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Elderberry is anti-bacterial,
anti-viral and modulates the
immune system:
anti-bacterial, anti-viral and
immunomodulatory non-clinical (in-vitro)
effects of elderberry fruit and flowers
(Sambucus nigra): a systematic review
Reedited with additions, January 2020 and November 2020
Updated for SARS-CoV-2 the virus which causes Covid-19
Julia Helen Wermig-Morgan
Submitted to Oxford September 2018
This dissertation has been submitted to the
University of Oxford in partial fulfilment of the
requirement for the award of the degree of MSc in
Evidence-Based Health Care
University of Oxford
MASTER’S PROGRAMME IN EVIDENCE-BASED HEALTHCARE
ABSTRACT
Background The black elderberry (Sambucus nigra) has played an important role historically as a
European herbal medicine, and features in the folk medicine of most European and West Asian
countries. More recent pharmacological studies have shown it to have anti-viral, antibacterial, anti-
inflammatory and immunomodulatory activities. Recent experimental evidence also suggests it is a
powerful antioxidant, and it is an ingredient in some food preservatives and disinfectants. The most
common use of the plant is for the treatment of colds and influenza, and some clinical randomized
controlled trials of extracts of the plant tentatively support its use as an anti-viral. However, the trials
are small so decisive conclusions cannot be made. Still, some commercial preparations of elderberry,
produced to pharmaceutical standards, exist. Research is required to investigate the properties of this
potential medicine.
Objectives To conduct a systematic review of the in-vitro trials of the antimicrobial and
immunomodulatory activities of elderberry.
Data sources, study eligibility criteria Google Scholar and twelve other databases were searched
for in-vitro anti-microbial and immunological trials of elderberry fruit or flower extract.
Interventions, study appraisal Only extracts of fruit or flower were included. Due to extreme
heterogeneity of the microorganisms, both bacterial and viral, and of the experimental techniques for
testing them only a narrative appraisal could be made of results.
Results 5870 papers were found on Google Scholar for “elderberry (Sambucus nigra)” and finally 21
papers were eligible for inclusion in this review. No other databases brought additional papers. Of 56
trials (15 publications) 49 (87.5%) on well prepared extracts of ripe fruit or flowers showed at least
some inhibition of microorganisms. However, one publication using extract made from a mixture of
all plant parts – leaves, wood, ripe and unripe fruit – showed no inhibition at all in all 10 trials. It was
clear from this that the plant parts used is crucial to its beneficial properties. The types of extract, the
outcomes, and the microorganisms tested were heterogeneous, making statistical pooling illegitimate.
They were therefore reported narratively. The immunomodulatory effects (from 6 publications) were
treated separately and appeared to show that the S. nigra stimulates the immune system without
exacerbating a cytokine storm, and it might dampen one. Two case studies of elderberry for Covid-19.
Limitations The heterogeneity of the trials and the fact none of them were blinded were the two
biggest limitations. Others are mentioned in the text, but in human trials randomized would have been
a higher level of evidence to those of in-vitro trials, however these only exist for colds and ’flu and it
appears to inhibit many other microorganisms. It would have been preferable if several replications of
each of the in-vitro experiments were conducted and forest plots could be made to verify results.
Conclusion Elderberry extract from fruit and flowers appears to show some inhibitory effect against
many microorganisms including those found as nosocomial pathogens (hospital “super-bugs”) e.g.
MRSA; HIV; Mycoplasmae; IBV coronavirus (an envelope virus), and influenza (and its bacterial
super-infections). These effects may be stronger in-vivo than in-vitro. The immunomodulatory effects
of S. nigra have been investigated and appear to show that the plant would be likely to stimulate the
immune systems of the weak or immune-compromised. It has also been investigated to see whether it
would exacerbate or mitigate a cytokine storm, and does not seem to make one worse and might
dampen it. Elderberry is also shown to have potential as an ingredient in a hospital disinfectant for
which in-vitro trials are sufficient, and without effective disinfectants modern medicine will become
impossible. The S. nigra inactivates two distinct envelope viruses and should be tested on Ebola, also
an envelope virus, as it is likely that it may inactivate that too. It should also be tested on SARS and
other novel coronaviruses such as COVID-19 which are all envelope viruses. Other species of
Sambucus appear to have very similar properties including inhibiting coronaviruses. Elderberry seems
to have potential as a useful medicine, particularly since there are reasons to believe resistance to it is
unlikely to ever develop. It might be possible to use it in combination with leeks (Gallium porrum)
which are also anti-viral. Elderberry has been shown to be effective against upper respiratory tract
diseases including some Coronaviruses, and has potential against the current pandemic of Covid-19.
Two case studies, included here, suggest it to be effective against Covid-19 caused by SARS-CoV-2,
and clinical trials are under way. In-vitro trials are planned to test it against other viral possible future
pandemics.
3
CONTENTS
ABSTRACT
AUTHENTICITY STATEMENT 4
ACKNOWLEDGEMENTS 5
INTRODUCTION 6
Aim 6
Distribution and historical use of black elderberry 6
Why it is important to undertake this review 11
How elderberry might work 14
Objectives of this review 16
METHODOLOGY 17
Criteria for considering studies for this review 17
Types of studies 17
Types of interventions 18
Types of outcome measures 18
Developing a search strategy 18
Measures of treatment effect 19
Dealing with missing data 19
Heterogeneity of studies 20
Assessment of reporting biases 20
Assessment of bias 20
Assessment of quality reporting 22
Checklist for quality of in-vitro trials 22
Is statistical analysis possible? 24
RESULTS 25
Missing data 25
Selection and analysis of studies 26
PRISMA diagram of search strategy 27
Heterogeneity of studies 27
Summary of Results Table 1 29
Summary of Results Table 2 33
Narrative summary of papers 34
Tests of efficacy on microorganisms 34
Composition of elderberry molecules and immunomodulatory effects 64
DISCUSSION 74
Summary 74
How this fits with other evidence 75
Other Sambucus species 77
Strengths and limitations 78
November 2020 Postscript 80
CONCLUSION 82
REFERENCES 83
APPENDICES:
APPENDIX 1 The conventional hierarchy of evidence in healthcare 91
APPENDIX 2a Influenza deaths and cytokine storms 92
APPENDIX 2b Immunomodulation and cytokine storms 93
APPENDIX 3 Sambucus nigra in other languages 96
APPENDIX 4 How to make elderberry extracts 97
APPENDIX 5 Table 1 from Hearst et al., 2010 100
APPENDIX 6 Case studies of elderberry cordial used as a treatment for Covid-19 102
4
DISSERTATION AUTHENTICITY STATEMENT
I confirm that I have read the Department of Continuing Education’s rules relating to
plagiarism as found in the 2015-2016 Student Handbook.
I confirm that the work presented here is my own except where otherwise indicated.
Date: September 2018
5
Acknowledgements
Many thanks to Dr Jason Oke and Dr Man-Mei Lee, Oxford statisticians, for their advice on
the handling of data.
Thank you, Morag Evans of The Thomas Sydenham Library, Dorset County Hospital for
carrying out a second literature search.
Thank you to the volunteer staff at Burton Bradstock Library for putting up with numerous
difficult requests from International Library Loan, and Robin Beachy and the administration
team, Rewley House, Oxford for dealing with many small requests.
Many thanks to Denise Bilton of Abbotsbury for formatting my poor typing efforts, and for
checking the spelling. You are a star of Norwegian and Croatian accents and complex tables.
Thank you to all my teachers so far in my journey, but above all thank you to my supervisor
Dr Jeremy Howick for invaluable advice and for encouraging me to carry on. The best of all
teachers.
Note: An attempt has been made to write this paper in plain English in the hope that it can be
of help to non-scientifically trained readers and to readers all over the world where English is
a second language.
6
Anti-bacterial, anti-viral and immunomodulatory non-clinical (in-vitro)
effects of elderberry fruit and flowers (Sambucus nigra): a systematic
review
INTRODUCTION
Aim
The purpose of this paper is to investigate whether elderberry might have antimicrobial
activity in-vitro which could have potential for medicinal use. Its effect on the immune
system is also investigated.
Distribution and historical use of black elderberry
There are several species of the Sambucus family that are commonly called elderberry. For
the purposes of this dissertation the term “elderberry” only refers to the plant Sambucus nigra
or its fruit.
The black elderberry is a shrub of the honeysuckle family (Caprifoliaceae/Adoxaceae) which
occurs throughout Europe (except north of 63° N) from Iceland across South West Asia as far
as Siberia. Elder is also native to North Africa, and has been introduced to many other
countries. It is naturalized in North America. It grows well in most soils and is commonly
found in woodlands, hedgerows and wastelands, even in many cities. It is popularly used in
the folk medicine of all the countries where it grows naturally. The most common medicinal
use is for infections and fevers such as influenza, and for its iron content in cases of anaemia.
In most of these countries it is also a common foodstuff (Royal Botanic Gardens, Kew:
database, n.d.). Elderberry is widely available, growing wild and cheap to buy.
The earliest record of medicinal plant use we have today is an Egyptian papyrus written in the
sixteenth century BC. It included Sambucus, the elderberry (Kay, 1996). Hippocrates in the
7
fifth century BC is said to have called elderberry his medicine chest, implying that he used it
for a great many conditions as did, later, Blochwich (Wells, 2010). Pliny the Elder wrote
about it in classical Rome, as did Galen of Pergamon and Theophrastus (Coxe, 1846; Hort,
1916). Pliny referred to its use for constipation. In the mid-17th century the German Martin
Blochwich wrote a whole book about elderberry’s medicinal properties, The Anatomy of the
Elder (Blochwich, 2010). In this he says of its use to treat fevers:
Common people use to take Rob of the Elder in vinegar, water, or Spirit of Elder
flowers on the first hints of fever, sweating in bed afterwards. Physicians do not
disapprove of these habits because experience has shown that they work well in
preventing even worse affections (i.e. more serious illness).
Blochwich is the first surviving comprehensive account of its many uses. Today many of
these uses are proving to be correct. Dutch 17th century physician Hermann Boerhaave, who
started the first real European medical school in Leiden, never passed an elder without raising
his hat, so great did he think its healing powers (Grieve, 1931).
Henry Box, a famous English herbalist in the early part of the twentieth century, said,
For colds, influenza, fevers, inflammation of the brain, pneumonia, stomach,
bowels or any part, this is a certain cure. I have never known it to fail, even when
given up at the point of death. … [Elderberry] will not only save at the eleventh
hour, but at the last minute of that hour. It is so harmless that you cannot use it
amiss, and so effectual that you cannot give it up in vain (Shook, 1974).
Elder flowers, berries and extracts appeared in most European pharmacopoeias from the 18th
century until about the time of World War II when antibiotics were discovered, and even
elderberry’s use for viruses, for which antibiotics do not work, was largely forgotten in many
countries including Britain. However, it has continued to be much used in Germany,
Scandinavia and Eastern Europe, particularly as a treatment for colds, ’flu, and upper
respiratory infections. Chrusbasik (2015) at a symposium on elderberry cites the
pharmacological effects to include “anti-viral, anti-bacterial, analgesic, immunomodulatory
and anti-proliferative”. He says that “… the putative effects […] include protection from
8
damage of the gastric mucosa, liver and pancreas”. German doctors currently recommend the
juice, which is sold in supermarkets, to pregnant women for its high iron content (Prof Dr S-D
Costa, Magdeburg: private correspondence). Kšonžeková (2015) suggests it is good for new-
borns because of its high anthocyanin content. Elderberry is used as a strong food
preservative (Mohammadsadeghi, 2013).
A problem with all the evidence described above, however, is that it is observational. As such
it suffers from all the well-known biases associated with observational data. This has started
to change over the last 50 years, with some emerging clinical trials. My extensive search
identified only five randomized trials and these mostly only for colds and ’flu, which I
describe briefly below, together with their main methodological failings:
1. Zakay-Rones et al. (1995) conducted in-vitro trials of elderberry on influenza, and also a
small RCT during an outbreak of influenza B in Panama. Children received two, and adults
four, tablespoons of Sambucol or placebo daily for three days. Their trial had 23 participants,
and they found that a significant improvement of the symptoms, including fever, was seen in
93.3% of the cases in the elderberry-treated group within two days, whereas in the control
group 91.7% of the patients showed an improvement within six days (p ≤ 0.001). The
numbers of patients were low; however such differences in recovery times have been found in
other small trials. There was no intention to treat analysis. This trial made no note of rescue
medication.
2. Zakay-Rones et al. (2004) conducted another RCT on 60 patients and found that influenza
symptoms were relived on average four days earlier, and rescue medication was significantly
less in those receiving elderberry extract compared with placebo. Patients received 15 ml
elderberry (Sambucol) or placebo four times a day for five days. The elderberry group showed
a pronounced improvement (VAS score nearer to 10) after a mean (+or –SD) of 3.1 days,
9
while a similar score was obtained after 7.1 + or – 2.5 days in the placebo group. This
difference was significant (p<0.001). Specified rescue medication was allowed and was
significantly less (p<0.001) in the elderberry group compared to the control group.
Compliance was carefully checked. The methodology appeared to be quite good, but there
was a financial conflict of interest since it was sponsored by manufacturers of the extract. One
fault was that symptoms were not assessed at the onset of treatment (baseline) to investigate if
the two groups were clinically comparable at the start of the study.
3. Kong et al. (2009) also conducted a pilot RCT with 64 participants on influenza and found
similar results. Patients were given a proprietary elderberry extract lozenge containing 175
mg extract or placebo, 4 lozenges a day for two days. Patients were followed for only 48
hours after treatment, which is very little for influenza, which usually lasts at least 7 days.
The patients should have had lozenges until they recovered. Nothing was said about whether
patients also used other treatments such as rescue medications. In the 48 hours, the mean VAS
scores of most of the symptoms of ’flu showed no significant differences between the group
(p>0.05); only the mean VAS score of fever revealed statistical relevance.
4. Tiralongo et al. (2016) conducted a randomized trial of 312 airplane passengers, 29 of
whom went on to develop colds (coronavirus) and found that elderberry supplementation
reduces cold duration and symptoms in air-travellers. Patients were given proprietary capsules
containing 300 mg elderberry extract, 2 capsules a day, 10 days before travel until 4/5 days
after reaching their destination. Placebo group participants had significantly longer duration
of cold episode days (117 vs 57, p= 0.02) and the average symptom score over these diagnosis
also significantly higher 583 vs 247, p=0.05). All of the trial group were given elderberry and
no account was made of the fact that it might inhibit the virus at such an early point of
replication that some of this group might not develop colds at all and it might have a
10
prophylactic effect. It assumed every patient was compliant in taking medicine, so it was
intention to treat analysis.
5. Levine et al. (2014) conducted a randomized trial of 54 patients and found that a herbal
product which included S. nigra is a safe and effective anti-bacterial and anti-inflammatory
mouth rinse, which significantly reduces the progression of gingivitis, dental plaque
formation and spontaneous bleeding. PeriActive proprietary mixed herbal extract was given,
15 ml in mouth for 60 seconds, 3 times daily for 14 days. A total of 59 patients were enrolled,
with 54 completing the two week study protocol. GI values increased (i.e. worsened) more
significantly among the controls compared to the treatment group. Number of bleeding sites
was significantly less in the treatment group (p= 0.001 vs p=0.304). Note that the gingivitis
did not clear up, but it just progressed less than the control. There was no intention to treat
analysis.
6. In addition, Konlee (1998) reports a case study (the sixth level of evidence) on a female
with HIV, and taking no HIV drugs, who used elderberry extract Sambucol, with olive leaf
extract and experienced a viral load drop from 17,000 to 4,000
*
.
In 2020, after this review was published on the internet, Mahboubi concluded from the same
evidence that clinical studies confirmed the efficacy of standard elderberry extract in
reduction of cold and ’flu duration and severity. However, the numbers of patients in these
studies are still small.
