Breakthrough method gives ‘snapshot’ of a plant’s metabolic effects

Scientists developed a new way to help understand what happens in the body when people consume a plant product and the many chemicals it contains. The American Chemical Society’s Journal of Natural Products published the method to quickly analyze the effects of a natural product, developed at Emory University.

As a test case, the paper focused on biotransformation of chemicals from the kratom plant by human liver cells in a laboratory dish. The researchers developed an automated method — based on high-resolution mass spectrometry and molecular network mapping — to gain a detailed, big-picture view of the resulting metabolites, or chemicals produced.

The new, streamlined methodology can be broadly applied to nutrition and dietary supplement research, filling a critical gap in the field.

“Plants evolved extraordinarily complex chemical defenses and signaling systems,” says Cassandra Quave, co-senior author of the study and professor of dermatology at Emory School of Medicine and the Center for the Study of Human Health. “Our new approach in molecular mapping gives us a way to follow how that chemical complexity is reshaped by human metabolism.”

“Our technique does not just look at how one compound in this plant is metabolized,” adds William Crandall, first author of the study and a PhD student of molecular and systems pharmacology in Emory’s Laney Graduate School. “It shows how dozens of compounds are metabolized at one time.”

“This method marks a major, transformative step in natural products research,” says Dean Jones, co-senior author of the paper and professor in Emory School of Medicine. “A process that used to require years of work now takes just days.”

The new framework provides a starting point to help standardize and deepen understanding of the effects of a natural product when it is consumed in a manner typical to traditional medicine, such as leaves brewed for a tea.

The breakthrough was driven by the passion and dedication of Crandall, who plans a career in natural products research, combined with the unique resources of his co-mentors, Quave and Jones.

“All these factors came together,” Jones says. “You can call it serendipity.”

Quave is a leading ethnobotanist, studying how plants are used as medicine in traditional cultures. She has built a natural product library of more than 3,000 extracts isolated from plants, including documentation of the medicinal uses of the plants. Her lab has discovered the actual mechanisms for how some of these natural products work on dangerous pathogens, including antibiotic-resistant bacteria.

Jones, professor of medicine in the Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, is director of the Emory Clinical Biomarkers Laboratory. He is a world leader in the development of high-resolution techniques for metabolomic analyses of nutritional and environmental factors in human health and disease.

Crandall enjoys tapping cutting-edge technology to explore the ancient secrets of plants.   

“I love the complexity and the endless possibilities of all the compounds in plants,” he says. “There are always more interesting things hidden way down in the weeds just waiting to be discovered.”

Around half of modern-day medicines trace their origins to a natural product — a chemical compound produced by a living organism such as a plant, animal or bacteria. Common examples include aspirin (based on a pain-relieving compound found in willow bark) and Taxol (a potent anti-cancer drug derived from the bark of the Pacific yew tree).

Plants are master chemists — a single species may produce hundreds or thousands of chemicals to help the plant function normally and defend itself from predators and diseases. The richness of this treasure trove, however, makes it challenging to research.

“Science is typically reductionist,” Crandall explains. “You approach a large system, like a medicinal plant, and you try to isolate a compound responsible for that medicinal activity.”

The problem with that approach, he adds, is it doesn’t consider all the interactions of the many different chemicals of a plant when it is consumed.

Despite the enormity of the challenge, Crandall worked to find a streamlined solution to this complex problem.

He chose kratom (Mitragyna speciosa) as his test subject due to the extensive research available on the plant and its active ingredients.

A plant from the coffee family native to Southeast Asia, kratom has deep roots in herbal medicine due to its reported mood-enhancing, pain-relieving effects. It is traditionally consumed by chewing the leaves or brewing them in water to drink as a tea.

Modern-day science isolated two major psychoactive ingredients in kratom: alkaloids with opiate-like effects known as mitragynine and 7-hydroxmytragynine.

In the United States, kratom leaves and products prepared using these active ingredients are sold as extracts, pills, capsules and suspensions. Kratom is often used to self-treat conditions such as pain, coughing, diarrhea, anxiety and depression, opioid-use disorder and opioid withdrawal, according to the U.S. Food and Drug Administration (FDA).

The reported side effects associated with kratom use are broad, including everything from insomnia to sedation, hallucinations to confusion and, in rare cases, death. The U.S. Drug Enforcement Agency has listed kratom as a “Drug and Chemical of Concern” and the FDA warns consumers not to use kratom because of the risk of serious adverse events, including liver toxicity, seizures and substance-use disorder.

