Artemisia samples arrived at Max-Planck for testing and a chemistry update

Finally. It took a while but the Artemisia samples have arrived in Germany and will hopefully be tested for in-vitro activity against SARS-Covid-2 quite soon. Now in the meantime, I’ve been separating the extract into various fractions. The idea behind this is that in case the original samples, currently in Germany, turn out to be active, then we need to concentrate that active compound(s) into a specific fraction. (To save time I will be sending these fractions early next week as well). So step-by-step we get rid of in-actives (which I always call rubbish) and we start to zoom in on the possible actives. Unfortunately, my semi-prep column died on me (and wasted quite a bit of my time), so I had to revert to some old-school techniques which actually works quite well. I am still drying some of these fractions, but to illustrate how it works, please have a look at Fig 1.

A afra tea Hexane fraction
Fig 1. The top chromatogram illustrates the fresh A. afra tea infusion (data 1) whilst the bottom chromatogram illustrates the non-polar fraction separated from the fresh A. afra tea infusion (data 2) 

Okay, so what are we looking at? One can divide the chromatogram in Fig 1 into roughly three sections.

Section 1: between 2-10 min, containing mainly polar compounds;

Section 2: between 10-15 min, containing your medium polarity compounds and:

Section 3: between 15-25 min, containing your non-polar compounds.

So what I’ve done here is to extract and concentrate the non-polar compounds into one fraction, and I’ve also managed to get rid of all the polar and a lot of the medium polarity compounds (they will be in the other fractions not shown here). So let’s say, for argument sake, that the active compound turns out to be non-polar, then we will expect this fraction to be a lot more active than the original tea infusion and all of the other fractions. The advantage of separating the extract based on polarity is that it also separates both UV-visible and UV-invisible compounds into these fractions – so you don’t loose any of your ‘invisible’ compounds without you knowing about it.

With a semi-prep column (which I’ve ordered) one can do a far better job than with this old-school technique that I’ve used, but it is still not too shabby.

If you find any of this interesting (strongly doubt of many people will do so), please add your email to this blog site for automatic updates, and please have a look at our crowd funding campaign website which is currently funding this work (and thanks to all who have donated – excluding the one troll).

The Artemisia Covid 19 Initiative – June 2020 update

Like they say ‘ the proof is in the pudding’. In this case, will the in vitro tests yield any useful results? Useful in this sense simply means a clear positive or negative result (keeping in mind the much dreaded false positives and false negatives). So I’ve send some samples over to the Max Planck Institute in Germany to test against SARS-CoV-2. At the moment the samples are somewhere in Dubai, so it finally cleared our customs in Johannesburg. The next potential hurdle might be the German customs, but hopefully it will reach its destination early next week. Here is the DHL tracking number for those interested to see where it is and when it arrives: 3544019883. (Interestingly, I found myself tracking this parcel as if being jealous about getting on a plane and being able to go places – this lock down is having a strange effect on people!)

But anyhow, I am currently fractionating (splitting up) extracts of Artemisia annua and A. afra, which is a bit of a time consuming process. The splitting up is quite quick, it is the liquid handling eg. drying, that is taking a lot of time (Fig 1). Usually one would wait for the in vitro test results but we simply don’t have the time (If it turns out to be inactive, I will focus on finding the antimalarial compound in A. afra – so the fractions will be useful to have either way).

semi-prep HPLC
Fig. 1. Semi-prep system that we use to fractionate extracts.

I’ve also noticed that the University of Western Cape has recently set up an in vitro test system, so I will definitely have to contact them – it is always good to confirm results (positve and negative) with independent research groups. So for me, next week will be in the lab doing good old chemistry, and stressing myself to bits about the in vitro tests.

If for some reason anyone out there happen to have a good second-hand semi-prep RP column (min diameter 10mm) and don’t have any use for it, please contact me (frankvanderkooy@gmail.com) – the column I’m currently using is near its end. And please have a look at our Crowd Funding website and circulate via your networks if you feel like it. And of course, thank you for those who have donated thus far!

 

Chemical variation and artemisinin content of A. afra and A. annua

As part of our ongoing crowd funding campaign to investigate whether Artemisia might become a useful treatment of Covid 19, we continue with the chemistry of these two species (if you have a dollar to spare please donate on the crowd funding website). Tea infusions (as described earlier) of three A. afra samples (collected here in Potch and Bronkhorstspruit, South Africa) and 3 A. annua samples (A3 variety grown in Germany and harvested in different years) were prepared. If all goes well, we will also be testing these samples against SARS-CoV-2. Results from Germany, from a different research group, indicated that extracts of A. annua does indeed show activity against the virus, whereas artemisinin was found to be inactive. So it is good news, but there is still a lot to do. With their permission I will upload the link to those results.  