7. Since this dissertation was first written, Hawkins et al. (2019) have published a meta-
analysis of randomized controlled trial including a total of 180 (relatively few) participants,
which found that supplementation with elderberry (S. nigra) substantially reduced upper
respiratory symptoms. The quantitative synthesis of the effects yielded a large mean effect
*
Konlee advocates using glucosamine sulphate and chondroitin with the elderberry and olive
leaf as a combination therapy.
11
size. These findings, they conclude, present an alternative to antibiotic misuse for upper
respiratory symptoms due to viral infections, and a potentially safer alternative to
prescriptions drugs for routine cases of the common cold or influenza.
While a review of these studies would have been possible in principle, there were very few.
Moreover, the limits of these small pilot trials meant that doing a review of “upstream” (in-
vitro) studies was required to inform future trials more than a review of these trials. Studies
are mostly on the effects of elderberry on colds and ’flu. They do not mention its effects on
bacterial super-infections to ’flu, nor do they include all the other microorganisms which the
extract may inhibit. It was felt that more research on the in-vitro properties of elderberry
would provide more information on these microorganisms, and on dosage and mode of action.
There is increasing interest in elderberry’s use in veterinary medicine. (Animals have very
similar immune systems to humans.) Trials have been conducted for its use in bronchitis in
chickens (a coronavirus) (Chen 2014) and Streptococcus in trout (Salehi 2015). This use, if it
transpires that it is effective, may reduce the use of antibiotics in farming, a major cause of
antibiotic resistance. Dr Mumcuoglu and her team also tested elderberry on a small troupe of
chimpanzees in a zoo, both prophylactically and on flu-like symptoms and found it reduces
the appearance of symptoms by two thirds (Burge et al, 1999). Chen (2014) found that S.
nigra extracts inhibit the infectious bronchitis virus at an early point during replication.
Why it is important to undertake this review
There are several reasons why it is important to undertake this review.
1. A major reason this review is important is that the increasing problem of antibiotic
resistance makes it imperative to investigate potential alternatives. Anti-microbial resistance
is becoming a very serious threat to modern medicine. There is now a strain of E.coli which
is resistant to Colistin which until then been considered the antibiotic of last resort. Therefore,
now there is a strain of the bacterium which cannot be treated by any pharmaceutically
12
produced medicine. A person cannot become resistant to antibiotics. Resistance is a property
of the microbe, not a person or other organism infected by a microbe (Centers for Disease
Control and Prevention [CDC], 2013). For this reason, in-vitro trials are a perfectly valid way
to test for anti-bacterial properties.
Howard ascribes several reasons for the increase of antibiotic resistance caused by: their
overuse; uncontrolled sale and prescription; use in livestock feed to promote growth; release
into the environment by pharmaceutical companies, and use in antibacterial soaps, all causing
antibiotic resistance and resistant strains which have spread (Howard, 2013 and CDC, 2013).
The Hearst (2010) paper is of particular interest in suggesting it might be effective in the
treatment of hospital acquired pathogens such as MRSA. The preliminary evidence cited
above suggests one alternative to antibiotics might be elderberry. This is something the World
Health Organization has been looking for.
2. Another reason why it is important to do this review is to investigate the potential anti-viral
properties of elderberry. For viruses such as influenza, neuraminidase inhibitors such as
Tamiflu and Relenza at first seemed very promising, but are proving to be a disappointment
(Jefferson et al., 2014). Elderberry has also been shown to be a neuraminidase inhibitor but
one which may have more promise and to which resistance is unlikely to ever occur due to the
shape of the bioactive molecule (Swaminathan, 2013). If elderberry is shown to work it could
be used for a long time, if not indefinitely.
3. Elderberry is thought to inhibit both bacteria and viruses, which could make it suitable for
use when there are often secondary infections, such as when influenza is followed by bacterial
pneumonia. It may also be effective for nosocomial infections (hospital “super-bugs”)
(Hearst et al., 2010). The results to this very important paper were lost in a printing error and
are published in Appendix 5 for the first time. It has potential as an ingredient in hospital
disinfectants.
13
4. For HIV and AIDS, drug resistance has also occurred to anti-retrovirals, even when they
are used in combination. Alternatives are needed. Preliminary research suggests elderberry
may aid the efficacy of other anti-retrovirals (Manganelli, 2005). Elderberry grows in a
number of African countries where the costs of drugs for HIV are prohibitive to many. Its
cheapness and availability would be a bonus if it were shown to be effective.
5. The immunomodulatory effects of elderberry need further investigation, particularly
concerning how it affects cytokine storms. Some research suggests that elderberry always
increases the immune effects whereas others think it may sometimes dampen them down
(Voldvik, 2015).
6. Elderberry could be a safe effective alternative for general practitioners to prescribe when
patients demand what would be ineffective – an antibiotic for a viral infection. Physicians
need something they can prescribe to patients who demand a prescription and which may be
better than placebo, and who do not understand that their cold, ’flu or mild virus will not be
treated by an antibiotic. Elderberry extract is a pleasant tasting medicine, usually in syrup
form, to which hot water can be added to make a soothing drink. Safety tests show no
unpleasant side effects or contraindications. It is safe to use in babies, children and the
elderly. Research in Norway, where it is commonly used, show no adverse consequences
when it is used in pregnancy (Rødahl, 2011). Therefore, elderberry extract could, potentially,
be a suitable medicine for GPs to prescribe when their patients are asking to be given
something for their viral condition. It is relatively cheap, might actually be effective, and
could discourage the over prescription of antibiotics when they are unnecessary. Moreover,
compared to the use of Tamiflu during a pandemic, it could afford savings to the NHS in UK
of around £400,000,000. Information from this paper could be enough to justify physicians
using elderberry when patients are demanding an antibiotic for a viral infection when nothing
else is appropriate.
14
7. Meanwhile, there have been a few clinical trials in this area (see Discussion); however,
these have been small (mostly fewer than 60 participants), and there are not many (my search
identified only five, and these mostly on colds and ‘flu). Hence, I believe that a systematic
review of in-vitro trials would be useful for informing the theoretical basis of future clinical
trials, for example by helping to inform the correct dose and whether it may inhibit a
particular microorganism, or have super-additive effects with other medicines. Further
research might shed some light on the mode of action of S. nigra.
How elderberry might work
For viruses: Dr Mumcuoglu was the first person to discover the constituents of elderberry that
neutralize the activity of hemagglutinin that is found on the surfaces of many viruses. Viruses
no longer have the ability to enter or pierce and replicate in the cell when hemagglutinin
spikes are deactivated (Vardini et al., 2015).
Elderberries probably produce most of their antimicrobial effects because they contain
anthocyanins, a sub class of flavonoids that are polyphenols. Mohammadsadeghi (2013) says
that
… the phytochemical analysis of the crude extract of this medicinal plant revealed
the presence of anthocyanin, vitamin A and C as well as a good source of calcium,
iron and vitamin B6. It contains sterols, tannins and essential oils and can easily
be considered healthy food.
Elderberry contains a number of these flavonoids, amongst them natural anthocyanins which
are thought to bind to bacteria and viruses. Amongst all fruits, elderberries are the most
concentrated source of anthocyanins (Mateus, 2004). Swaminathan et al. (2013) used mass
spectrometry to show that the anthocyanin derived from Sambucus nigra fruit extract,
cyaniding-3-sambubiocide, binds to neuraminidase residues. Swaminathan’s paper showed
that this anthocyanin binds remote from residues Asp151 and Glu119, two residues known to
regulate neuraminidase resistance. They found that this provides the potential for the
15
development of a new class of anti-virals without this susceptibility. Binding of the
anthocyanins in elderberry to bacteria and other viruses have not been studied in so much
detail, and maybe should be if pharmaceutical companies want to develop new medicines,
rather than use the whole plant from nature as could be done. However, herbalists consider it
better to use whole plant extracts which can act synergistically.
In addition to polyphenols, lectins are found in many plant extracts including S. nigra and
often show anti-viral activity by binding to viral proteins or host receptors, preventing their
interaction (Chen, 2014).
The European Medicines Agency (2013) Committee on Herbal Products (HMPC)’s
monograph on Sambucus nigra L., fructus (the fruit) lists all the then known constituents of S.
nigra, the components that may contribute to pharmacological activity and the research
papers which found these constituents. The reader is directed to this monograph for details of
the constituents, but briefly they comprise the following flavonoids: 8 kinds of anthocyanins,
flavonol ester, flavonol glycosides; lectins and cyanogenic glycosides in fruit and seeds; 0.1%
essential oils in fresh fruit; 13 fatty acids; organic acids; vitamins; minerals and 7.5%
carbohydrates.
Whilst pharmaceutical companies are interested in finding the one bioactive substance that
binds to a microorganism or is in other ways bioactive, naturopaths and herbalists consider
the whole combination of constituents to be acting synergistically. It is possible that one of
the bioactive molecules (flavonoids) inhibits one microbe but another molecule inhibits
another.
Sidor et al. (2015) include an even more comprehensive list of constituents, and the molecular
structure of the constituents of elderberry. Anthocyanins are polyphenols, and it would seem
that the elder plant contains a very long list of these and other bioactive molecules. It may
16
well be that the large number of components of the plant explains why it appears to have a
number of beneficial effects on the human body, as well as binding to and inhibiting a wide
variety of microorganisms.
Ferreira et al. (2020) have also written an account of the chemical composition of Sambucus
nigra, fruit and flowers and its related bioactivities.
Varro, in his paper “Herbal medicine: from past to the future” (2000), refutes the viewpoint
that herbal medicines will be ultimately rejected, and the prediction is made that herbs are
ultimately destined to become part of mainstream medicine. They are cheaper than
pharmaceuticals and bring medical help within the reach of even the poorest populations.
What is needed is more and good quality evidence about them. Herbal medicine is also
available in countries or at times (such as war) when there are no pharmaceutical companies.
Large clinical trials are usually not possible for those producing small amounts of herbal
products because they are too expensive. It is to be hoped that by gathering together the
increasing number of cheaper in-vitro trials, some charitable or governmental agency, such as
WHO or the Welcome Trust will see the merit in funding more of these and in human
randomized controlled trials in the expectation of solving the problems of ever increasingly
ineffective pharmaceutically prepared medicines.
WHO estimate that 75% of the world’s population fulfil their health care necessities by using
herbs or plant-based medicines (Shah et al. 2020).
Objectives of this review
To investigate whether extracts of elderberry (S. nigra) fruit or flowers have antibacterial,
anti-viral and immunomodulatory effects compared with controls when tested in-vitro.
17
METHODOLOGY
Criteria for considering studies for this review
Only trials of S. nigra are in this study. Other species of the same genus are not included.
Types of studies
The only studies considered for this review are in-vitro trials. There is growing veterinary
interest in animal studies in the use of elderberry for the treatment of particularly farmed
animals suffering from diseases caused by microorganisms. However, for clarity these have
not been included.
Other systematic reviews of in-vitro trials were consulted to try to duplicate methods (Witt et
al., 2007; Tabatabaei-Malazy et al., 2012; Zusman et al. 2013). However, these, unlike those
in this paper, had enough similarity to allow for statistical comparisons. None of these papers
addressed the problem of bias.
It was decided to only include trials using the flowers and fruit of the elder. This is because
they are commonly used as food, are pleasant to taste, and therefore make a very acceptable
medicine even for children. If they can be shown to be better than placebo, then they would
make a good medicine for doctors to prescribe for common infections, including many viruses
when they now have nothing they can give.
It was initially decided in the protocol to include trials on microorganisms, and those that
show the immunomodulatory effects. This decision meant that one in-vitro trial showing that
elderberry extract killed the Toxoplasma gondii parasite was not eligible for inclusion (2015
in Revista de Biologica Tropical, no authors listed) even though this finding is of considerable
interest. Another paper studied the antimicrobial effects of elderberry, but also found that it
18
inhibits the yeast Candida albicans. This observation, too, is outside the scope of this paper
(Mohammadsadeghi, 2013).
Types of interventions
The interventions were all extracts of fruit or flowers of the elder, some prepared in the
laboratory, and others available commercially. This meant that the finding that the bark of the
elder, mentioned in the historical use of the plant and used in one or two recent trials, was not
eligible for inclusion even when it inhibited feline immunodeficiency virus (Manganelli et al.
2005).
Types of outcome measures
The method of measuring the antimicrobial effects of elderberry extract differed considerably,
as did the assessment of its effects on the immune system. These are described in individual
papers. This, unfortunately, increased the heterogeneity of results and increased the
impossibility of using meta-analysis on the papers. The field might be improved by some
standardization of methods, e.g. in some experiments the highest non-cytotoxic concentration
of extract was found, in others the end point was the dose of tissue culture which will infect
50% of the cells challenged with inoculum.
Developing a search strategy
Trials were searched in computerized databases, by checking bibliographies of relevant
articles, and by contacting manufacturers and researchers.
Firstly, similar papers involving herbal interventions were read to see what databases were
searched. The Cochrane library is one of the sources of such papers, but other herbal trials
were also searched.
In the protocol the databases to be searched were based on those used for herbal medicines:
19
• AMED
• CINAHL
• CISCOM
• EMBASE
• HERMED
• International pharmaceutical abstracts
• Medline
• Medline veterinary science search.
• Napralert
• Socpus
• Google Scholar
• WHO
• LILACS
• German private database Phytodoc
• Kew
• Web of knowledge /web of science
• Index Copernicus.
The large number of databases was because this subject crosses several disciplines.
Measures of treatment effect
There was no consistency between authors of the papers in the way in which they measured
treatment effect. For instance, some studies noted percentage of inhibition of bacteria in
liquid culture, others millimetres of inhibition bacteria on an agar plate. Viruses were
sometimes measured by plaque assay, for example. In other experiments they might be
measured by end point assays with TCID50 (this is the tissue culture infective dose which
will infect 50% of the cell monolayers challenged with inoculum); therefore, results, even of
the same microorganism, could not be entered into meta-analysis. This is a limitation of the
paper.
Dealing with missing data
Unlike in human trials where some patients go missing, data tends not to be missing within
in-vitro trials. If there are any missing data or missing papers they will be sought via inter
library loan, and via correspondence with authors. It is likely that some pre-internet papers
might not be found.
20
Heterogeneity of studies
Prior to examining the studies which qualified for inclusion in this paper, it was hoped that
there would be duplication of microorganisms tested, and that methods would be duplicated
also, so that experiments could be grouped and then meta-analysis could be conducted.
Assessment of reporting biases
The following has been included dealing with the system set up by the author to assess
reporting of bias. It should be noted, however, that not one in-vitro trial even mentioned the
possibility of bias, let alone did anything to mitigate it. Publication bias could be particularly
easy in the case of laboratory experiments where there are no human subjects, and where
experiments could be easily repeated without any external person knowing. Publication bias
could explain why there are few reports of negative results.
It would be possible to mitigate bias in in-vitro trials if all the trials to be conducted were
registered and numbered beforehand. The treatment and control experiments could be set up
randomly, and separate people used to set up experiments and record inhibition of the
microorganisms. Wishful thinking on the part of the experimenter can exaggerate treatment
effects in in-vitro trials.
Assessment of bias
It is extremely rare to do a systematic review of in-vitro trials. For this reason until recently
there were no methodological indices for the evaluation of such trials. JHWM found a group
of post-graduate medical research students discussing this online who provided an evaluation
and published it in Arthroscopy 30(2), 2 February 2014. They adapted a MINORS scale and
published it in Management of the contaminated anterior cruciate ligament graft (Khan et al,
2014). It is believed no other scale exists for assessment of bias in in-vitro papers. Since this
paper referred to ligament grafts it was not quite appropriate for the papers in this review,
21
which mostly refer to the effect of elderberry extracts on microorganisms. Therefore this
scale was adapted further, slightly, so that it applies to the research in this review. This made
a 12 point assessment of bias which may be able to be used again if a similar systematic
review of in-vitro trials is conducted in the future. JHWM took into account the work and
items assessed by Jeremy Howick on extraction sheets when choosing items to report.
