Blackbrush: Scapegoats or Shaky Goats?

Last week, news broke about newly published research demonstrating the presence of an amphetamine isomer in nine of 21 weight management dietary supplements tested. These supplements listed Acacia rigidula  extract as an ingredient and source of several amines, including the -methylphenethylamine found by FDA scientists. The manufacturers insist the amine is naturally occurring in A. rigidula, but the researchers said there is no evidence the compound is natural in the plant.

A. rigidula, also known as blackbrush, is native to Texas. In 1998, researchers from Texas A&M investigated the amine content in the plant and published results in Phytochemistry (49(5):1377-1380).  Beverly Clement, Ph.D., the lead scientist on this study, was a synthetic organic (natural products) chemist working at Texas A&M on research in toxicology. She spoke to INSIDER about what prompted the study of A. rigidula amine constituents and framed her team's findings.

Various species of acacia plants are found in Texas, where it is a part of goats' diets. Clement explained goats in south Texas and northern Mexico develop a fatal syndrome called the "guajillo wobbles," also known as "limber leg." Clement noted the syndrome is particularly a problem during drought years. "Originally, it was suspected that this was due to the lack of other vegetation and the goats being forced to consume the leaves of the various acacias in the area, guajillo (Acacia berlandieri) being one of the more common acacias," she said. "-methylphenethylamine was identified as the potential causative agent in guajillo that was responsible for the wobbles; Cattle are not affected by this possibly because they may get supplemental feeding and are not as likely to browse solely on acacias."

Also during drought years, some white tailed bucks failed to shed their antler velvet, and hunters frequently report testicular atrophy in these deer, according to Clement, who added the Texas AgriLife Extension Labs at Texas A&M observed testicular atrophy, reproductive decline, etc., associated with consumption of these acacias especially during droughts in goats, and diminished reproductive potential in cattle (seriously altered calf crops).

"This suggested that drought might further stress the plant and cause them to expend energy on more exotic defense mechanisms, which may be reflected in the animals that consume them," she noted.

Clement was asked to analyze the plants. "This particular research is not funded by the USDA, because the wobbles are considered to be a regional rather than national problem."

The early season guajillo was unremarkable to Clement and her team. "The extraction was clean (bright green extract, easily partitioned with acid and base extraction)," Clement reported. "The analysis was relatively straightforward; we found terpenes, a few simple phenols and scant traces of -phenethylamine, but no detectable N-methyl--phenethylamine, which had previously been reported." However, work on late season guajillo was a different story. "The initial methanol extract was a very dark green, almost black color," she noted, adding the team realized these extracts had to be handled much more carefully due to extreme vulnerability to degradation. "After we began using an argon blanket, this degradation was no longer a problem; argon is a much more forgiving inert atmosphere than nitrogen." The end result was positive identification of N-methyl--phenethylamine; N,N-dimethyl--phenethylamine; and tyramine in these fractions. 

Because of their results with guajillo, Clement and her team analyzed other acacias, including A. rigidula. Blackbrush was collected during the late summer, during harsh drought conditions; it was identified as A. rigidula by a trained botanist and transported to Clement's laboratory, where it was kept in a freezer until subjected to analysis. "We did not have any early season blackbrush to use as a comparison since the South Texas researchers were dealing specifically with guajillo and analysis of these additional acacias was just a side interest," she noted.

Clement explained the leaves were extracted using a soxhlet extraction system employing methanol as the extraction solvent. "Because of prior experience with the guajillo extractions, the soxhlet was purged with argon to minimize exposure to 'air.'  Following extraction, the extract was concentrated on a rotary evaporator (vacuum released to argon again to minimize exposure to 'air').  The residue was dissolved in dichloromethane and extracted with 10-percent HClit was not as critical at this point to keep the acidic extract under argon to prevent side reactions. The HCl extracts were combined, neutralized with NaOH and extracted with dichloromethane (argon blanket again necessary). We had tested the extraction and neutralization procedure with tyramine to make certain that the conditions allowed recovery of this phenolic amine and related material." She further noted the extracts were combined, concentrated and kept under argon in the freezer until analysis with GC/MS (gas chromatography/mass spectrometry.) "Dichloromethane was used rather than ether," she said. "Initial tests suggested that dichloromethane would not be a problem with b-phenethylamine; we could get high-purity dichloromethane and did not have to worry about solvent grade ether.

On the specifics of the GC/MS analysis, Clement explained: "We used an on-column injection technique and scanned full scale from 45 m/e to 400 m/e.  The GC went through a ramp cycle from near room temperature on up to around 200 °C (I dont remember the exact temperatures or rate of heating.)  Injection volume was 1 uL . There was approximately a five-minute delay before collection of the data commenced to allow the dichloromethane to go undetected." She further noted once the spectrum was collected, her team of well-trained students would undertake the initial analysis on the runs; they used computers to analyze the spectrum under each peak and come up with possible matches. "That pointed us in the direction of most all of these amines," Clement said.

However, this computer analysis was not sufficient proof of identity. "I pretty much feel that computer identification alone is not worth the paper it is printed on. Therefore, after the discoveries were made, a great deal of effort was spent synthesizing small quantities of authentic material for direct comparison." Clement performed these syntheses herself, as none of her students had the synthetic training. "If the synthesized material matched published literature values, I felt confident that I had authentic material for comparison," she said, noting no effort was made to resolve any racemic material. "Confirmation of the identity of the amine was made when this authentic amine was injected upon the GC/MS following the same conditions produced a peak with the same retention time and the same fragmentation pattern as was seen our extract. I was only then confident to report the presence of that particular alkaloid. We did not shoot a blank before each run nor did we inject a blank after each run."

When enough material was available, the team would run two separate extractions of a blended sample and then compare  the two extracts by GC/MSperformed by different students, each protective of their own sample, according to Clement. The samples were subjected to multiple injections, and the GC/MS profiles were reproducible; the peak profiles, the relative size (area) and MS profiles associated with these peaks was reproducible between the two samples and the runs were repeatable.

Clement, now a professor at Blinn University, College Station, Texas, concluded blackbrush, at least the drought stressed plant, is a toxic mess. "The phenols alone would probably boost basal metabolism while inhibiting digestive enzymes," she cautioned, adding the variety of phenethylamines and tyramines detected should trigger a blinding headache if consumed.  "Im not even considering potential synergism that might occur. While we were specifically looking for the amines and related alkaloids, there are lots of other less than friendly chemicals present as well."

FDA said Clement's published rsults on A. rigidula amines lacked sufficient data; the agency's own analysis did not find the amphetamine isomer in verified samples of A. rigidula, and their publication suggests the plant could be a scapegoat for the presence of this compound in the supplements tested. However, Clement's work on plants that contribute to shaky goats and other animal health issues claims the compound is found in late season, drought-stressed acacia species, including A. rigidula.

Find the FDA study on A. rigidula products here.

Find Clement's published study on amines in A. rigidula here.