Richard Bogren, Liu, Zhijun | 12/17/2009 9:06:54 PM
For more than 15 years, Dr. Zhijun Liu with the LSU AgCenter’s School of Renewable Natural Resources has been investigating plants for medicinal properties. He started by looking at plants that traditionally have been used as folk remedies to treat diseases such as hypertension, diabetes and cancer.
After cancers are treated by surgery, chemotherapy or radiation therapy, the next task is to prevent them from recurring. Remnant cancer cells that survive the initial treatment or have metastasized can still grow when the environment is favorable. But first, they must make new blood vessels.
Preventing cancer recurrence requires long-term management using a regimen that is safe with no or minimal adverse effects. Current methods of preventing cancer regrowth are often highly toxic at effective doses, so researchers are looking for agents that inhibit angiogenesis – the process by which new blood vessels initiate and grow.
For the past 10 years, Liu’s focus has been on angiogenesis inhibitors, hoping to find compounds that will prevent the growth of blood vessels and can be used to treat cancers and other maladies, such as obesity and psoriasis. While the pharmaceutical industry searches for single-entity drugs that target angiogenesis, Liu and other researchers are searching for anti-angiogenic agents from botanical sources.
“Inhibiting angiogenesis can prevent cancer – and perhaps even fat tissue – from developing beyond the simple limits of existing blood vessels,” Liu says. But he admits turning these discoveries into practical therapies presents major obstacles, which are typical in the use of botanical extracts for health care and disease treatments. “To be effective, the active ingredients must be concentrated enough to achieve a therapeutic effect,” he says.
Liu and his colleagues have identified a number of such botanical agents. One that has shown the greatest promise is Chinese sweet leaf tea. In research studies, the scientists have found that sweet leaf tea water extract at a dose of 0.45 grams per pound of body weight completely blocked blood-vessel initiation and growth in a human tissue-based angiogenesis test.
In his search for plant-based therapeutics, Liu first screens plant extracts by fractionating them – dividing the material into smaller segments. Then, he further subdivides the fraction that holds the most promise for delivering an effective compound. Finally, he purifies the fraction to produce an effective concentration of the compound.
“We use bioassay-directed isolation to trace down the molecules responsible, then expand to similar chemical structures,” Liu says.
Through laborious isolation, Liu identified gallic acid as a contributor to angiogenesis inhibition. Then he began looking for derivatives – analogs of gallic acid from other sources. With Chinese sweet tea, for example, the extract did a better job of inhibiting angiogenesis than gallic acid alone.
Native to the Guangxi-Guizhou regions of southwestern China, sweet leaf tea for years has been a beverage, mainly for its naturally sweet taste said to be 300 times sweeter than cane sugar. This human-use history, plus the scientific discovery of its anti-angiogenic property, prompted a multi-institutional investigation led by the LSU AgCenter and supported by the National Center for Complementary and Alternative Medicine of the National Institutes of Health. The ongoing two-year project focuses on pre-clinical evaluation of the sweet leaf tea extract, with the goal of spinning it off into human clinical investigations as a cancer-preventative agent.
“Based on bioactivity, we determined that the effect was not because of a single molecule,” Liu says. “Three different chemicals in an appropriate proportion behave synergistically. This finding is extremely encouraging because it is now possible to re-compose the three synergistic molecules into a new formulation that is intellectually protectable, better quality-controlled and lower in effective human dose among other benefits.”
Liu believes he and his colleagues – Eugene A. Woltering, who is the James D. Rives Professor of Surgery and Neurosciences at the LSU Health Sciences Center in New Orleans, and Peiying Yang, assistant professor in the Department of General Oncology at the University of Texas M. D. Anderson Cancer Center in Houston – are on the threshold of a breakthrough.
“I used to start with the plant and not stop until I found the molecule,” Liu says. “Now, I realize it may not be one compound but several compounds. My interest now is to see if they’re synergistic. That’s the beauty of natural plants – they produce synergistic compounds.”
Although the enormous advantages of using safe botanical extracts as cancer-preventative agents are yet to unfold, the greatest challenge is to standardize an extract so batch-to-batch variations are measured and controlled. “This is especially challenging given the natural variations of botanical materials and the nature of mixed compounds in a single-leaf extract compared to single-entity drugs, such as aspirin,” Liu says.
“Without quality control, botanical extracts often lack the reproducible results of clinical efficacy,” he adds. “Our investigation placed great emphasis on achieving standardization – using multiple chemical markers that have proven combined anti-angiogenic activity.”
Liu’s laboratory team has developed a chemical fingerprinting analysis to assess the similarity of overall chemical constituents among different batches against a “standard extract” with a defined efficacy. Any batch that falls outside 90 percent similarity to this standard extract is disqualified.
The researchers have found one fingerprint chromatogram that monitors the quality profile of the sweet leaf tea extract. They have conducted laboratory tests with a variety of human tumors and found the extract repeatedly and consistently inhibits the initiation and growth of new blood vessels regardless of each tumor’s angiogenic potential.
“This is very encouraging because it shows efficacy against solid tumors of various types in which growth or recurrence depends on new blood vessel formation,” Liu says. A positive anti-tumor efficacy result using appropriate animal models is a “gold standard” for beginning human clinical trials.
In a preliminary study with mice highly susceptible to infection, two groups 25-39 days old were injected with human breast cancer cells to develop a tumor. Immediately after the injection, one group of mice received the standardized sweet leaf tea extract via tubes to the stomach – a process called oral gavage – and a control group received only water. The result showed tumor volume continued to increase in the control group but remained constant in the treated group starting the fourth day after tumor growth began.
“This encouraging finding is a proof of concept that the sweet leaf tea extract is orally active in stopping tumor growth, possibly by inhibiting angiogenesis that deprives the implanted cancer cells from accessing blood vessel networks to feed their growth,” Liu says.
Liu and his colleagues are continuing animal studies to determine the minimal effective dose in a multiple-dose study and variations in pancreatic cancer. They’re also planning a toxicity and safety study of the standardized sweet leaf tea to define a safe dose range.
“However, all indications are that it is safe for short-term use and predicted safe in longer-term use as a cancer-preventative agent,” he says.
Liu’s colleagues also have conducted studies to investigate the mechanisms of action. They used endothelial cells – the cells that become angiogenic to produce new blood vessels – from human umbilical veins and found that the sweet leaf tea extract markedly inhibited the expression of a basic growth factor that stimulates and promotes angiogenesis. And the suppression depended on the concentration of the extract.
“These results suggest that the observed anti-angiogenic property of sweet leaf tea works by inhibiting receptors on the cell rather than the growth factors themselves,” Liu says. “Moreover, the sweet leaf tea extract has the ability to slow the growth of endothelial cells and disrupt blood vessel formation.”
In addition to their work with the extract, the researchers are trying to determine if and how the multiple constituents of the sweet leaf tea extract combine and work together to produce the anti-angiogenic effect.