown).

Qualitative and quantitative analysis of a typical HPLC ngerprint of showing a wide variety of polyphenols such as arbutin, gallic acid, and, most important, 3,4,5-tri-O-galloylquinic acid together with its higher molecular weight derivatives (having a higher degree of galloylation). Right: the phenolic compounds and the 3,4,5-tri-O-galloylquinic acid in  remained very stable over time. Quantitative analysis at di erent time points upon storage at 40°C is shown.


“Magical state” of embryonic stem cells may help overcome hurdles to therapeutics

June 13, 2012

“Magical state” of embryonic stem cells may help overcome hurdles to therapeutics

Salk researcher’s findings suggest a potentially favorable time to harvest stem cells for therapy and may reveal genes crucial to tissue production

LA JOLLA, CA—With their potential to treat a wide range of diseases and uncover fundamental processes that lead to those diseases, embryonic stem (ES) cells hold great promise for biomedical science. A number of hurdles, both scientific and non-scientific, however, have precluded scientists from reaching the holy grail of using these special cells to treat heart disease, diabetes, Alzheimer’s and other diseases.

In a paper published June 13 in Nature, scientists at the Salk Institute for Biological Studies report discovering that ES cells cycle in and out of a “magical state” in the early stages of embryo development, during which a battery of genes essential for cell potency (the ability of a generic cell to differentiate, or develop, into a cell with specialized functions) is activated. This unique condition, called totipotency, gives ES cells their unique ability to turn into any cell type in the body, thus making them attractive therapeutic targets.

red fluorescent reporter moleculesThe Salk researchers found that embryonic stem cells cycle in and out of a state from which they can develop into any kind of tissue. Here, red fluorescent “reporter” molecules indicate that these early embryonic cells are exhibiting genetic activity indicative of this flexible state.

Image: Courtesy of the Salk Institute for Biological Studies

“These findings,” says senior author Samuel L. Pfaff, a professor in Salk’s Gene Expression Laboratory, “give new insight into the network of genes important to the developmental potential of cells. We’ve identified a mechanism that resets embryonic stem cells to a more youthful state, where they are more plastic and therefore potentially more useful in therapeutics against disease, injury and aging.”

ES cells are like silly putty that can be induced, under the right circumstances, to become specialized cells-for example, skin cells or pancreatic cells-in the body. In the initial stages of development, when an embryo contains as few as five to eight cells, the stem cells are totipotent and can develop into any cell type. After three to five days, the embryo develops into a ball of cells called a blastocyst. At this stage, the stem cells are pluripotent, meaning they can develop into almost any cell type. In order for cells to differentiate, specific genes within the cells must be turned on.

Pfaff and his colleagues performed RNA sequencing (a new technology derived from genome-sequencing to monitor what genes are active) on immature mouse egg cells, called oocytes, and two-cell-stage embryos to identify genes that are turned on just prior to and immediately following fertilization. Pfaff’s team discovered a sequence of genes tied to this privileged state of totipotency and noticed that the genes were activated by retroviruses adjacent to the stem cells.

Nearly 8 percent of the human genome is made up of ancient relics of viral infections that occurred in our ancestors, which have been passed from generation to generation but are unable to produce infections. Pfaff and his collaborators found that cells have used some of these viruses as a tool to regulate the on-off switches for their own genes. “Evolution has said, ‘We’ll make lemonade out of lemons, and use these viruses to our advantage,’” Pfaff says. Using the remains of ancient viruses to turn on hundreds of genes at a specific moment of time in early embryo development gives cells the ability to turn into any type of tissue in the body.

From their observations, the Salk scientists say these viruses are very tightly controlled-they don’t know why-and active only during a short window during embryonic development. The researchers identified ES cells in early embryogenesis and then further developed the embryos and cultured them in a laboratory dish. They found that a rare group of special ES cells activated the viral genes, distinguishing them from other ES cells in the dish. By using the retroviruses to their advantage, Pfaff says, these rare cells reverted to a more plastic, youthful state and thus had greater developmental potential.

Pfaff’s team also discovered that nearly all ES cells cycle in and out of this privileged form, a feature of ES cells that has been underappreciated by the scientific community, says first author Todd S. Macfarlan, a former postdoctoral researcher in Pfaff’s lab who recently accepted a faculty position at the Eunice Kennedy Shriver National Institute of Child Health and Human Development. “If this cycle is prevented from happening,” he says, “the full range of cell potential seems to be limited.”

It is too early to tell if this “magical state” is an opportune time to harvest ES cells for therapeutic purposes. But, Pfaff adds, by forcing cells into this privileged status, scientists might be able to identify genes to assist in expanding the types of tissue that can be produced.