Like the original MINORS scale, the Khan scale consists of 12 items. Each item is scored
from 0-2; for most items, 0 indicates that the item is not reported, 1 indicates that the item is
reported but inadequately, and 2 indicates that the item is sufficiently reported:
Assessment of bias
1.
Clearly stated purpose.
0, 1, 2
2.
Adequate control groups.
0 Control groups not adequately described.
1 Control group negative only.
2 Positive and negative control group.
3.
Clear description of the culture
and test material.
0, 1, 2
4.
Justification of number of trials
conducted.
0, 1, 2
5.
Appropriate statistical analysis
and scoring.
0, 1, 2
6.
Unbiased assessment of
outcome.
0 No description of outcome and evaluator.
1 Qualitative /subjective methodology or
blinded evaluator.
2 Qualitative and blinded or quantitative.
7.
The organism type and
quantity clearly described.
0, 1, 2
8.
Culture method clearly
described.
0, 1, 2
9.
Timing described beforehand.
0, 1, 2
10.
Preparation of the intervention
clearly described and
standardized.
0, 1, 2
11.
Risk of selective reporting.
High 0, Medium 1, Low 2.
12.
Other sources of bias.
High 0, Medium 1, Low 2.
22
Assessment of quality of reporting
The quality of reporting was considered as a separate issue to the assessment of bias.
However, bias is the most important issue in assessing a paper.
The author was unable to find a published checklist for the assessment of quality of reporting
of in-vitro trials and it is believed none exists. Therefore, the ARRIVE checklist (Kilkenny et
al. 2010) for reporting in-vivo animal experiments was adapted. Some questions concerning
welfare and health of animals did not apply at all and the resultant checklist, therefore, has
fewer items. It was felt to be clearest to convert the results from a scale out of a maximum
score of 14 to a percentage. This scale may be used in future systematic reviews of in-vitro
trials.
CHECKLIST FOR QUALITY OF REPORTING OF IN-VITRO TRIALS (modified
from ARRIVE guidelines checklist Carol Kilkenny et al. 2010).
ITEM
RECOMMENDATION
TITLE
1
Provide as accurate and concise a description of the
content of the article as possible.
ABSTRACT
2
Provide an accurate summary of the background,
research objectives, including details of the
microorganisms or strain, key methods, principal
findings and conclusions of the study.
INTRODUCTION
Background
3
a) Include sufficient scientific background (including
relevant references to previous work) to
understand the motivation and context for the
study, and explain the experimental approach and
rationale.
b) Explain how and why the microorganisms and
model being used can address scientific objectives,
and its relevance to human biology.
Objectives
4
Clearly describe the primary and any secondary
objectives of the study, or specific hypotheses being
tested.
METHODS
Study design
5
For each experiment, give brief details of the study
design including
a) The number of experimental and control groups.
23
ITEM
RECOMMENDATION
b) Any steps taken to minimize the effects of
subjective bias.
Experimental
procedures
6
For each experiment and each experimental group,
including controls, provide precise details of all
procedures carried out.
a) Drug formulation
b) Timing
c) Substrate and cells
Microorganisms
7
a) Provide details of the microorganisms used and
strain.
b) Provide further relevant information like source of
microorganism.
Sample size
8
a) Number of microorganisms used.
b) Indicate the number of independent replications of
each experiment, if relevant.
Experimental
outcomes
9
Clearly define the primary and secondary
experimental outcomes assessed.
Statistical
methods
10
a) Provide details of the statistical methods used for
each analysis.
b) Describe any methods used to assess whether the
data met the assumptions of the statistical
approach.
Results
11
Report the results for each analysis carried out, with a
measure of precision (e.g. standard error or confidence
interval).
DISCUSSION
Interpretation /
scientific
implications
12
a) Interpret the results, taking into account the study
objectives and hypotheses, current theory and
other relevant studies in the literature.
b) Comment on the study limitations including any
potential sources of bias, any limitations of the
laboratory model, and the imprecision associated
with the results.
c) Describe any implications of your experimental
methods or findings for the replacement,
refinement or reduction (3 Rs) of the use of in-
vitro methods in research.
Generalisability /
translation
13
Comment on whether, and how, the findings of this
study are likely to translate to other species or systems
including any relevance to humans.
Funding
14
List all funding sources (including grant number) and
the role of the funder(s) in the study.
When assessing the standard of the paper, there should be one mark for each numbered
question if a good example, or part thereof for part questions. Fewer marks for poorer
24
examples or queries like pharmaceutical company funding. The total should be converted to a
percentage.
Is statistical analysis possible?
In a systematic review of the effects of a medicine, it is desirable to be able to do a statistical
analysis of all the results together, preferably to facilitate forest plots (also known as
blobbograms) of the results. This is a graphical display of results obtained from a number of
scientific studies addressing the same question, along with the results obtained by collating
them. Confidence intervals are included and also a solid vertical line to represent no
treatment effect. If the collated results differ significantly from the no effect line then one can
be confident the treatment has an effect.
However, in all these in-vitro trials on the effects of elderberry (including the flower) on
microorganisms, there was too much heterogeneity to make statistical analysis possible. Two
Oxford statisticians, Mei-Man Lee and Jason Oke, were consulted to see whether any way
could be found to do statistical analysis. Many of the trials give p-values. These are
calculated probability of finding the observed, or more extreme, results when the null
hypothesis of a study question is true. Apparently, an old statistical technique was to pool the
p-values. However, this technique is no longer considered valid, and so was not deemed to be
suitable in this case. The statisticians decided that only a narrative report of results could be
used on the papers in this study. It would be ideal, and the cheapest way to get more certain
answers, if all the in-vitro trials in this review were repeated several times until it was
possible to do forest plots of them. It would be even better if this were done in different labs.
The lack of statistical analysis is a limitation of the paper.
25
RESULTS
Missing data
Only those databases openly available or available via Oxlip could be used. Therefore,
CISCOM, International pharmaceutical abstracts, Medline veterinary science search and
SOCPUS were, in the end, unable to be used. Some pre-internet papers might have been
missed.
For all the databases the search terms were: elderberry (Sambucus nigra) + in-vitro. The
exception was Google Scholar which was searched in detail for all papers concerning
elderberry (Sambucus nigra). There were approximately 5870 of these. All the citations for
inclusion were to be found on Google Scholar, although one was missed and picked up by the
librarian in her search. All the other databases found only a few papers each, with much
multiplication between databases. None found a paper not to be found on Google Scholar.
Searches were conducted by JHWM. The full search of Google Scholar was conducted on 8
April 2016 with smaller additional searches until 14 September 2016. Searches of the other
databases were conducted 20 April 2016. A later small search repeating the first was done 21
September 2016. A very brief additional search was done in May 2018.
Morag Evans, Master’s student in librarianship and employee of the Thomas Sydenham
Medical Library, Dorset County Hospital, searched Medline and Embase databases using
search terms elderberry (Sambucus nigra) in-vitro. This search was conducted 6 June 2016
and found two interesting papers.
N.B. A limitation of the study is that the second search of databases was not as
comprehensive as that conducted by JHWM. This was because a second researcher with
26
sufficient time was not available, and only Medline and Embase are available in Thomas
Sydenham library. However, this represents what is available to physicians in Dorset.
The reference sections of papers related to those qualifying for inclusion in the study were
searched to see whether this revealed other qualifying papers. Proceedings of a symposium
on elderberry were ordered via inter-library loan. Researchers in the field were also contacted
to enquire as to whether they have work in progress.
Selection and analysis of studies
JHWM made the final selection of the studies, although supervisor Dr Jeremy Howick was
consulted over one or two papers and as to whether the original parameters could be altered.
The results of the qualifying papers were carefully read, analysed and put onto an extraction
sheet on Microsoft Office Excel. All published papers which came within the parameters of
the question, and which could be found, whether or not they had a positive result, were
included in the study.
One important paper, Barak et al. (2002), proved very difficult to find, even with
correspondence with Israel. It was finally found via International Inter-Library Loan.
The key table missing from the Hearst et al. (2010) was finally found after considerable
“detective” work. One in Spanish is proving difficult to obtain. Only the abstract has been
found. No other missing data are known about. In the case of in-vitro trials there are no
recorded missing data within the papers, unlike in human trials where patients drop out.
27
Figure 1.
Heterogeneity of studies
There was considerable heterogeneity in the studies. Different extracts of elder were used,
some laboratory made, some available commercially. A large number of different
microorganisms were used, and since some were bacteria and some viruses, the procedures
for the in-vitro experiments differed. Even with the same microorganism, differing styles of
experiment were set up. As a result of this, after consultation with statisticians, it was decided
that only a narrative of results could be produced, and no meta-analysis was possible.
Screening
Identification
Eligibility
Included
28
For the sake of clarity, each of the papers which qualify for inclusion in the study will be dealt
with separately with results and discussion together. For papers concerning the effect of
elderberry on microorganisms the main results tables will be shown with p values when
available. A brief outline will be given, the microorganisms studied, the extract used, the
control used, results tables showing the amount of inhibition observed, the likelihood of bias
in the papers, and the quality of reporting. The papers will be summarized and appraised.
More weight will be given to papers considered to have greater clinical importance. There is
also a summary of results shown on a separate table at the beginning.
The chosen papers will be put into two sections: a) tests of effects of elderberry extracts on
microorganisms; b) immunomodulatory effects and molecule identification causing them.
5870 papers were found on Google Scholar with search term: elderberry (Sambucus nigra).
Eventually twenty-one papers qualified for inclusion in this review, one of which was found
by Morag Evans. Some papers tested the effects of elderberry on both bacteria and viruses, so
these could not be separated. All the citations for inclusion were to be found on Google
Scholar, although one was missed and picked up by the librarian in her search. All the other
databases found only a few papers each, with much duplication between databases. None
found a paper not to be found on Google Scholar. In all 15 papers were found which related to
the antibacterial activity of elderberry, and 6 papers relating to its effect on the immune
system.
Of 56 trials (15 publications) measuring inhibition of microorganisms using extract of flowers
or ripe fruit, 49 (87.5%) showed a positive effect. 10 trials of one lab made extract unlikely to
contain any flavonoids showed no inhibition. There were 6 immunomodulatory trials but it
was not possible to quantify these.
29
SUMMARY OF RESULTS TABLE 1
RESEARCH PAPERS TESTING MICROORGANISM INHIBITION BY ELDERBERRY EXTRACT
(FULLER RESULTS ARE GIVEN IN TABLES WITH EACH INDIVIDUAL PAPER)
Author/Year*
Microorganism tested by
elderberry extract
Intervention
Extract type
Comparator
Was there
inhibition?
Special
observations
Results
Signific-
ance
Krawitz 2011
Streptococcus pyogenes
Streptococcus Group C
Streptococcus Group G
Branhamella catarrhalis
Influenza A Kan
Influenza B mass
Staphylococcus aureus MRSA
Staphylococcus aureus MSSA
Haemophilus influenzae
Haemophilus parainfluenzae
Fruit Proprietary
elderberry
extract Rubini
(2 strengths)
No treatment
"
"
"
"
"
"
"
"
"
✓
✓
✓
✓
✓
✓
✓
More concentrated
Inhibits more
Varying amounts
Inhibited
99% →35%
e.g.
10% elderberry decreased Strep
pyogenes ≥70% compared control
20% elderberry decreased Strep
pyogenes = 99% compared control
Elderberry 1:100 decreased
Influenza Kan 70% compared
control.
Elderberry 1:100 decreased
Influenza Mass 75% compared
control
p<0.05
p<0.07
p<0.03
Zakay-Rones 1995
Influenza virus strains:
A/Beijing 32/92 H3N2
A/Shandong 9/93 H3N2
A/Singapore 6/86 H1N1
A/Texas 36/91 H1N1
B/Panama 45/90
B/Yamagata 16/88
B/Ann Arbor 1/86
A/Sw/Ger 2/81 H1N1
A/Tur/Ger 3/91 H1N1
A/Sw//Ger 8533/91 H1N1
Fruit
Proprietary
elderberry
extract.
Sambucol
No treatment
"
"
"
"
"
"
"
"
"
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
All strains inhibited.
Each strain inhibited
by different amount.
Number of TCID50 of virus
inhibited by dilution of Sambucol
1:8 conc. 1:16 conc.
Beijing 40 40
Shandong 40 ND
Singapore 4 ND
Texas 2 ND
Panama 2 ND
Yamagata 20 10
Ann Arbor 7 3.5
Sw/Ger 81 8 4
Tur/Ger 2 1
Sw/Ger 91 8 2
n/a
Chatterjee 2004
Helicobacter pylori
Elderberry fruit
from proprietary
elderberry
extract Optiberry
Clarithromycin
✓
More concentrated
extract works better
0.25% elderberry 30% inhibition of
Helicobacter pylori compared to no
treatment.
1% elderberry >90% inhibition
compared no treatment.
1% elder+Clarithrom = 100% inhib
p<0.05
30
SUMMARY OF RESULTS TABLE 1
RESEARCH PAPERS TESTING MICROORGANISM INHIBITION BY ELDERBERRY EXTRACT
(FULLER RESULTS ARE GIVEN IN TABLES WITH EACH INDIVIDUAL PAPER)
Author/Year*
Microorganism tested by
elderberry extract
Intervention
Extract type
Comparator
Was there
inhibition?
Special
observations
Results
Signific-
ance
Hearst 2010
Staphylococcus sp.
Listeria monocytogenes
Bacillus subtilis
Salmonella poona
Enterococcus faecalis
E. coli
Staphylococcus aureus MSSA
Staphylococcus aureus MRSA
Staphylococcus epidermidis
Pseudomonas aeruginosa
Serratia marcescens
Klebsiella aerogenes
Fruit and flower
tested
Lab made extract
Positive control
Ciprofloxacin
and
no treatment
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
Ciprofloxacin
inhibits more
e.g. (Zone of inhibition in mm)
Elder flower Cipro
S. sp. 15 25
L. monocytogenes 12 25
B. subtilis 12 27
S. poona 12 25
E. faecalis 14 18
E. coli 12 22
S. aureus MSSA 13 18
S. aureus MRSA 17 22
S. epidermidis 13 22
S. aeruginosa 9 24
S. marcescens 11 20
K. aerogenes 12 26
n/a
Arjoon 201
Mycoplasma mycoides subsp.
capri
Fruit. Two
proprietary
elderberry
brands
Elderberry-G
Elderberry-365
Compared to
activity against
Escherichia coli
and Bacillus
subtilis
✓
Different brands
fruit extracts inhibit
to different degree
Min inhibitory concentration
e.g. E. coli M. mycoides
mg/mL mg/mL
Elder-G 0.0038 0.38
Elder-365 0.1 0.001
n/a
Chen 2014
Infectious bronchitis virus
Fruit
Lab made
Solvent alone
✓
Inhibits at an early
point during
replication of virus
No cytopathogenic effect below
MOI 0.1
Elder +MOI 0.1 of virus = x 10-6
Elder +MOI 1.0 of virus = x 10-4
n/a
Harokopakis 2006
Porphyromonas gingivalis
Actinobacillus actinomycetem
comitans
Flower
Lab made
Negative
✓
✓
Dental infections
inhibited all
proinflammatory
activity
Control-1:32elder
P gingivalis pg/ml
e.g. TNF cytokine ≈ 5250 ≈1000
Actin. act ≈ 7000 ≈1500
p<0.05
31
SUMMARY OF RESULTS TABLE 1
RESEARCH PAPERS TESTING MICROORGANISM INHIBITION BY ELDERBERRY EXTRACT
(FULLER RESULTS ARE GIVEN IN TABLES WITH EACH INDIVIDUAL PAPER)
Author/Year*
Microorganism tested by
elderberry extract
Intervention
Extract type
Comparator
Was there
inhibition?