In this study all six samples were analysed with our UPLC system using the same method and settings that was used for the stability tests. I have also developed a very sensitive MRM method for the quantification of artemisinin on our LCMS with a sensitivity level of about 1 – or so picogram (on column). Still need to do all the calculations, so at the moment it is only a qualitative result. Now, A. afra is quite interesting because I could never find any trace of artemisinin in any of the samples that I’ve analysed over the years. The main idea behind looking for artemisinin in A. afra is that it grows very well in the sub-Saharan Africa region, so finding a individual plant that produces artemisinin might eventually produce a better growing and higher yielding variety (after plant breeding of course) than A. annua (which is from China). There is a global shortage of artemisinin, and the world needs a lot more in order to fight malaria effectively.  

So first up, the 3 A. afra samples (Fig 1) 

 

3X Afra comparison
Fig. 1: Overlaid chromatograms of tea infusions prepared from 3 different A. afra samples. 

This is quite lovely. Many similarities, but also big differences in concentrations and some chemical differences. It might be a bit difficult to see but look at the peak near 20min. Sample 1 does not contain this compound at any appreciable level whilst the other 2 have high levels of this compound. Lets say for example that we find sample 1 inactive, sample 2 reasonably active and sample 3 by far the most active, then it would be quite possible that the main active compound is this peak at 20min. If this is the case we can isolate, identify and test the purified compound against the virus in order to confirm this. So it can all be quite easy, but from experience I can tell you, it rarely is. But lets stay positive, and fist test against the virus.

Figure 2 illustrates the A. annua samples. Same story applies.

3X Annua comparison
Fig. 2: Overlaid chromatograms of tea infusions prepared from 3 different A. annua samples. 

Artemisinin content

In short, large quantities in all three A. annua samples and not even a trace in any of the  three A. afra samples – as expected (I also injected 10X more of the A. afra samples than the A. annua samples). However, analysing a different A. afra sample which was used in two clinical trails (malaria and schistosomiasis) I did find relatively large quantities of artemisinin (this is also reported in their publications). Now, how can this be? According to the authors, this particular A. afra sample was bought at Kirstenbosch, Cape Town and it was germinated and grown close to A. annua in Senegal. It might be that this particular A. afra specimen is our missing link and it do indeed contain artemisinin, but I don’t know. According to two of the authors, hybridization between the two species is unlikely. Another possibility is that A. afra were contaminated by the pollen of A. annua plants which was grown nearby, or it might even be something simple as using a ‘dirty’ grinder.

Point is, I am not convinced that this is my missing link so and I am still ‘fighting’ with the authors about this issue. We’ll eventually figure it out.

Forthcoming attractions: Sending those samples to be tested against the virus, fractionation of samples (just did not get the time this week), accurately quantifying artemisinin, and winning my ‘fight’ with the authors of the clinical trials.

 

Stability of Artemisia afra tea infusion and its artemisinin content

Quite an eventful week, with the lowest recorded maximum temperatures in over a decade (those Europeans do not know what cold is), and some ‘building issues’ that prevented me from getting to the lab. But what would life be without challenges.

So I’ve analysed the Artemisia afra tea and compared it to Artemisia annua. The phenolic profile looks quite stable (figure 1) but there are differences between the profiles of the two species (figure 2).

Artemisia afra stability
Fig 1. Artemisia afra profiles of the same tea infusion sample analysed at 3 different time points. Data 1=fresh tea, data 2= 7 hours old and data 3 = 14 hours old.
Annua afra comparison
Fig 2: Tea infiusions from Artemisia annua (data 1) and A. afra (data 2). Clear differences can be seen, not only in quantity, but also identity of compounds. 

This is fully expected. Chemical differences between species is well known but also within species (depending on where, how, when etc samples were collected). We have a number of A. annua and A. afra samples and will be conducting a test to see how much the chemical intra-and interspecies variation will be.