And yet, an estimated 1.7 million Americans aged 12 and older used kratom in 2021, according to the Substance Abuse and Mental Health Services Administration’s National Survey on Drug Use and Health.

Consumers, and regulators, are for the most part flying blind when it comes to evaluating the potential toxicity and benefits of different kratom products.

Even individual kratom plants may vary widely in their chemical makeup, Crandall notes, depending on factors such as the age of the plant and the environmental conditions in which it was grown.

For the current study, kratom leaves from different plant samples were steeped separately in hot water to create tea-like mixtures. The mixtures were then evaporated into powdered extracts.

The extracts were chemically characterized using high-resolution mass spectrometry, a standard technique to identify unknown compounds.

“Mass spectrometry is a powerful tool,” Crandall says. “You can identify molecules of extremely low abundance and extremely high abundance and everything in between.”

In simple terms, the molecules within a sample are converted into gas-phase ions so they can be manipulated by electric fields. That allows for the separation of the ions according to their mass-to-charge ratios.

Crandall drilled down further by using a technique known as tandem mass spectrometry, which fragments ions in the sample to determine their structural similarity, based on how they break apart.

The fragmentation patterns reveal unique molecular “fingerprints” related to a chemical’s structure. These results can be run through existing datasets of molecular fragmentation patterns to find a match, much like a detective might run fingerprints from a crime scene through a national database.

The results showed the extracts from different plants had three distinct chemical profiles.

Each extract was added to a laboratory preparation of human liver cells. The preparations were then incubated to allow biotransformation to take place.

Liver cells are an established tool used in laboratory studies of metabolites, since the liver is the primary organ for the metabolism of any substance consumed. A reductionist approach, for example, looks at how enzymes in liver cells interact with a single chemical compound to produce perhaps a dozen different metabolites.  

The current study, however, investigated the simultaneous interactions of liver enzymes and around 100 chemical compounds contained within each kratom extract. Each of these compounds in turn transforms into dozens of metabolites.

Crandall repeated the mass spectrometry analyses for the metabolized samples.

That gave him “before” and “after” data for the biotransformation process.

Crandall tapped existing software to visualize the biotransformation data as a molecular network. He then used the computer language Python to develop a practical, streamlined tool to automate the process of analyzing the resulting, highly complex network maps.

“I specifically designed my networks to show the relationship between precursor compounds and their resulting metabolites,” he says. “And I added layers of criteria that increase the confidence in their accuracy.”

These verification criteria include delineation of a chemical formula; its function, as determined by the fragmentation pattern; and the intensity of the chemical’s signal, to pinpoint its strength within the sample.

The multilayered, molecular network provides an unprecedented view of all the chemicals in kratom leaves and the metabolites generated by their metabolism.

Previously, trying to tease out all these relationships in a natural product was hit and miss.

“It was like taking a random walk through a universe of chemicals,” Jones explains. “Will Crandall developed a way to show all these interactions in a single snapshot. He’s opened the door to answer questions that were previously almost unanswerable regarding the potential benefits and toxicity of natural products.”

The results of the kratom analyses demonstrated how some of the most important bioactive compounds of a plant can be created in the liver during metabolism, even if they are not present in the plant itself.

The results also revealed different metabolites resulting from the three, chemically distinct, extracts of kratom leaves.

“This helps explain why botanical products like kratom can have variable, and sometimes unpredictable, effects on individuals using different formulations of these products,” Quave says.

While the current paper focused on kratom, the new analytical framework sets the stage for investigating the potential benefits and toxicity for a range of natural products.

Crandall envisions a database mapping out the precursor compounds and metabolites for various natural products. This repository could serve as a foundation for further research in animal models and, eventually, clinical studies in humans.

“I hope our method sparks more researchers to study the effects of natural products as a whole, because that’s how people typically consume them,” Crandall says. “We now have the technology available to both streamline and advance the rigor in natural products research to benefit human health.”

Co-authors of the paper include Emory graduate students Jaclyn Weinberg and Grant Singer; Ken Liu, a scientist in Emory Department of Chemistry; Choon-Myung Lee, an Emory research associate; and Edward Morgan, Emory emeritus professor of biochemistry.

The research was funded by the National Center for Complementary and Integrative Health and the National Institute of Environmental Health Sciences at the National Institutes of Health.

Story by Carol Clark.