Special
observations
Results
Signific-
ance
Mohammadsadeghi
2013
Staphylococcus aureus
Bacillus subtilis
Pseudomonas aeruginosa
Escherichia coli
(Candida albicans) – a yeast
Fruit
Lab made
Okogun method
Negative
✓
✓
✓
✓
(✓)
Varying amounts
inhibited. Very
strong against C.
albicans
Minimum inhib. conc. (μg/mL-1)
S. aureus 5.000
B. subtilis 5.000
P. aeruginosa 3.320
E. coli 1.900
(C. albicans) 0.625
n/a
Karimi 2014
H9N2 influenza
Fruit proprietary
elderberry
extract
Sambucol
Amantadine and
negative controls
✓
Compared
favourably with
Tamiflu and
Amantadine
Dilution Viral m seg. copy no.
Control 1 1/E+7
Control 2 1/E+7
2^(-11) 1/E+7 see graph
2^(-6) 1/E+6 page 53
2^(-5) 1/E+2
p<0.05
Karakas 2012
Streptococcus pyogenes
Staphylococcus aureus
Staphylococcus epidermis
Escherichia coli
Pseudomonas aeruginosa
Salmonella tyhimurium
Serratia marcescens
Proteus vulgaris
Enterobacter cloacae
Klebsiella pneumoniae
Extract lab made
but badly
Leaves, wood
ripe and unripe
fruit used
5 antibiotic
controls
Very badly prepared
extract so results
may be invalid
Needs re-running
with good extract
All negative. No negative figures
recorded on this paper. Just positive
results for other herbs.
n/a
Fink 2014
HIV
Fruit
Lab made
With Enfuvirtide
✓
Super additive
effects with
antiretroviral
Elderberry concentrate compared
with Enfuvirtide alone
μg/ml Inhibition of HIV
1000 80%
200 65%
n/a
Rodino 2015
Staphylococcus aureus
Escherichia coli
Pseudomonas fluorescens
Bacillus cereus
Enterococcus faecalis
Flowers and fruit
Lab made
Negative and
chloramph-
enicol (CAM)
✓✓
✓✓
✓
✓
Fruit more effective
than flowers
Zone of inhibition mm
Flower Fruit CAM
S. aureus 9 11 20
E. coli 20
P. fluorescens 15 10.5 30
B. cereus 11 15
E. faecalis 12 19
p<0.05
32
SUMMARY OF RESULTS TABLE 1
RESEARCH PAPERS TESTING MICROORGANISM INHIBITION BY ELDERBERRY EXTRACT
(FULLER RESULTS ARE GIVEN IN TABLES WITH EACH INDIVIDUAL PAPER)
Author/Year*
Microorganism tested by
elderberry extract
Intervention
Extract type
Comparator
Was there
inhibition?
Special
observations
Results
Signific-
ance
Kinoshita 2012
Influenza A
Fruit
Lab made and
fractions
Ostelamivir
(Tamiflu)
(in-vivo)
✓
Stronger effect in
animals than in-vitro
Stimulates immune
system
Anti-viral activity: elder + influenza
(IC50.μg/mL)
A (during infection) 720 ±79
B (after infection) 3,600±590
n/a
Ramírez 2015
MRSA
Unknown
Unknown
✓
Full paper not
obtained
Abstract concludes there was
inhibition of MRSA
n/a
Roschek 2009
Influenza H1N1
Fruit
Lab made
Negative
✓
Identified active
component and
synthesized
Elderb. conc. % inhib.
Compared no treatment
(μg/ml) of infection
150 22
600 75
1000 94
R squared
= 0.92
* Authors + et al.
The degree by which the microorganisms were inhibited varied considerably between those microorganisms and dependent on the extract used and its
concentration.
33
SUMMARY OF RESULTS TABLE 2
PAPERS TESTING IMMUNOMODULATORY EFFECTS AND MOLECULE IDENTIFICATION OF ELDERBERRY EXTRACTS
Author/Year*
What did experiment test?
Conclusion
Barak 2001
Tested healthy immune system with
fruit / Sambucol
Activates healthy immune system
Possible that it causes cytokine storm
Barak 2002
Tested cytokines
Lower stimulation index LPS
Less likely causes cytokine storm
Frøkiær 2012
Assessed immune modulating activity
May exert anti-viral and immune enhancing effects
Voldvik 2015
Tested NF-KB activity controlling
immune response
Elderberries may have dampening effect on the production of cytokine
storm
Ho 2015
Looked at molecule causing
immunomodulatory effect
Molecule identified and synthesized
Olejnik 2015
Is elderberry denatured in gut?
Inhibits pro-inflammatory pathway, not denatured in gut
* Authors + et al.
Immunomodulatory means “modulates the immune system”. In medicine modulate means 1. To regulate or adjust to a certain degree: physiological
mechanisms that modulate the body’s metabolic rate. 2. Biochemistry. To act on (a receptor, for example) as an activator, an inhibitor, or both.
34
Narrative summary of papers
Table numbers and figures are those in the papers to which they refer.
a) Tests of efficacy of elderberry on microorganisms
Krawitz et al. (2011). Inhibitory activity of a standardized elderberry liquid extract
against clinically-relevant human respiratory bacteria pathogens and influenza A and B
viruses
Figure 1
Effect of the elderberry extract on bacterial growth. Visualization of Gram-positive and Gram-negative
bacterial strains exposed to different concentrations of Rubini elderberry extract during growth in liquid
culture. Every biological experiment was independently repeated at least three times with two replicates
per trial. Standard deviation is indicated. The p < 0.05 was observed for all bacterial species tested.
This paper tested elderberry extract against influenza and the most common super infections
after this virus.
The method used in this study was that the antimicrobial activity of the elderberry was
determined by bacterial growth experiments in liquid cultures using the extract at
concentrations of 5%, 10%, 15% and 20%. The inhibitory effects were determined by plating
35
the bacteria on agar plates. In addition, the inhibitory potential of the extract on the human
pathogenic H5N1-type A influenza virus isolated from a patient and an influenza B virus
strain was investigated using MTT (a colorimetric assay) and focus assay.
This experiment analysed a standardized, commercially available extract of elderberry, at
varying concentrations, against cell cultures of two different strains of influenza and three
Gram-positive bacteria and one Gram-negative bacterium all responsible for infections of the
upper respiratory tract. The bacteria tested were Streptococcus pyogenes, Streptococcus
group G, Streptococcus group C, Branhamella catarrhalis and Streptococcus mutans. The
infections caused by these bacteria can include pneumonia, acute otitis media and acute
sinusitis in young children, bronchopulmonary disease in older immunocompromised adults,
occasionally meningitis, and also sepsis and death. It is to avoid these diseases, the more
serious super infections after influenza, which are the main reason medication is needed
during an influenza outbreak. The authors also tested MRSA, MSSA, Haemophilus
parainfluenzae, and Streptococcus mutans. The results for these were negative.
Neuraminidase inhibitors Tamiflu and Relenza only treat the influenza viruses. If a treatment
were effective for both influenza and the bacterial super-infections that sometimes follow a
bout of influenza, it would be particularly beneficial.
The elderberry extract used for these trials was Rubini, a commercially available extract
made by BerryPharma. Full strength Rubini was called 1.0 and different dilutions used.
Dilution 0.1 inhibited bacteria > 70% when a no treatment control counts as 100%. Dilution
0.2 inhibited about 99% of some bacteria only about 35% of others. Like most of the in-vitro
trials the inhibition was shown to be concentration dependent, the greater the concentration
the greater the inhibition of the bacteria. P<0.05.
36
Figure 2
Impact of the elderberry extract on propagation of IV. A) Focus size reduction assay. MDCK cells
were infected with the two virus strains as indicated and incubated for 48 h in the presence of the extract
(1:100). Foci were detected by immunochemistry. B) Pre-treated MDCK cells were infected with pre-
treated virus and were propagated for 48 h in the presence of the extract. As control untreated virus and
cells were used for infection. The mean titre of three independent experiments is given as percentage of the
control (black) set to 100%. Standard deviation is indicated (KAN-1: p < 0.07, Mass: p < 0.03).
This was also true of the inhibition of the influenza virus, which was tested at a far greater
dilution and which as only inhibited by 30% (KAN-1) p<0.07and 25% (MASS) p,0.03. The
trial concluded that Rubini elderberry extract is active against human pathogenic bacteria as
well as influenza viruses.
37
Funding of the trials was partially by BerryPharma, the manufacturers of Rubini. This raises
the possibility that the results might only have been published because they were positive. It
would have been possible to suppress all publication if the results were not what the firm
wanted due to the financial conflict of interest.
By scoring the assessment of risk of bias it was felt that the possibility was fairly high,
scoring a fairly low number of 14/24. The instructions for carrying out the trials were clear,
but the report said that each trial was carried out at least three times. It should have been an
exact predetermined number; otherwise the trial could be repeated until the required score
was obtained. As in all the trials there was no blinding and the observer who took the results
could be the person who set up the experiment.
The most significant flaw in the paper is that the negative data where elderberry had no effect
against MRSA (skin and soft tissue infection sepsis), MSSA (which can lead to septicaemia),
Streptococcus mutans (dental and heart disease) and Haemophilus parainfluenzae (often
found in people with common obstructive lung disease) were not given. All these results
were not even mentioned in the abstract. Only very careful reading of the full text and
accompanying tables (not published together) would ever reveal that these further trials had
been conducted.
It was a good experiment and could be replicated, but was very badly let down by the way the
negative results were hidden. The elderberry did not inhibit four of the nine bacteria initially
tested. This gave a false impression to anyone who did not read the paper, with its hard to
find supplements, with extreme care.
38
Zakay-Rones et al. (1995). Inhibition of several strains of influenza virus in-vitro and
reduction of symptoms by an elderberry extract (Sambucus nigra L.) during an
outbreak of Influenza B Panama
ND above means no data. No significance given.
Only the in-vitro trials of this paper were examined for this appraisal. This paper tested ten
strains of influenza virus, both types A and B, and including strains found in animals. A
small in human randomized controlled trial, described above, showed significant
improvement in symptoms. Standardized elderberry (Sambucol) was used.
The viruses were grown on Madin-Darby canine kidney cells, and then titrated in the absence
of trypsin to receive a limited number of virus replication cycles. Then the final titration of
virus that gave complete cytopathic effect was used to test the protective effects of Sambucol,
and higher concentrations. The TCID50 inhibited by the elderberry was calculated from the
titre of MDCK. Then haemagglutination tests of the viruses were done using phosphate
buffered saline and sheep red blood cells. This is a rapid test to determine influenza virus in
a sample. Haemagglutination reductions were then done with Sambucol, and reduction of the
haemagglutination titre was assessed by comparison with controls.
39
Inhibition of infectivity was also determined by titration of the viruses with MDCK. The
final dilution that gave complete lysis was calculated, and then the viruses at concentrations
producing 100% complete cytopathic effect calculated. Then the infectivity was inhibited
with different concentrations of Sambucol.
The inhibition of the viruses was dose dependent. The dose needed to inhibit the viruses
varied considerably between strains of the influenza. It would be extremely interesting to
calculate whether there is a correlation between the dose of Sambucol needed to inhibit the
virus in-vitro, and the seriousness of the different strains, as calculated by mortality rates in
patients. However, Sambucol was shown to be anti-viral in-vitro.
It should be noted that SRBC incubated with Sambucol were agglutinated by influenza
viruses, suggesting that the extract inhibits the haemogglutinin of the virus itself and does not
interfere with the glycoconjugate receptor on the erythrocytes. In easier language the
elderberry would not appear to attack the patient’s cells, just the virus. This potentially very
important finding should not be overlooked.
The risk of bias was rated as 16/24, partially because of the closeness of the experimenting
team to the Sambucol company. The quality of reporting was rated as 75% but because of
financial conflict of interest negative results could be unreported and this could be less.
This paper comes from the University Hadassah medical school in Jerusalem. It should be
noted that Madeleine Mumcuoglu is the senior author, and the elderberry extract used was
Sambucol. Dr Mumcuoglu was one of the first scientists to study elderberry with proper
experiments, rather than just relying on its historic and folkloric use. Her work in this field is
of considerable interest. However, as well as being a scientist she is also the founder of the
Sambucol company which has become quite a big industry in Israel, growing and processing
the plant to pharmaceutical standards. This work may well be done with the upmost
40
integrity, but the close academic and commercial link raises the possibility of reporting and
publication bias.
Chatterjee et al. (2004). Inhibition of Helicobacter pylori in vitro by various berry
extracts, with enhanced susceptibility to clarithromycin
As this paper points out, 50% of the earth’s population is infected with Helicobacter pylori,
which has been implicated in the aetiology of chronic gastritis and peptic ulcer, in both adults
and children.
41
Several antimicrobial agents are effective against H. pylori, which is commonly treated with
more than one of these at the same time. Clarithromycin is a key component of many
therapeutic agents recommended for the eradication of H. pylori, but the treatments are often
accompanied by side effects, and resistance to Clarithromycin occurs. The tests used to see
whether the elderberry did indeed inhibit the bacteria was a bought one, CLO, which would
guarantee uniformity and its ability to be replicated.
The berries tested in this paper including elderberry (S. nigra) are all ingredients of the trade-
marked supplement Optiberry. There is no mention of funding of the experiment which was
conducted at a university. However, the lack of mention itself leads one to think it might
have been funded by the manufacturers, and therefore open to all the biases this brings,
including publication bias. The overall bias was scored at 19/24, a medium risk of bias. The
quality of reporting of the trial was rated as 73%. The results of the tests were not reported
with a degree of precision, but shown on a hard-to-read bar graph. This showed that
Clarithromycin after exposure to 1% elderberry inhibited the bacteria by nearly 100%, the
berry alone at concentrations of 0.25% by less than 30%. Elderberry at 1% alone inhibited the
bacteria about 95%. In all the papers listed here the concentration of the elderberry is
important.
The strain of H. pylori used was American type culture strain 49503. This strain was chosen
based on a previous study that demonstrated that a tissue culture model, the lactate
dehydrogenase leakage and superoxide anion production was greater than in other strains. In
other words, it is the strain which does the most damage. This is a good feature of the paper
and should be emulated in choosing the “worst” strains of bacteria and viruses for other trials.
Optiberry is a blend of six berry extracts; all the different berries were shown to inhibit H.
pylori. This is an indication that is possible that other berries may be found which have anti-
42
bacterial and anti-viral effects for other microorganisms. This gives further hope for
alternatives to antibiotics when more resistance occurs.
As with the other in-vitro trials, the results were of great interest but repetition of the trials is
needed to confirm them. Also, as with the other trials, no attempts were made to mitigate
against possible biases.
Hearst et al. (2010) Antibacterial activity of elder (Sambucus nigra L.) flower or berry
against hospital pathogens
The thirteen bacteria tested include the most unpleasant hospital pathogens. They are listed
in Appendix 5, together with the zone of inhibition recorded. These results have never before
been published. In the case of MRSA the zone of inhibition exhibited by 5 µm disc with
Ciprofloxacin is larger (approx. 22mm) than the natural product extracts. However, it must be
noted that the inhibitory elder flower extract exhibited against MRSA is a formidable 17 mm
despite being supplied in 100 fold dilutions of extract. Significance was not given for this
paper. It is possible some bioactive molecules could have been lost in the complicated
preparation of the extracts. Both elderflower and elderberry inhibited the bacteria, but in this
trial the flower was more effective.
Bacterial broth culture was plated to form a “lawn” of the test bacterial growth on agar
surface. Then sterile disks which were impregnated with plant extract were transferred to the
“lawns” and incubated. The zones of clearance were recorded to indicate antibacterial
activity. Ciprofloxacin was the control.