Artemisinin content

The main bioactive compound in A. annua is artemisinin. I’ve set up a very sensitive MRM method on our new MS equipment to quantify artemisinin in these samples. The A. annua material that I used for a first test did yield significant amounts of artemisinin – no surprise there. But the interesting thing is that I could not find any trace of artemisinin in the A. afra sample. I’ve tested a number of A. afra samples in the past mainly to see if I could find any artemisinin but I’ve always came up empty handed (I’ve even published a paper on this way back in 2009). The first report that a specific A. afra sample contains artemisinin was published in the clinical trial paper a couple of years ago and I’ve actually managed to get some of that material. This material is currently being analysed on our LCMS and it will be very interesting to see if we can confirm those published results.

So there is a lot of things that needs to happen this week. I need to test the chemical variation of all the samples. I need to quantify artemisinin in all these samples and I need to fractionate selected samples to be tested against the corona virus (if all goes well, the antiviral testing will be done at Basel university within a couple of weeks).

Stability of Artemisia tea infusions Part 2.

We are now 24 hours later, so how does the tea infusion perform regarding stability? It actually looks quite okay, to be honest. But just to clarify again what we are looking at. The equipment that we used only detects compounds that absorb UV light, in other words, compounds such as the well known active ingredient, artemisinin, will not be detected. In the figure below you will be able to see a wide range of compounds, some of which we have identified in the past as chlorogenic acid derivatives and a couple of flavonoids and coumarins. All of these compounds are called Phenolics, so strictly speaking we are at the moment only looking at the Phenolic stability of the tea infusion.

Figure 1. Overlayed chromatograms of the fresh A. annua tea infusion (data 1) and after 7 hours (data 2) and 14 hours (data3).

The Phenolic compounds do indeed appear to be remarkably stable, but does this now really say anything? Well, as long as we do not know if the tea infusion is active against Covid 19, and if so, which compound(s) are responsible for activity, it doesn’t really mean much. But nevertheless, we need to gather data, so this exercise will be repeated with the inclusion of an internal standard and using a mass spectrometer (MS) to detect and quantify artemisinin and related compounds. The MS depicted in the photo above has recently been bought by our Faculty for an eye-watering R3.3 million and is currently being installed. If all goes well, we will be able to start using it early next week.

Stability of Artemisia tea infusions. Part 1

As part of our Artemisia crowdfunding campaign we decided to create this website so that we can report on our progress. The main idea here is to give a short overview of the tests that we are conducting, the reason why we are conducting these tests, and also to give the public an opportunity to make comments or suggestions (this site will be updated as we go along).

The first test that we are currently conducting is to check if the Artemisia annua and Artemisia afra tea infusions, prepared in a similar way than in the clinical trials, will be stable over a 12-16 hour period. We need to get our head around the chemistry and stability of these infusions for quality control purposes. In the clinical trials, 5g of plant material was added to 1L of boiling water, it was left to infuse for 10min, after which it was filtered and administered over a 1 day period to the participants in the trials (a 12 hour period). We used the same ratio (100mg/20mL of boiling water), used a 0.45 micron syringe filter to filter the infusion into HPLC vials for HPLC analysis (equipment depicted above). The detector that we use is a PDA detector which detects UV-absorbing compounds. The figure below illustrates the chemical profile of a fresh A. annua tea infusion. The x-axis is time in minutes, and the y-axis the intensity of UV absorbance by the different compounds.

Figure 1. Chemical profile measured at 254nm of a fresh A. annua tea infusion. x-axis is time and y-axis the intensity. Each peak corresponds to an unique molecule.
Figure 2. The same chemical profile as in figure 1, but with the baseline highlighted to illustrate the hundreds of minor compounds present in the tea infusion.

The current plan is to compare the chemical profiles of the fresh tea with the profile of a 16 hour old tea. If any of the molecules disappear then we have stability issues. There is now obviously a number of problems.

  1. We do not know which of these compounds are the ‘actives’, and therefore even if a number of compounds degrade, we will still not know if it will have an impact on the activity.
  2. The analytical instrument only detects UV absorbing compounds. There are many compounds that will not be detected. The well known antimalarial compound, artemisinin, does not absorb UV and will not be detected with this technique.
  3. Based on the clinical trial publications, we do not know how the tea infusions where stored eg. fridge, at room temp, exposed to light or not etc. and what the influence of this will be on the chemistry.
  4. etc.

In order to get a better idea about the chemistry and the stability of the tea infusions we therefore need to conduct a number of tests. HPLC with UV detection is one method, this will be followed mass spectroscopy specifically to look at artemisinin and related compounds. And finally we will conduct Nuclear Magnetic Resonance spectroscopy in order to get a more ‘complete’ picture of the chemistry of the Artemisia tea infusions.