“There’s tremendous hype over the practical applications of embryonic stem cells in clinical situations,” he says. “The struggle in labs throughout the world is that the smallest changes in environmental conditions could subtly and unpredictably have an effect on these cells. So, the more we know about the basic requirements needed for these cells to be able to generate a full range of tissue types, the better off we’ll be.” While the findings shed light on the basic biology of embryonic stem cells, Pfaff says there is still a “long way to go” in terms of their practical, clinical value.

Other researchers on the study were Wesley D. Gifford, Shawn Driscoll, Karen Lettieri, Dario Bonanomi, Amy Firth, and Oded Singer, from the Salk Institute; and Helen M. Rowe and Didier Trono of Ecole Polytechnique Federale de Lausanne in Switzerland.

The work was supported by the National Institute of Neurological Disorders and Stroke (R37NS037116), the Howard Hughes Medical Instituteand the Marshall Heritage Foundation.
About the Salk Institute for Biological Studies:
The Salk Institute for Biological Studies is one of the world’s preeminent basic research institutions, where internationally renowned faculty probe fundamental life science questions in a unique, collaborative, and creative environment. Focused both on discovery and on mentoring future generations of researchers, Salk scientists make groundbreaking contributions to our understanding of cancer, aging, Alzheimer’s, diabetes and infectious diseases by studying neuroscience, genetics, cell and plant biology, and related disciplines.

Faculty achievements have been recognized with numerous honors, including Nobel Prizes and memberships in the National Academy of Sciences. Founded in 1960 by polio vaccine pioneer Jonas Salk, M.D., the Institute is an independent nonprofit organization and architectural landmark.





Todd S. Macfarlan, Wesley D. Gifford, Shawn Driscoll, Karen Lettieri, Helen M. Rowe, Dario Bonanomi, Amy Firth, Oded Singer, Didier Trono and Samuel L. Pfaff

New Technique to Study How Proteins and Ligands Interact

New Technique to Study How Proteins and Ligands Interact

ImageA new imaging method, called Transient Induced Molecular Electronic Spectroscopy (TIMES), allows researchers to investigate protein-ligand interactions without introducing disturbances to their binding. Credit: Tiantian Zhang, Tao Wei, Yuanyuan Han, Heng Ma, Mohammadreza Samieegohar, Ping-Wei Chen, Ian Lian, and Yu-Hwa Lo

A team of researchers has developed a more accurate and less disruptive method to study how proteins and the small molecules that bind to them, known as ligands, interact. The method, called Transient Induced Molecular Electronic Spectroscopy (TIMES), could be used as a tool to better understand protein chemistry and to accelerate drug discovery and development.

“This new tool will enable researchers to investigate a larger variety of chemical and biochemical reactions than was previously possible,” said Yu-Hwa Lo, electrical engineering professor at the University of California San Diego and senior author of the study. The work, a collaboration between UC San Diego and Lamar University in Texas, was published recently in the journal ACS Central Science.

Protein-ligand interactions are of particular interest to biopharmaceutical researchers because of their applications in drug discovery. During the early stage of drug discovery, researchers screen millions of small molecule drug candidates, such as ligands, in order to find the ones that can most effectively combine with a target protein.

But one of the biggest challenges in early drug discovery is that there is currently no good way to detect protein-ligand interactions without requiring chemical or structural modifications, Lo explained. These modifications can affect protein-ligand binding, potentially skewing the measurement, he said.

Lo and colleagues developed TIMES, a new high-resolution imaging technique that can accurately characterize protein-ligand interactions without perturbing their binding. In the TIMES technique, proteins, ligands and protein-ligand complexes flow through a device containing an electrode surface connected to a low-noise electric amplifier. As molecules approach the electrode surface, electric signals are produced. The signals can be analyzed to determine exactly how proteins and ligands bind.

Using TIMES, researchers were able to accurately measure the interactions between a protein called lysozyme with a ligand called N-acetyl-D-glucosamine (NAG) and its trimer, NAG3. While NAG and NAG3 are relatively similar in size and chemical makeup, experiments showed that NAG3 had almost a thousand-fold greater binding affinity to lysozyme than NAG, which agreed well with results published in the literature.

“That’s another advantage of this method. It can actually differentiate the degree of binding — for example, what percentage improvement in binding — between similar molecules. Current methods typically just tell you whether or not binding occurs,” Lo said.

An area of improvement with the TIMES method involves increasing the sensitivity of the technique. “We’re working towards being able to do single molecule detection with this method,” Lo said.

Full paper: “Protein-Ligand Interaction Detection with a Novel Method of Transient Induced Molecular Electronic Spectroscopy (TIMES): Experimental and Theoretical Studies.” Authors of the study are: Tiantian Zhang, Yuanyuan Han, Ping-Wei Chen and Yu-Hwa Lo of UC San Diego; and Tao Wei, Heng Ma, Mohammadreza Samieegohar and Ian Lian of Lamar University.

This research is funded in part by the National Science Foundation (grant ECCS-1610516) and Vertex Pharmaceuticals, Inc.