This paper should have been of great significance and importance because it tests the thirteen
strains of bacteria, including MRSA against elderflower, elderberry and the antibiotic
Ciprofloxacin to see how much these substances inhibit the bacteria. However, the results
were omitted and the importance of the work, which did seem to show that the elderflower
43
and berry do inhibit all of these nosocomial (hospital) pathogens to varying degrees, was lost.
The fact that it does seem to inhibit a wide range of bacteria which causes serious infections
is highly important when more and more bacteria are developing antibiotic resistance, and
ciprofloxacin is contraindicated for most uses in children. In the future it could become
impossible to do many now common operations due to hospital pathogens.
The risk of bias of this paper was rated as 20/24, fairly moderate. The quality of reporting
should have been good, but because of the failure to publish the results due to a printing error
this fell dramatically. This was not the fault of the authors. The whole paper should be
republished in the original journal and on Google Scholar free access. Otherwise future
researchers will not discover it.
The extracts used were prepared in the laboratory, so there was no possibility of bias due to
financial interests. Caroline Hearst, the principal researcher, seems to have been badly let
down by the Journal of Medicinal Plant Research failing to publish the results of one of the
most important of these in-vitro trials. Research which could be lifesaving in the future is
being hidden from public view. Even contacting the corresponding author proved very
difficult. No physician in an emergency would ever find this paper. The abstract is
insufficiently full and explicit but the authors do call their findings seminal and indeed, if
correct, they are. This paper deserves to be well known because it represents hope in solving
the serious problem of hospital pathogens, particularly MRSA. It could also uphold the
attempts to use S. nigra as an ingredient of disinfectant which might even be able to be used
in operating theatres in the future.
This is possibly the trial which most merits repeating a number of times at different
institutions, and doing forest plots to verify whether the results are valid, and it would be of
benefit to add Clostridium difficile to the list of bacteria tested. No scientific experiments and
their findings can be considered valid unless they are replicable by other scientists.
44
Arjoon et al. (2012). In-vitro efficacy of antimicrobial extracts against the atypical
ruminant pathogen Mycoplasma mycoides subsp. capri (goat)
MINIMUM INHIBITORY AND BACTERIOSTATIC CONCENTRATIONS A
aN = 3 replicates; MIC/MBC concentrations reflect mean values across trials. Abbreviations: N/A (not
applicable); BC (bactericidal); NI (no inhibition by solvents at the lowest dilution [1:10]). Efficacious
compounds with a measureable inhibitory concentration or bactericidal activity are bolded.
bColumns marked N/A or BC indicate that MBCs could not be determined due to lack of inhibition (N/A)
or bactericidal activity (BC).
cConcentration reflects total alkaloid content.
POST-INHIBITION ANALYSIS A
Compound
E. coli
B. subtilis
M. mycoides capri
Elderberry (365)
bactericidal
resistant
bactericidal
Elderberry (G)
bacteriostatic
bacteriostatic
bacteriostatic
aDefinition of terms: “resistant”-species was resistant to the compound; no analysis
performed; “bacteriostatic”: compound was shown to be bacteriostatic by growth upon
subculture; “bactericidal”: compound was shown to be bactericidal by lack of growth upon subculture.
N = 2 replicates. Tested against other herbal products and other bacteria. No significance given.
Mycoplasma is a genus of bacteria that lack a cell wall around their cell membrane. This
means that, without a cell wall they are unaffected by many common antibiotics such as
penicillin or other beta-lactam antibiotics that target cell wall synthesis. Mycoplasmosis is a
common infection in human and veterinary medicine. This experiment is on goat
Mycoplasma. However, in humans Mycoplasmae have a negative effect on fertility. M.
45
hominis causes male sterility /genital inflammation, and low birth weight preterm infants are
also susceptible to Mycoplasma infections. They are becoming a major problem in sexually
transmitted diseases.
This paper tested antimicrobial susceptibility by performing broth microdilution assays. The
lethal or inhibitory effect of each extract was determined by subculture into neat growth
medium. Amongst the commonly used medicinal (antimicrobial) plant extracts tested were
two commercially available brands of elderberry extract.
The two extracts of elderberry tested here were inexpensive and easy to obtain in the USA.
They were assessed as to whether bacteria were resistant to them, or whether they were
bactericidal (killed the bacteria) or bacteriostatic (stopped bacteria reproducing, while not
necessarily killing them). Elderberry 365 was found to be bactericidal to E. coli, B. subtilis
was resistant to it, and M. mycoides susp. capri was bacteriostatic. Elderberry Gaia was
bacteriostatic with all three bacteria. It is clear that the brand of elderberry used, if it is a
commercial brand, is important, for a hard to treat group of bacteria.
This experiment appeared to present a possible treatment. The risk of bias score was 22/24,
very low risk. The reporting quality was 73%.
The paper concludes that the in-vitro efficacy of plant extracts at their available
concentrations on atypical bacteria is vastly under studied. This is the only paper found on
this subject. The paper suggests that the plant extracts be used to disinfect agricultural and
milking equipment, but in future they could also be used to help disinfect hospitals and
operating theatres. This might make medicine possible in a hypothetical future when
antibiotics no longer work.
This experiment, like all the other in-vitro trials, was not blinded, when blinding could be
very cheaply and easily arranged with different people setting up the trials and reading the
46
assays. This simple measure could greatly add to its validity, as would repetition at different
institutions. The global problem of antibiotic resistance requires a cooperative response.
Chen et al. (2014). Sambucus nigra extracts inhibit infectious bronchitis virus at an
early point during replication
Pre-treatment of IBV with S. nigra extracts dramatically reduces viral titers. Cells and virus were
treated with 4.0 x 10-3 g/ml of S. nigra extract as indicated below. Infection was done at an MOI of 0.1.
Quantitation of virus titers at 24 h p.i. was done by plaquing in duplicate using neutral red staining.
Independent infections with IBV were performed three times, with two replicates per assay.
47
Treating IBV with S. nigra extracts prior to infection is necessary for full virus inhibition and works
synergistically with treating cells after infection. Cells and virus were treated with 4.0 x 10-3 g/ml of S.
nigra extract as indicated below. Infection was done at an MOI of 0.1. Quantitation of virus titers at 24 h
p.i. was done by plaquing in duplicate using neutral red staining. Independent infections with IBV were
performed three times, with two replicates per assay.
MOI means multiplicity of infection – the ratio of virus or bacteria to infection targets (cells).
This is an experiment about a chicken coronavirus, but since viruses often pass from animals,
particularly chickens, to mutate to become a human virus and, since it shows the inhibition of
a virus in-vitro by elderberry, it qualifies to be included in this paper. The elderberry extract
was prepared in the lab, and so there was no financial commercial conflict of interest. The
total score for assessment of bias was 19/24, medium. Like all the trials there was no
blinding and it would seem that the quality of trials could easily be better if there were better
training in conducting these trials. The highest non-cytotoxic concentrations of the extracts
were found. Two other herbal extracts were also tested but these, known to be anti-viral for
some viruses, did not inhibit IBV. To screen for infectious bronchitis virus (IBV) inhibition,
48
cells and viruses were pre-treated with extracts, followed by infection in the presence of
extract. Viral cytopathic effect was assessed visually following an additional 24 hour
incubation with extract. Cells and supernatants were harvested separately and virus titres
were quantified by plaque assay.
The importance of this experiment was not just to show that elderberry inhibited IBV. It was
also shown that the inhibition occurred at an early point during replication. This was done by
variation of this screening protocol by shortening it. It is possible that elderberry also works
to inhibit other viruses in the same way. This should be investigated and may throw further
light on the way in which elderberry works, and how it may be an important anti-viral.
Also very important to note is that pre-treatment of IBV, an envelope coronavirus, with S.
nigra results in extensive membrane change to the virus, likely rending it non-infectious. The
plant has now shown to inactivate two enveloped viruses which are chemically distinct.
(Chen 2014). This makes JHWM hypothesize that it may also inactivate Ebola, which
acquires an envelope by budding from the plasma membrane.
Work on veterinary uses of elderberry is being carried out in many countries. It is cheap, and
as has been shown, because of the shape of the bioactive molecules, resistance is unlikely
(Swaminathan, 2013). This use in animals is to be encouraged, as it could lessen the use of
antibiotics in farming.
In the recent journal Feed Additives 2020, Yasmin et al. discuss the use of elderberry as a
food additive, one of a number of herbal extracts, with use as anti-virals for farm animals to
reduce the amount of antibiotics used in the food chain.
The quality of reporting of the paper was rated at 78%. No exact results were listed, which
were given in graphs and no statistical validity was given. Otherwise, this is an interesting
paper which, as it concludes could potentially lead to effective treatments or prevention of
IBV or similar corona viruses. This was not an experiment which lends itself to statistical
analysis.
49
Harokopakis et al. (2006) Inhibition of proinflammatory activities of major periodontal
pathogens by aqueous extracts from elderflower
x
The chronic disease states such as periodontitis not only lead to loss of teeth, but can also
ultimately contribute to heart disease. In this experiment, the bacteria causing periodontitis
Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans were tested.
This was, in a sense, an immunomodulatory experiment because monocytes /macrophages or
neutrophils were incubated with the bacteria or purified components of them, with or without
elderflower extract and then they were assayed for cytokine production, integrin activation or
induction of the oxidative burst. The elder flower extract was found to potentially inhibit all
pro-inflammatory activities tested. The mechanism whereby this occurred was investigated
and discovered. It should be noted cytokine release was inhibited by S. nigra not increased.
This adds weight to evidence that elderberry will not cause a cytokine storm.
50
The funding for this paper was via a public health service grant. This is, indeed, a very cheap
treatment for a problem which occurs in an extremely high percentage of the world
population and which is a major cause of tooth loss. The elderflower extract can be made
from dried elderflowers. A year’s supply of the dried flowers costs approximately £5 in U.K.
The paper rated as being 21/24 at risk of bias, and since the work was publically funded at a
school of dentistry the risk of publication bias was low. The quality of reporting was
assessed as 75%. The whole experiment would benefit from being repeated at other schools
of dentistry. The elderflower extract is so benign that human trials hold very little risk, and it
is anticipated that the treatment could be adopted without the huge costs usually associated
with clinical trials.
Since elderflower (often used as a drink) did appear to display useful anti-inflammatory
properties, these could be exploited therapeutically for the control of inflammation in human
periodontitis.
Mohammadsadeghi et al. (2013) The Antimicrobial Activity of Elderberry (Sambucus
nigra L.) Extract Against Gram positive Bacteria, Gram Negative Bacteria and Yeast
This experiment used laboratory produced elderberry extract (to a precise recipe) to test on
agar plates whether it inhibited five bacteria (and also Candida albicans yeast). The bacteria
51
tested were Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia
coli, Salmonella typhi and Candida albicans (which is a yeast). These are important
pathogenic microorganisms involved in wound, burn and skin infections; enteritis; typhoid
and also yeast candidiasis. It inhibits candidiasis very well. All the microorganisms were
inhibited by the extract.
Agar plates were infused with different concentrations of the extract and the minimum
inhibitory concentration was found. This was very low for the yeast. The concentration
needed to inhibit S. aureus and B. subtilis was fairly high, but it was more effective against P.
aeruginosa, E. coli and S. typhi. The control was extract without inoculum and inoculum with
extract. There was no statistical analysis of results.
This was a paper from an Iranian medical school, but overseen by a veterinary school. It
would appear that elderberry is much used in veterinary medicine in Iran. It would be highly
desirable if it were more used for this purpose in the rest of the world, rather than antibiotics
being fed to healthy animals in farming – to which U.N. attributes much of the problem of
antibiotic resistance.
The research was considered to be at very low risk of bias 23/24 and the reporting quality
high 90%.
Karimi et al. (2014). The effect of Echinacea purpurea and Sambucus nigra L. on H9N2
avian influenza virus in infected chicken embryo
52
This was a veterinary paper from the veterinary department of an Iranian university. The use
of chicken embryos is considered to be an in-vitro technique, one which is often used in
immunology, and also because influenza often mutates and moves from chicken to humans,
the study is included. It was of particular interest because the results were compared directly
with Amantadine, commonly used as an anti-viral although it can also be used with
Parkinson’s disease. It is associated with some unpleasant side effects when it damages the
central nervous system. Amantadine showed no anti-viral effect against the virus.
Elderberry is considered safe as a food and without side effects, so in this respect it was also
53
superior to the Amantadine. In effect, it concluded elderberry compared quite favourably
with both Amantadine and Tamiflu.
The study was performed in two steps. First, the Neutralization index (NI) based on a
standard embryo dose 50 (EID50) method was used to evaluate the efficacy of the
extract/Amantadine. Serial dilutions of the virus were treated with a steady amount of the
diluted extracts prior to inoculation of the embryonated eggs. The inoculated eggs were
incubated for 48 hours at 37° C. In the next step, an equivalent of 500 EID50 of the virus
was treated with a serial dilution of the extracts before inoculation. The titre of the virus in
the allantoic fluid was evaluated using haemagglutination (HA) and the Real
time/Polymerase chain reaction tests (qRT/PCR). The results confirmed the significant
virucidal effects of the elderberry (and Echinacea) against H9N2 avian ’flu virus.
The trial was rated as 23/24 risk of bias, very low, and the quality of reporting at 90%.
Therefore, this experiment, unlikely to be read by physicians considering a prescription for
’flu virus, appearing as it did in an obscure Croatian veterinary journal, nevertheless deserves
closer attention.
Karakas et al. (2012). Biological screening of various medicinal plant extracts for
antibacterial and antitumor activities
This screening of a great many commonly used Turkish medicinal plants should have been of
great interest. It scored 20/24 as risk of bias and elderberry was tested against Streptococcus
pyogenes; Staphylococcus aureus; Staphylococcus epidermis; Escherichia coli; Pseudomonas
aeruginosa; Salmonella typhimurium; Serratia marcescens; Proteus vulgaris; Enterobacter
cloacae and Klebsiella pneumoniae. There were five antibiotic controls. However, whilst
some plants were shown to have antibacterial activity, the results for S. nigra were negative
and not even on the accompanying table. All the same, when the trial details were read
carefully it was clear that the elder plant extract used was prepared from all parts of the plant,
54
and anyone understanding the bioactive components of the plant would not have expected
these extracts to work, drawn as they were from picking any part of the elder: leaves, bark,
stem, fruit (ripe or otherwise). It is the flowers and ripe fruit that contain flavonoids. It is also
clear from this and other experiments that the concentration of flavonoids in the extract is
critical to how well they work.
This trial, using as it did clearly identified strains of bacteria, should be re-run using well
prepared elderberry fruit extract. As the trial stands, the results cannot be considered valid.
Fink et al. (2014). HIV type-1 entry inhibitors with a new mode of action
55
This paper says it is “mining” compounds known in nature to find anti-virals because of the
resistance to anti-viral strains of infections, particularly in HIV-seropositive and AIDs
patients.
A newly developed direct binding assay with mass spectrometry technology (direct analysis
in real-time time-of-flight mass spectrometry) was used to show that compounds in extracts
56
of elderberry and other herbs (cinnamon and green tea) bind to and block HIV type-1 (HIV-
1) infection in target cells.
The anti-HIV-1 activity of the elderberry was evaluated using HIV-foci reduction assay. The
amount of inhibition was dependent on extract concentration, and the results were compared
to the inhibition of HIV-1 by Enfuvirtide, an antiretroviral. The elderberry was being tested
for its potential to be developed into a new pharmaceutical antiretroviral, but may also have
potential to be used by poor HIV sufferers who cannot afford drugs, particularly in countries
where the plant grows.
The paper was considered to be at medium to low risk of bias at 19/24 risk of bias, and the
quality of reporting 65%. The direct binding assay would make manipulation of data
extremely difficult so it might score even higher than these estimates.
The elderberry extract together with Enfuvirtide, an antiretroviral, revealed significant super
additive effects. It appeared to magnify the effects of the antiretroviral by a factor of about
six. This was not an experiment for which statistical analysis was possible.
Fink et al. (2009) reported that the flavonoids and A-type proanthocyanidins present in
elderberry extracts block HIV entry and infection in ghost cells (Vardini et al, 2015). Their
paper, together with Manganelli (2005), which showed that elderberry bark has significant
anti-viral effect on feline immunodeficiency disease, and Konlee (1998) means that the
elderberry plant may prove to be a very important medicinal plant in the future when present-
day anti-retrovirals for AIDS become less and less useful due to resistance. Many AIDS
sufferers are extremely poor, particularly in sub-Saharan Africa and they cannot afford any
medicines. In the USA a year’s treatment of Enfurvitide costs about 25,000 dollars.
Elderberry will grow in some African countries, and this may offer some hope to patients if
further trials do indeed show it to be effective. The aim of the authors of the paper is to
synthesize a new antiretroviral of the active molecule in elderberry. However, this should not
57
just be developed as an expensive drug; the knowledge about a plant, which can be free to
many, should be widely advertised.
Rodino et al. (2015) Comparative studies in antibacterial activity of Licorice,
Elderberry and Dandelion
Figure 1. The inhibition zones (mm) of the extracts obtained by percolation tested by disc diffusion method
(results are shown as mean ± SD from triplicate).
Figure 2. The inhibition zone of the extracts obtained by ultrasonication, tested by disk-diffusion method.
Bars with the same letters are not significantly different, according to Tukey test (P <0.01). (Results are
shown as mean ± SD from triplicate.)
58
This experiment tests elderberry and other herbs against Staphylococcus aureus (causes skin
infections, pneumonia, endocarditis and toxic shock); Escherichia coli (can cause bloody
diarrhoea, urinary tract infections); Pseudomonas fluorescens (causes septicaemia and
peritonitis); Bacillus cereus (causes diarrhoea and vomiting) and Enterococcus faecalis (UTIs
endocarditis, bacteraemia and wound infections). The control was the absence of plant
extract.
Ethanolic elderberry extract was shown to have an antimicrobial effect against E. faecalis, E.
coli and P. fluorescens. It did not perform best amongst all the herbs tested. The efficacy of
the plant extracts used depended on the way in which the plant extracts were prepared. Both
fruit and flowers were tested.
This paper, too, is an attempt to find substances which have antimicrobial activity, when
acquired resistance to pathogens to antibiotic products existing on the market keeps
increasing. Varro (2000) seems to be correct that plants are the medicine of the future.
This Romanian paper was funded by some official body, but since this body is identified only
by initials, it is not certain whether it is a commercial pharmaceutical company or a
government research body. In Romania elderberry has continued to be used in medicine
extensively, whereas in the UK its use had been largely forgotten since World War II.
The plant material used was prepared in such a way that it would be difficult to reproduce it
exactly. Otherwise, the experimental technique was considered medium to good and the risk
of bias low at 19/24 and the reporting quality, with some omissions 60%. Not all the results
were given in the paper, and results were only shown as graphs. This was a veterinary
research paper from a country where financial interests from “big pharma” are in all
probability low. Therefore the results do show that there is likely to be genuine antibacterial
activity by elderberry, at least against some bacteria, if not all.
59
Kinoshita et al. (2012) Anti-influenza virus effects of elderberry juice and its fractions
CJ-E means elderberry juice. No control in-vitro, so no significance given.
This paper included a small in-vitro trial with an in-vivo trial in mice. It goes just a small
way to answering the question as to whether the anti-viral activity in a live animal would be
as great as it has been shown to be in-vitro. It showed a relatively strong effect in influenza
infected mice, whereas its anti-viral activity was weak in a cell culture system. Its conclusion
is that elderberry enhances the immune system. Ostelamivir was used as a control.
The in-vitro results showed that elderberry possess anti-viral activity, and also suggested that
elderberry would interact with the early stages of viral replication including viral attachment
to the penetration into host cells. The in-vitro activity was assayed using MDCK cells in
bovine serum culture and then plaque assays were conducted.
The risk of bias was rated as 20/24 and the quality of reporting as very high at 95%.
In-vivo, the authors concluded that stimulating the immune system contributed mainly to the
anti-influenza activity of elderberry, whereas Ostelamivir (Tamiflu) does not have this effect
and the anti-viral activity of this drug in animals was lesser. It is an important finding to have
elderberry directly compared with a commercial neuraminidase inhibitor, with the plant
having the greater effect. They may however be wrong in the other conclusion as other
experiments suggest that viruses are inactivated by elderberry.
60
The paper was written mainly by the research and development division of the Kikkoman
Corporation in Japan, a major manufacturer of soy sauce, one of several companies showing
interest of elderberry as a medicine, as such, there may be financial conflict of interest.
The authors are suggesting anti-viral activities are relatively higher in-vivo that in-vitro, but
this finding needs further investigation. If it is, then the potential for elderberry may be even
greater than supposed from the rest of this review.
Ramirez et al. (2015) Actividad antibacteriana de extractos de plantas contra
Staphylococcus aureus resistente a meticilina (SARM)
We have as yet been unable to obtain the full copy of this paper, but from the Spanish
abstract it was clear that more plants (one of which tested was elderberry) had shown some
anti-bacterial activity against MRSA, duplicating some of the findings in the Hearst et al.
(2010) paper. This important finding for such a problematic bacterium deserves greater
investigation, and it is regrettable that one paper which found this was published without the
most important table, and the second paper is available only to those fluent in Spanish who
manage to obtain a copy.
61
Roschek et al. (2009). Elderberry flavonoids bind to and prevent H1N1 infection in-
vitro
This was not an experiment for which statistics were appropriate.
This in-vitro experiment did not just show that influenza H1N1 is inhibited by elderberry
flavonoids. It went a step further and identified the main active components and went on to
synthesize it. As such, as is showed in some of the acknowledgements, it is an attempt by the
pharmaceutical industry to find a new anti-viral that it can manufacture and patent. However,
the very attraction of elderberry as a medicine is that it can grow over much of the world, is
cheap, pleasant to taste and un-patentable. It might not be necessary – in fact it may be
counterproductive – to synthesize and patent an active molecule in the elderberry when other
components of the berry may also have a beneficial role as an antimicrobial. The risk of bias
of this paper was rated as 14/24, fairly biased, and the quality of reporting was 65%, fairly
low.
The direct binding assay technique was good at identifying the component of the berry which
binds to and prevents H1N1 infection, however, this was the only, ’flu virus, or any virus
62
tested. In experiments from other scientists elderberry has been shown to inhibit many
viruses, but it has by no means been proven that the active component is the same in every
case. It is possible, as herbalists claim, that the efficacy of herbs relies on the complexity and
number of different molecules in the whole plant and that these work synergistically to get
results. Elderberry contains a number of flavonoids and it is likely that different ones bind to
different viruses and bacteria. H1N1 influenza infections can indeed be very serious, but the
MAGPIE trials showed that there can be very cheap answers to serious health problems. The
paper concludes that the anti- H1N1 activities of the elderberry flavonoids compare
favourably to the known anti-influenza of Ostelamivir (Tamiflu) and Amantadine. Lectins in
elderberry are also antimicrobial.
It was extremely ingenious to synthesize a bioactive molecule, but it may not be the most cost
effective way to obtain a molecule widely available in nature, and some other of the
flavonoids in elderberry may have a bioactive role against different microorganisms.
Keyaerts et al. (2007). Plant lectins are potential inhibitors of coronaviruses by
interfering with two targets in the viral replication cycle
This paper has not been included in the main tables of results, despite the fact that it tests a
very interesting subject. It is an in-vitro trial of 33 different plant lectins on severe acute
respiratory syndrome (SARS), the novel and serious coronavirus. It is not included because it
only includes the effect of one lectin from each plant whereas plants contain many bioactive
molecules.
Lectins are any class of proteins, chiefly of plant origin, which bind specifically to certain
sugars and so cause agglutination of particular cell types. It is the authors’ hypothesis that it
is these plant lectins which bind to and destroy the cell walls of the virus. In the case of those
plants which contained mannose specific lectins, inhibition and inactivation of the virus was
63
shown. However, the lectin which they chose to test, derived from S. nigra, showed no such
viral inhibition. But the European Medicines Agency (2013) monograph on S. nigra clearly
lists a great number of different bioactive molecules in S. nigra including more than one
lectin. It is not clear, therefore, why only one molecule was tested which showed no binding
of that lectin to the SARS virus. It is likely the authors of the paper were preparing to
artificially synthesise those chosen molecules. Most of the plants chosen to be tested in this
paper are poisonous to humans. However, of great interest is the fact that the lectin derived
from leeks (Allium porrum) DID inhibit the SARS virus. This means that in countries where
they grow it, drinking leek soup may prove to be an effective treatment for coronavirus.
JHWM’s recommendation, when there is no better medicine for SARS and other novel
serious coronaviruses, would be to consume leek soup and elderberry cordial (because
Sambucus does inhibit several coronaviruses) and this might give a double opportunity to
disrupt virus replication. This would be a gentle and safe treatment regime for all ages and
all levels of fragility of patients in the absence of other more effective antivirals.
Statistical pooling of results
The extreme heterogeneity of the methods, elderberry extracts used and microorganisms
tested means that statistical pooling was deemed by statisticians to be impossible. However
Table 1 summary of results gives a summary of the research papers, the microorganisms
tested and whether or not they were inhibited by the elderberry extract.
Postscript
Since this review was first written, Torabian et al. (2019) conducted more in-vitro tests to
study the effects of elderberry extract on the treatment of ’flu. They found that elderberry
exhibits multiple modes of action against influenza infection.
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b) Composition of elderberry molecules and immunomodulatory effects
It is claimed that elderberry has an immunomodulatory effect. This section will investigate
that claim.
Appendix 2b gives a fuller explanation of the implications of elderberry’s
immunomodulatory properties.
Barak et al. (2001). The effect of Sambucol, a black-elderberry-based, natural product,
on the production of human cytokines. 1 Inflammatory cytokines
Figure 5
The effect of Sambucol and LPS on production of a) IL-1beta, b) TNF-alpha, c) IL-6, d) IL-8 in a donor
(K.L.).
65
66
67
This study was aimed to assess the effect of Sambucol products on the healthy immune
system – namely its effect on cytokine production. In this experiment, elderberry extract was
shown to stimulate cytokine production, particularly TNF-α. It produced more TNF-α than
did lipopolysaccharides, commonly used as a control because of its known stimulation of the
immune response. TNF-α is a cytokine which can, under different conditions, produce both
favourable and unfavourable effects. The paper acknowledges this; however, this is only on
very close reading, and the conclusion only points out the positive effects of immune
enhancement. It concludes that Sambucol activates the healthy immune system by increasing
inflammatory cytokine production. Sambucol might therefore be beneficial to the immune
system activation, and in the inflammatory process in healthy patients with various diseases.
Sambucol could also have an immuno-protective or immuno-stimulatory effect when
administered to cancer patients or AIDS patients.
The paper rated a 19/24 as a risk of bias, a medium risk. It also scored 70% on quality of
reporting. There was no funding from the pharmaceutical company; however there are strong
cultural links between Sambucol, an Israeli started company, and the Hadassah medical
organization who wrote the paper.
*
*
Dr Madeleine Mumcuoglu of the Hebrew University of Jerusalem sold the company in 2006 and has no more
financial connection with the company. However she retains a strong interest in encouraging elderberry
research.
68
Correspondence to the authors to enquire if they had studied Sambucol further to see whether
it stimulates a cytokine storm received no reply. It is 15 years since this paper was written
and if any studies on this question have been conducted, they have not been published except
the one below which was extremely difficult to obtain. A statement that there is no evidence
is not the same as evidence that there is no cytokine storm.
Correspondence about his paper with Professor Brian Angus, professor of immunology at
Oxford University, received this reply: “The fact that it stimulates more cytokine effect than
bacterial endotoxin (LPS) is not a good thing.” I.e. In the opinion of Professor Angus there is
a danger that with a microorganism which has the potential to stimulate a cytokine storm,
elderberry could make matters worse.
This following paper by Barak et al. (2002) was exceedingly difficult to obtain, not available
in any form from Oxford University Library and was available only from the British Library
via International Inter-Library Loan.
Barak et al. (2002). The effect of herbal remedies on the production of human
inflammatory and anti-inflammatory cytokines
69
This paper is from the same team and laboratory as the previous paper, and rates very similar
scores for assessment of risk and quality of reporting. One problem with the publication bias
was the very difficulty of obtaining a copy of the paper at all. Very few physicians would
ever persevere to the extent necessary to obtain it.
It too points out that the cytokines tested TNF-α, IL-1β, IL-6 and IL8 are multi-potential
mediators of the immune response. It says that unfavourable effects may occur at very low or
very high concentrations of these cytokines. In this paper, unlike the previous one, elderberry
is shown to produce a lower stimulation index than lipopolysaccharides. This would seem to
make the danger of cytokine storm lower than it appeared before. Again it suggests
Sambucol be used for healthy patients with influenza, or in immune-depressed patients, such
as those with cancer receiving chemotherapy, or with HIV patients.
It should be noted that this paper showed the presence of an anti-inflammatory effect by
elderberry extract, as did Harokopakis et al. (2006).
The Sambucol company later issued their statement that there is no evidence it produces a
cytokine storm
Frøkiær et al. (2012). Astragalus Root and Elderberry Fruit Extracts Enhance the IFN-
β Stimulatory Effects of Lactobacillus acidophilus in Murine-Derived Dendritic Cells
This is a veterinary research paper. There is considerable interest in the use of elder in
veterinary medicine because it is cheap and it is thought it can be used instead of antibiotics
to prevent farmed animals and fish from catching diseases or to treat them if they are
suffering. Diseases spread quickly in farmed creatures. These have very similar immune
systems to humans with the same cytokines. Therefore, it is relevant to report an in-vitro trial
such as this which uses murine (mouse) derived dendritic cells.
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As the authors say,
Dendritic cells are professional antigen presenting cells that respond to a
microbial signal through pathogen recognition receptors such as toll-like
receptors (TLRs) and orchestrate the responses of other immune cells.
Thus dendritic cells are model cells that can be used to assess the immune modulating
activities of the elderberry. They concluded that elderberry fruit extract increases the IFN-β
inducing activity of Lactobacillus acidophilus in dendritic cells, suggesting that they may
exert anti-viral and immune enhancing activity.
The risk of bias for this trial was rated as 18/24.The quality of reporting was rated at
80%.The team for this experiment received help from NATUREX, a company sourcing raw
products for health materials, even though there was no patent involved. Other than this
possible source of the bias, risk of bias was low and the preparation of all cultures and
extracts clear and replicable, the paper accurately says they were the first to conduct this
exact experiment, but they omit any reference to other work on the immunomodulatory
effects of elderberry. It was in an open access journal, freely available online, which is in its
favour. The financial assistance from NATUREX was clearly acknowledged, but this might
imply that if there were negative results the paper might not have been reported. There is no
similar organization to the all trials campaign for veterinary in-vitro trials, and negative
results could have easily been “buried”.
The use of elderberry in farming is to be encouraged so that fewer antibiotics are used.
Voldvik, Vibeke (2015) Master thesis. Effekt av svarthyllbær (Sambucus nigra) og dets
innholdsstoffer på in vitro modulering av NF-κB-aktivitet
This is a full master’s thesis written in Norwegian, and it has not been possible to translate
and read the whole dissertation. Therefore it has not been possible to critically appraise it
properly. However, the English abstract does go some way to answering whether elderberry
might cause a cytokine storm and dangerous inflammation.
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NF-κB (nuclear factor κ-light-chain enhancer of activated B cells) is a protein complex that
controls transcription of DNA, cytokine production and cell survival. It plays a key role in
controlling the immune response. Lawrence (2009) says that NF-κB is a prototypical pro-
inflammatory pathway.
Vibeke Voldvik tested methanol extracts of all the elderberry cultivars grown in Norway.
Raw and heat-treated berries were tested, and she found that heat treated berries had a higher
total phenol content and higher total monomeric anthocyanins than those not so treated. It
was found that different quantities of the polyphenols which are believed to have the
immunological effects were found in different varieties.
Human monocytic cells were stimulated with either lipopolysaccharides or the different
elderberry extracts and changes in the NF-κB activity measured.
The conclusion found was that elderberries cultivated in Norway, had a questionable effect
on the ability to induce basal NF-κΒ activity, but a potential inhibiting effect on LPS-induced
NF-κΒ activity. Elderberries may therefore have a dampening effect on disease and disorders
by causing elevated NF-κ activity. This conclusion needs further investigation because it
could be that elderberry dampens inflammation and may dampen cytokine storms.
This dissertation is therefore of great interest as it may show that elderberry could be the
answer to how to lower deaths due to cytokine storms in “killer” strains of influenza, SARS,
MERS and other similar diseases.
It is highly recommended that immunologists who specialize in cytokines should examine the
effects of elderberry on cytokine activity in detail to see whether it could cause or dampen
down a cytokine storm, since the answer could potentially save millions of lives in the case of
a pandemic such as the one which occurred in 1918.
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N.B. Sambucus nigra Haschberg, an Austrian variety, is widely used in commercial orchards
because it is believed to contain the greatest concentration of anthocyanins.
Ho, Giang Thanh Thi et al. (2015). Structure-activity relationship of
immunomodulating pectins from elderberries
This paper looks in detail at the structure of the molecule which has an immunomodulatory
effect in elderberries.
Olejnik et al. (2015). Anti-inflammatory effects of gastrointestinal digested Sambucus
nigra fruit extract analysed in co-cultured intestinal cells and lipopolysaccharide-
stimulated macrophages
This paper will not be appraised in full, because it is a little off topic, not concerning the
effects of elderberry on microorganisms. However, it does shed further light on whether
elderberry is an anti-inflammatory in other diseases, such as inflammatory bowel disease,
rheumatoid arthritis, chronic asthma etc. and therefore whether it could potentially have an
anti-inflammatory effect in the case of a cytokine storm. By creating an artificial alimentary
tract it also partially answers the question as to whether the extract would be totally digested
and denatured in humans and therefore lose its therapeutic ability, or whether it continues to
be effective after digestion. It found that it was indeed effective after digestion.
The results obtained showed that S. nigra fruit has the ability to inhibit pro-inflammatory
pathway in LPS to stimulate macrophages by down-regulating the expression of pro-
inflammatory genes and to reduce enhanced production of mediators crucial for the initiation
and progression of inflammation, which is a significant risk factor to develop various human
diseases. (LPS-lipopolysaccharides these are found in the cell membranes of bacteria). This
inhibition of the pro-inflammatory pathway is the major finding of the paper.
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This work, funded by the Polish ministry of science – a country where elderberry is much
used – does not appear to be at significant risk of bias. It was conducted in a reputable Polish
university. The artificial alimentary tract created was extremely ingenious.
These Voldvik and Olejnik and Harokopakis papers suggest that, whereas on first
examination it appeared that elderberry might exacerbate inflammation in the case of serious
disease with danger of cytokine storm causing death, on the contrary, the medicinal plant may
actually mitigate against such an outcome. However, immunologists certainly need to
examine and confirm this conclusion.
Since this dissertation was first written Torabian et al. (2019), already mentioned in the
previous section, found that elderberry does possess immunomodulatory properties through
stimulation of cytokines.
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DISCUSSION
Summary
Elderberry inhibits many microorganisms in-vitro, and seems generally safe.
A wide variety of bacteria and viruses were tested to investigate whether they were inhibited
by elderberry extract in-vitro. It appeared that different brands of extract inhibited
microorganisms by differing amounts. Generally, more concentrated solutions inhibited
microorganisms more than less concentrated solutions. Nosocomial infections (hospital
“super-bugs”) including MRSA were tested against no treatment and an antibiotic. These
were inhibited compared to no treatment, but not so much as by the antibiotic. Mycoplasmae
were also inhibited. Different strains of influenza were inhibited by differing amounts.
Evidence suggests inhibition of a virus by the extract may occur at an early point during
replication.
Elderberry has been shown to inactivate two distinct envelope viruses. The plant disrupts
virion structure by compromising the membrane, likely rending it non-infectious. This makes
JHWM hypothesize that it is probable that it may inactivate Ebola, which is also an envelope
virus, and that it would be highly desirable to test this as soon as possible, since effective
medicines need to be found to treat this deadly microorganism.
In-vitro trials of SARS-CoV-2 (which causes Covid-19) on elderberry are planned but are yet
to be a carried out. However, two case studies and verbal reports are highly suggestive that
elderberry may be effective for this virus.
Whilst in-vitro trials found that elderberry extract appeared to compare favourably with
Tamiflu and Amantadine, very badly prepared extract did not inhibit bacteria and it seems
that the method of extraction and therefore the flavonoid content is crucial. In HIV, one
experiment appeared to show super additive effect with antiretrovirals. Attempts are being
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made to identify and synthesize active components of elderberry. A direct comparison
between the antimicrobial properties of elderberry extract in-vivo and in-vitro suggests that it
inhibits more in in-vivo than in-vitro. However, the risk of bias in this field is considered
fairly high and must temper any conclusions which can be made.
In investigations of elderberry extract on the immune system, initial experiments suggested
that it might cause a cytokine storm. But later experiments by the same team refuted this
finding. It is thought that it might exert antimicrobial and immune enhancing effects, but may
actually dampen a cytokine storm. It was found to inhibit the pro-inflammatory pathway, and
not be denatured in an artificial gut.
How this fits with other evidence
When considering elderberry as a potential medicine, it is generally considered to be a safe
food plant, consumed regularly in many European counties. Rødahl (2011) reported in a
master’s thesis that that there are no increased risks of adverse pregnancy outcomes after it
has been used in pregnancy. It can be used in all ages from babies to the elderly and infirm.
This refers to the consumption of flowers or heat treated berries.
The evidence in this review provides a theoretical basis for the emerging clinical evidence in
this area. For example:
• Vlachojannis et al. (2010) is a review which concluded that elderberry may provide
antioxidant, anti-viral and antiproliferative effects in-vitro. It also says that
antibacterial and anti-inflammatory effects seem possible, but this needs finding
further support. However, many of the antibacterial experiments that have now been
conducted are not reported here, and many of these have great clinical importance.
Only anti-viral effects are reported against colds and ’flu and not the anti-HIV trials,
despite the poverty in many countries, which makes it very important to find cheap
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alternatives to expensive antiretrovirals. There are no reports of exact results or
comparison with antibiotics or other neuraminidase inhibitors.
• Sidor et al. (2015) is a review which looks in detail at the antioxidant and health
benefits of elderberry as a food. Amongst other properties it says it helps prevent the
incidence of cardiovascular diseases and diabetes. The authors say that elderberry is a
good source of protein. It has medicinal properties associated with the presence of
polyphenols, which are compounds with potential antioxidant properties. They can
greatly affect the course of disease processes by: counteracting oxidative stress;
exerting beneficial effects on blood pressure; reducing glycaemia; stimulating the
immune system; potentially reducing tumours; increasing the activity of antioxidant
activity enzymes in the blood plasma, including also glutathione, and reducing uric
acid levels. It points out that the flowers contain tenfold more flavonols than do the
fruit. However, with all these healthy properties, the paper makes no mention that it
might be antimicrobial and might help in cases of bacterial and viral disease.
• Porter et al. (2017) review the anti-viral properties of elderberry and recommend it as
an anti-viral, particularly for colds and flu. They found that the evidence from clinical
trials for these uses rate as level B (good) on the Natural Medicines Standard
Evidence-Based Validated Grading Rationale. This finding adds weight to the
possibility that it might already be suitable to prescribe elderberry for colds and ’flu
where no suitable alternative exists.
• Mahboubi (2020) has done a review of the use of elderberry fruit and flowers as an
alternative treatment for colds and ’flu.
• Ulbricht et al. (2014) is a systematic review which deals with trials on all parts of S.
nigra rather than just the flowers and heat-treated fruit. It must be noted that other
parts of the plant contain cyanides and should only be used with caution under
medical supervision. (Unripe, un-heat-treated fruit also contain a small amount of
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cyanides so elderberry should always be eaten cooked for at least five minutes, as this
denatures these cyanides. Cooking also increases the phenol content). The review
includes trials of herbal products including elderberry with other herbs, even though it
cannot be determined which of the herbs had the supposed beneficial effects. It
critically appraises papers including those studying other conditions, not just
antimicrobial effects, or immunological effects. However, it omits many in-vitro trials
suggesting elderberry inhibits a wide range of microbes, not just those causing colds
and ’flu. It is, nonetheless, a good paper summing up many of elderberry’s properties.
• Burge et al. (1999) small trial on chimpanzees is the only evidence that elderberry
may act prophylactically.
• Yixi Xie et al. (2015) have proposed the structure-activity relationship and
mechanisms behind the antibacterial activities of flavonoids. The proposed
antibacterial mechanisms of flavonoids are as follows: the inhibition of nucleic acid
synthesis; inhibition of cytoplasmic membrane function; inhibition of energy
metabolism; inhibition of the attachment and formation of biofilm; inhibition of the
porin on the cell membrane; alteration of the membrane permeability, and attenuation
of the pathogenicity.
Other Sambucus species
It would appear that other Sambucus species from around the world are used in traditional
medicine in very similar ways to S. nigra in its native habitat. There is Sambucus canadensis
in North America, Sambucus peruviana in South America and Sambucus FormosanaNakai
which is used in traditional Chinese medicine.
• Weng et al. (2019), a paper concerning a different species of Sambucus, does not fall
within the main dissertation; however, it is of interest. Human coronavirus NL63
(HcoV-NL63), one of the main circulating HcoVs worldwide, causes respiratory tract
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illnesses like runny nose, cough, bronchiolitis and pneumonia. A severe respiratory
illness outbreak of HcoV-NL63 was reported in a long-term care facility. The study
investigated the antiviral activity of S. FormosanaNakai. The results revealed that the
extract displayed strong anti-HcoV-NL63 potential. It is believed that the stems of
the plant were used and the findings suggested that bioactive molecules might be
caffeic acid in this case. This paper was trying to make a new pharmaceutical drug,
rather than using the constituents of the plant synergistically. However, it does
suggest that Sambucus species do have anti-coronavirus potential.
Strengths and limitations
A strength of this paper is that it is the first systematic review of studies testing the potential
of elderberry extract to contribute to treatments of influenza (and its bacterial super-
infections), and to bring together the many other microorganisms which it might inhibit
including nosocomial pathogens (hospital super-bugs) such as MRSA; HIV; Mycoplasmae,
and envelope virus IBV. Another strength of this paper is its development of a risk of bias
tool for assessing bias of in-vitro studies; this is a methodological advance in an under-
researched area.
However, the study was not without limitations. For example, a limitation to knowledge
about elderberry is that much is observational, and there are few randomized controlled trials.
The lack of a comprehensive literature search by a second researcher was a limitation to this
study. Two papers of interest, one which became an included trial, were missed by the author.
The second researcher only used Medline and Embase – the only databases available to
physicians in the library of the main Dorset hospital – in a fairly brief search. At the end of
writing it was found that one in-human case study had been missed (now included), and it is
possible that other papers of interest were never found. A second author conducting a
thorough search might have found missing data. All papers found were on computer
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databases. It is possible that those written before the invention of the internet could have been
missed.
It would have been desirable to have an already well used method for assessing in-vitro trials.
However, methods for doing this from in-vivo and one in-vitro PhD trial was an attempt to
overcome this limitation. These scales were close to existing scales, but it may be beyond the
scope of a Master’s student to create these, which have not been verified elsewhere.
The lack of all but a few, small, in-human trials is a major limitation to knowledge and the
conclusions which can be drawn about elderberry’s effectiveness. Most of these trials had
methodological flaws. As shown in Appendix 1 a systematic review of elderberry’s use in
humans would be the best evidence. In-vitro trials are the lowest form of evidence. For use
as a disinfectant they are sufficient.
Another limitation was heterogeneity. The heterogeneity of the microorganisms tested means
that the trials conducted cannot be bunched together with forest plots to form more robust
conclusions. There was also heterogeneity in the manner in which inhibition of
microorganisms was tested. From the trials that do exist, no statistical analysis was possible.
Each of the different microorganisms should be tested several times. Cos et al. (2006) wrote a
paper suggesting how to develop a stronger in-vitro proof of concept when investigating anti-
infective potential of natural products, but their recommendations do not yet seem to have
been widely adopted. In addition, lack of blinding was a major issue since human nature
wants to find positive results and it is difficult to be totally objective when conducting in-
vitro trials.
Another limitation in this field, as with many other tests on other pharmaceuticals, is that
many of the trials on elderberry were conducted by companies who manufacture extracts of
the berry, or those closely associated with them. There was a financial conflict of interest in
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many of the trials, if not the one on nosocomial infections. This greatly increases the chance
of bias. Bias is very easy within the field of in-vitro trials. Negative results can be concealed,
trials rerun without recording, and it is easy to slightly exaggerate or underestimate the
readings of a test. There are few large profits to be made in the field of herbal medicines as
compared with most pharmaceuticals, as they cannot be patented. This is probably why there
are comparatively few trials, and it is for this reason governmental, international or charity
funding is required to test herbals in a search for an answer to problems such as antibiotic
resistance and emerging diseases.
It would be of benefit to repeat the in-vitro trials on the inhibitory effects of elderberry
several times in order that more robust conclusions, with statistical pooling, could be made.
Further tests by a team of immunologists (which could be in-vitro trials) need to be
conducted to ensure that elderberry does not increase the chances of a cytokine storm. This is
the only potential danger from elderberry that has been found, and recent work suggests it
does not promote these cytokine storms. The case study of someone having a cytokine storm
suggests elderberry mitigated one. However, this needs to be conclusively eliminated as a
possibility.
November 2020 Postscript
Elderberry inhibits other coronaviruses and preliminary anecdotal reports suggest it may be
effective for SARS-CoV-2 which causes Covid-19. At the time of writing two clinical trials
have been arranged on elderberry for Covid-19. These were stimulated by scientists reading
this paper on Researchgate.net amongst others. One is to be conducted by WHO in Iran,
where there is very limited access to pharmaceuticals. One trial will be conducted by the
Sambucol company in Germany where elderberry is commonly used by doctors.
The author (JHWM) is currently organizing more in-vitro trials of elderberry against 12-14
known viruses which are thought may cause future pandemics. SARS-CoV-2 will also be
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tested in-vitro. These trials are to be conducted at a very reputable medical school and
carried out by an experienced microbiologist.
Full publication of this dissertation and its findings will not be until these known dangerous
viruses can be included in results.
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CONCLUSION
Elderberry extract shows promise to be an effective and cost-effective means to fight
infections. However the evidence is limited by being in-vitro, and by methodological
problems listed. Further trials are therefore warranted to confirm whether elderberry extract is
“better than placebo” in humans, and also to confirm that it is effective as part of a hospital
disinfectant.
WHO estimate that 75% of the world population are reliant on medicinal plants for their
health care. Bioactive molecules of elderberry are absorbed into the bloodstream and body,
and it seems the plant has great potential in the future fight against disease in much the same
way as it has been used throughout a long history. All species of the elderberry appear to
have very similar properties.
The author has been corresponding with doctors and scientists in universities throughout the
world during this 2020 pandemic.
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APPENDIX 1 – THE CONVENTIONAL HIERARCHY OF EVIDENCE IN
HEALTHCARE
The conventional hierarchy of evidence in healthcare is shown in the figure below.
Figure 2 Conventional hierarchy of evidence in healthcare (Source: Booth et al., 2004)
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APPENDIX 2
a – INFLUENZA DEATHS AND CYTOKINE STORMS
Some influenza strains can kill many people. In 1918 millions of people died of Spanish ’flu,
and the pathogen was an ancestor of H1N1. For most patients, ’flu causes only mild to
moderate symptoms. In H1N1 the typical course is an acute airways disease with fever, often
with sore throat, dry cough or runny nose. In 1918 there was severe fatigue and/or loss of
appetite; the young were more susceptible, and often also had nausea and vomiting. It is
thought that this was because a cytokine storm was initiated and the young have stronger
immune responses. Most ’flu patients are better in about a week. Therefore it might not
seem very important that treatment with elderberry appears to get individuals better a few
days earlier; for a whole population, however, this treatment can have large economic
relevance. Influenza viruses can easily mutate and disease experts fear we may have another
pandemic with severe disease and a high death rate for which an effective treatment is
needed. Pharmacologically synthesized neuraminidase inhibitors such as Tamiflu
(Oseltamivir) and Relenza (Zanamivir) are proving to be disappointing, despite seemingly to
have had early promise (Jefferson et al., 2014).
Secondary infections can occur after milder ones. One common result of a cold or ’flu virus
is that the patient goes on to suffer, as Blochwich would say, “a worse affection”, i.e. a more
severe upper respiratory infection such as bronchitis or bacterial or viral pneumonia. This
occurs particularly in the old, weak or immuno-compromised who can be killed by such
infections, common among which are the upper respiratory infections Streptococcus
pyogenes and Branhamella catarrhalis. Recent research reported in this paper suggests
elderberry is effective against these microorganisms as well as the original cold or ’flu.
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Cytokine storms can occur during viral infections, and can kill. Another problem with some
influenza viruses such as swine ’flu H1N1 and the 1918 Spanish ’flu is that young and
healthy adults can be particularly badly affected and may be likely to die. David Woodland,
editor of the journal Viral Immunology, suggested that segments of the virus might cause a
“cytokine storm” (Liebert, 2009). This is when an overreaction of the immune system can
cause very high levels of cytokines (inflammatory mediators) to be released, which can lead
to massive inflammatory reactions and pulmonary oedema. In this condition a patient’s
hyper-activated immune system causes potential fatal damage to the lungs. This could
explain why in Spanish ’flu, some of the ’flu viruses and the SARS and MERS epidemics, it
was the young with strong immune systems who were particularly affected. Therefore in the
case of influenza infection it is not primarily immune strengthening that is needed, but also
immunomodulation and the combating of viruses, and secondary possible bacterial infections.
Trials have suggested that elderberry has significant immunomodulatory effects
(Swaminathan et al.(2013).
N.B. An immunomodulator is defined as a chemical agent that modifies the immune response
or the functioning of the immune system (as by the stimulation of antibody formation or the
inhibition of white blood cells) (Merriam-Webster dictionary online).
Elderberry was in European pharmacopoeia until the time of World War II. There are reports
of its being used during the 1918 pandemic. However, due to the complications of searching
historic medical records, particularly in different languages, it has not been possible to locate
contemporaneous reports of its use, and whether it reduced or increased the death rate. This
might make an interesting and valuable question for medical historian.
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b – IMMUNOMODULATION AND CYTOKINE STORMS
Immunomodulation can be increasing or damping down the immune response. In the case of
the old, weak or immunocompromised such as those with HIV increasing the immune
response when they are suffering from an infection can be very beneficial. However, as is
mentioned in Appendix 2a, during the 1918 influenza pandemic it was the young and healthy
who were most likely to die. This was because that particular ’flu virus produced a cytokine
storm. In simple terms the immune system went into “overdrive” and starting killing the
organs of its own body.
As Wikipedia says about cytokine storms,
When the immune system is fighting pathogens, cytokines signal immune cells
such as T-cells and macrophages to travel to the site of the infection. In addition,
cytokines activate those cells, stimulating them to produce more cytokines.
Normally, the body keeps this feedback loop in check. However, in some
instances, the reaction becomes uncontrolled, and too many immune cells are
activated in a single place. The precise reason for this is not entirely understood
but may be caused by an exaggerated response when the immune system
encounters a new and highly pathogenic invader. Cytokine storms have potential
to do significant damage to body tissues and organs. If a cytokine storm occurs in
the lungs, for example, fluids and immune cells such as macrophages may
accumulate and eventually block off the airways, potentially resulting in death.
The real danger during a ’flu pandemic is that it might be a strain to set off a cytokine storm.
It is an inconvenience to be ill for a few days, but what is needed from an anti-viral (or anti-
bacterial) medicine is that it kill or inhibit the microorganism, but above all not exacerbate, or
provoke a cytokine storm. During the SARS outbreak, also, it was cytokine storms which
killed.
There are a great many human cytokines and they cannot be labelled as being either good or
bad. They may have more than one action, and can be good in one circumstance and bad in
another. An example of this (Dinarello, 2007) is that interferon-gamma (IFN-γ) is essential
for defence against several intracellular microorganisms such as Mycobacterium tuberculosis,
but is also a major cytokine in the pathogenesis of several autoimmune diseases.
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The following statement was posted on the Sambucol website by the developer:
Does elderberry cause a cytokine storm to be produced?
Fri, 09/14/2012 – 14:48 – developer
Cytokines are produced by the immune system when it is under attack from
viruses and pathogens. Cytokines signal immune cells to travel to the site of
attack or infection. This is a normal body process that is kept under control by a
feedback loop.
Sometimes this process can become uncontrolled and too many immune cells are
stimulated in a single place. This is often referred to as a cytokine storm.
Cytokine storms can cause significant tissue and organ damage.
There is no evidence in the literature to indicate that elderberry extract causes
cytokine storms. We recommend that Sambucol products, which contain
elderberry extract be used only for the indications on the product labelling.
Postscript. Elderberry used for a case of Covid-19 appeared to actually dampen a
cytokine storm (JHWM).
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APPENDIX 3 – SAMBUCUS NIGRA IN OTHER LANGUAGES
The old herbals’ and apothecaries’ name for elderberries was “fructus sambuci”.
Arabic: Balasan, baylisan, basham, al khaman, sabuqah
Bulgaria: Bez
Denmark: Hyld
France: Sureau
Finland: Selja
Germany: Holander, Holunder, Hollunder, Holder
Greece: Sampoukou
Iceland: Yllir
Ireland: Ruis
Italy: Sambuco, Sammucu, Savucu, Zambucu
Norway: Hyell, Hylle, Svarthyll
Japan: Niwatoko No Mi
Mexico: Canilolerro, Sauco (blossoms)
Poland: Bez Czarny
Portugal: Sambuco, Sambugo, Sabugueiro
Romanis: Soc, Socul
Russia. Buzina Chornaya
Spain. Sauc, Sauco
Sweden, Hyll, Flader
Turkey: Belesen
Other names for the elderberry plant include common elderberry, black elder, bore tree, bou
tree, boortree, common elder, eldrum, ellhorn, European alder, hylder, hylantree, hylder, pipe
tree. These tend to be found in older literature. (Norms Farms, 2014)
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APPENDIX 4 – HOW TO MAKE DIFFERENT ELDERBERRY EXTRACTS BOTH
AT HOME AND IN THE LABORATORY
Homemade Elderberry Rob (Cordial)
Rob is the old name for a fruit juice or cordial.
Pick a basket of ripe elderberries. (Unripe ones are bad for you.). Strip the berries from their
stems and weigh them, then wash them. Use an equal weight of sugar; this can be white or
brown.
Boil the berries in a large pan then pass through a sieve, muslin or old fashioned mouli-
legume (the easiest option). Then put the resultant liquid back in the pan and add the sugar
and boil until a syrupy consistency is reached. Bottle in sterilized bottles.
Take about two tablespoons diluted in hot water three times a day for a cold or ’flu or to
prevent an infection. This recipe works just as well to inhibit or cure a viral infection as does
pharmaceutically prepared extract. For Covid-19 higher doses are needed and for a bad case 3
litres were used in a week
For those proficient in bottling (in USA called canning) the juice can be bottled without the
sugar, however it does keep better with the added sugar, which will make it last a few years.
Care has to be taken not to miss the ripe berries as if you are not quick to pick them, birds
will strip the trees very quickly. Children enjoy hot elderberry rob which is not unlike
Ribena.
Elderberry makes a very good homemade wine and retains its anti-bacterial and anti-viral
properties so can be made if preferred. Elderberry’s inhibition of microorganisms is dose
dependent, and in the opinion of JHWM the doses recommended by most pharmaceutically
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prepared extracts is too low and it is likely that the concentration of bioactive molecules in
three mugs of this homemade elderberry rob a day is higher.
Elderflower tea
Elderflowers can be dried at home or bought in many health food shops. Two dessert spoons
in a pint of boiling water makes a pleasant tea. This is good for bacterial and virus infections,
particularly those of the teeth and gums.
Elderflower cordial (after Anne Mcintyre, Healing Drinks)
This is a light, fruity tasting refreshing drink and is also effective against summer colds.
• 1.2 litres (2 pints) water
• 1.3 kg (3 lbs) sugar
• 1 lemon sliced
• 25 large elderflower heads
• 75 g (3 oz) citric acid
• Sparkling or still mineral water to dilute
Place the water in a large pan and bring to the boil. Add the sugar and lemon and remove
from the heat until the sugar dissolves. Place on the heat again and bring to the boil. Add the
elderflower heads and citric acid. Bring to the boil once more, remove from the heat and
allow to stand until cool. Strain and bottle in clean bottles with corks. This can be drunk
immediately. Stored in a cool place it should keep approximately three months. When
serving, dilute with five parts waters and add ice.
Preparation of plant material as done by Hearst et al. (2010)
Collect fresh (S. nigra L.) flower or ripe berry and freeze dry. Homogenize 10 g of the elder
flower or berry freeze-dried sample in 200 ml of solvent mixture (50:50 water: 96% ethanol),
and stir for 72 h at 4°C; filter, centrifuge at 9000 x g for 10 min and repeat extract as above
the pelleted (plant residue) left behind, at least 3 more rounds. Carefully combine the
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supernatants obtained each time, then filter using Buckner flask and Sintered glass filter bed
apparatus and pack up to 0.5 cm depth with a mixture of Celite (Merck) and activated
charcoal (10:1 w/w) to minimize plant pigments. Collect the filtered cold aqueous ethanol
extract over anhydrous sodium sulphate for drying, concentrate using a rotary evaporator
(Buchi) and freeze-dry the resulting aqueous residues.
For bacterial assay purposes a recorded weight of freeze-dried powder of elder flower or
berry extracts is reconstituted with an equal weight/volume of sterile 0.1% (w/v) peptone
saline to give a known concentration for each extract solution, filter-sterilized through a
0.22μ syringe filter before microbiological challenge.
Elderberry extract prepared to be rich in polyphenols (after Fink)
Extract elderberry fruits (S. nigra) with 80% (v/v) ethanol in two stages (2 h in each stage) at
70°C. Filter and centrifuge the extracted slurry and collect and vacuum-distil the supernatant.
Load the material onto an ADS5 polymer adsorbent column, wash with distilled water and
then elute with 80% (v/v) ethanol. Take the eluate to dryness. Prepare into stock solutions
and dilute appropriately.
Bark preparation (after Manganelli)
Collect fresh bark of S. nigra from the wild and boil for 20 min about (1 g/10 ml). Filter the
extract and then lyophilize (freeze-dry). The lyophilized extract is then melted in bi-distilled
water at the appropriate dilution. These are for laboratory in-vitro trials. For human use
elderberry extract can be made in syrup, sugar free or in ethanol.
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APPENDIX 5 – TABLE 1 FROM HEARST ET AL., 2010
The growing amount of laboratory evidence that elderberry may be effective against
microorganisms has never been collated before, and due to a publishing error the work of
Hearst et al. (2010) which, as has been said, seems to suggest that elderberry is even effective
against nosocomial pathogens was never published properly at all. Since the results were
never published they were recovered by communication with the authors and are printed for
the first time in this appendix. It should be noted that Hearst et al. (2010) did not test
Clostridium difficile and this is an omission. If the trial is rerun this problematic hospital
infection should be included in the trial.
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102
APPENDIX 6 - CASE STUDIES OF ELDERBERRY CORDIAL USED AS A
TREATMENT FOR COVID-19
Case One
A female aged 67, with autoimmune diseases, contracted Covid-19 in early March 2020 from
someone ill for about one week who had travelled out of a high infection area after the
beginning of lockdown. This was before confirmatory tests were available; however, the
symptoms were consistent with those described for Covid-19. The most reliable symptoms
were a high fever and sore throat.
About seven days after contact with the infected person she suddenly noticed the sensation of
having a very hot back. Later a sore throat developed and she felt very tired. She
immediately self-isolated, but remained able to walk up and down stairs once a day
throughout the duration of the infection and was able to occasionally walk her dog in the
garden, without meeting anyone. On day three of feeling unwell, she experienced the same
feelings in her lungs as she had had when she previously suffered from pneumonia. Her
temperature rose to 103°F, 39.5°C during some of the illness, and was always quite high.
From the onset of feeling unwell, she began to drink large quantities of hot, diluted elderberry
cordial and also other fluids, and she had light meals.
The feeling of suffering from pneumonia was most intense in the late evening of day three of
her illness. She remained awake and propped up on pillows with her legs stretched out on a
sofa, and she continued to drink large quantities of elderberry cordial. After less than an hour
the feeling of having pneumonia subsided. When the worst symptoms had subsided, she
went to bed, and slept on her stomach with her head turned to one side. She slept for several
hours with the help of melatonin.
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She was able to maintain relatively normal day and nights and to do simple cooking, hygiene
and self-care during the daytimes, spending most of the days lying on the sofa, reading,
watching television and emailing others.
On day five she experienced a feeling which she believes was the beginning of a cytokine
storm. She had nearly drowned in a swimming pool as a child and experienced the same
feeling of fluid entering the lungs. She remained propped up on pillows on the sofa for two
or three hours, consuming large quantities of elderberry cordial, until the feeling of fluid
entering the lungs stopped. At this stage (of the presumed cytokine storm) she feared the
virus might kill her.
When it was over, she found that her hands and feet were severely swollen as were other
parts of her body. Her wedding rings had cut into the flesh of her fingers. After only a few
hours, when the feeling of drowning had gone, she went to bed and slept (with the
melatonin). As throughout the week, she slept on her stomach with her head to one side.
This, she felt, was the safest way to keep breathing. She lives by the sea, and had the
windows open throughout the week to provide as much oxygen as possible from the sea
breeze.
By day seven she felt recovered, and went to the Accident & Emergency department of her
local hospital and had her wedding rings cut off; they had cut into her flesh about 0.5cm all
the way round her finger. During the week she gained about 20 pounds in weight and she
had only consumed light meals, therefore this is assumed to be fluid. She used 3 litres of
homemade elderberry cordial, before dilution, over the course of the illness.
Case two, confirmed by tests.
A female aged 72 who suffers from severe asthma. This patient had been feeling extremely
tired for two weeks before beginning to feel very unwell. A test at the local test centre
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confirmed that it was Covid-19 but her local hospital sent her home until such time as she
could no longer cope alone, with instructions to call an ambulance when she got very bad.
She had a dry cough with no mucus. 24 to 48 hours later she felt very bad and had great
difficulty breathing. On day three of feeling unwell she was given one litre of elderberry
cordial and had to make it last for the duration of her illness, so she only used it when the felt
most ill. Gradually the cough began to ease. For the first few days of her illness she had very
bad asthmas attacks and was using three times the usual amount of her asthma medications.
She nearly called the ambulance fearing she would die; this was at night and she decided to
wait until morning. By then, on day three or four she had begun to take the elderberry, and
the asthmas and other symptoms gradually eased. In all, she did not leave her home for four
weeks, slowly recovering. Her health returned to almost the level it had been before Covid-
19, although she was left a little tired in the following weeks.
