by MD, of - Akuna

PROVINCE OF ONTARIO
CANADA
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)
)
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I, JORDANA ENIG STOCKHAMER, a Notary Public in and for the Province of Ontario, by Royal
Authority duly appointed, residing in the City of Vaughan, in the Regional Municipality of York, in
the Province of Ontario, DO HEREBY CERTIFY that the paper writing hereunto annexed, each
page of which bears an impression of my seal, is a true and correct copy of A CASE-CONTROL
STUDY OF THE EFFECTS OF PHYTOTHERAPY WITH THE ALVEO FORMULA ON
IMMTINITY IN CHILDREN signed by Josef Richter, MD, PhD, of the Department of
Immunology, Regional Public Health Institute, Usti nad Labem, Czech Republic, the same having
been compared by me with the.original document, an act whereof being requested I have granted
under my hand and notarial seal of office to serve and avail as occasion shall or may require.
DATED at Toronto, Ontario, this 26tr day of August, 2010
Public in and for the
Province of Ontario

an health se1ices, treatment and health procedures worldwide are provided by the practices
of CAM (2).
The public's growing interest in botanical formulas is notable. There has been a wide
application of phytotherapy in the past decade (9, 10, 13, 15, 18, 19, 27'and 29). The general
public is becoming more concerned with the use of various herbal formulas, herbal extracts
and mixtures. Many clinical case studies based on a wide scale of herbal products have
already been published (1, 2,7,8, and l9).
This is what inspired us to conduct a study on the effects of the botanical formulation
ALVEO. We were mostly interested in the response of clinical changes and the changes in
seeretory immunity after the administration of Alveo. After studying its individual
components we suspected the possibility of positive effects. To observe its impact we selected
two groups of children: one group consisted of children being treated at our clinic for chronic
upper respiratory tract infections and the other was a control group of healthy children'
ALVEO is a botanical tonic manufactured by Akuna Health Products Inc. in Canada-
It was used in the recommended dose of 28 ml per day. The study was conducted between
September 2006 and September 2007. Following are the partial results of this study.
Study methodolory:
The case group (Group A) consiste d of 27 children ( 14 boys and I 3 girls) undergoing longterm
treatment at the Department of Microbiology and Immunology; the control group (Group
B) consisted of 35 children (18 boys and 17 girls) from an elementary school in Usti nad
Labem, Czech Republic. The children in Group A were between the ages of 6 and 13 with the
average age being 10.95 years; the children in Group B were between the ages of 9 and 14
with the average age being 10.74 years. Group A was given I ounce or 28 ml of ALVEO
daily on an empty stomach or half an hour after a meal. Every child consumed I bottle of
product in Bn average of 35 days. The children in Group B received a placebo which
resembled Alveo in appearance and contained the same amount (950 ml) of liquid but no
active ingredients. The length of application was averaged 34 days.

The clinical disposition of each child was recorded on a daily basis using a
questionnaire with an emphasis on the symptomatology of infections mainly of the upper
respiratory tract (cough, phlegm discharge, temperature, rhinitis, pharyngitis, and otitis).
Collected data was processed and duration of illness observed. Anthropometric measurements
were taken for each child and hiVher BMI was calculated'
At the same time, the saliva of each child was collected prior to and after application
using the standard method of gathering unstimulated saliva. These specimens were stored in
standard plastic test tubes at less than 70 o C until processing. The exact time of collection
was observed when the effects of circadian rhythmicity were not present (24). The levels of
albumin and IgA in the saliva were determined by immunonephelometry using the Behring
and the Dade Behring BN II antisera. The obtained results were grouped according to the
following levels: Albumin at 50rng/L of saliva being the norm; 100mg/L being a sign of
irritation; 500 mg/L a sign of infection; and amounts greater than 500 mg/L signiffing an
infection in progress. As for the IgA, normal amounts range between 20 and 150 mgll of
saliva.
The ratio of IgA to albumin was then calculated at all levelsin relation to the
individual groups observed. The preference of this index being a reduction in the amount of
lgA transferred into the saliva as a result of infection.
Data analysis:
Differences in findings for the individual groups observed were compared for each of the
groups before and after the application of Alveo and later also in interrelation to the placebo
groups and the control files. The average values of the findings were calculated as well as the
absolute values and later also the logarithmic values of the mean-root-square errors at95% Cl
by means of statistical software such as Microsoft Excel,Instat 3 and Prism 4 (USA). The
differences were then evaluated using the Smirnov-Kolmogorov test, the t-test, the A}'{OVA
test, the Tukey-Kramer test.
Ethics:

This case study was conducted in accordance to the Declaration of Helsinki (Fourth revision
1996) and the guidelines for clinical testing in the Czech Republic. Permission was obtained
prior to the start of the study from all the parents of the children tested with the formula.
Results:
Table I contains the findings before and after the application of the formula and the placebo
over the course of 4 weeks (equal to the consumption of one bottle of product). The
comparison in the amounts of IgA and albumin in the saliva of the placebo controlled group
did not show any significant changes before or after application. On the contrary, the children
who were given the ALVEO formulation and who suffered from chronic upper respiratory
tract infections showed statistically a highly significant decrease in the amount of IgA and
albumin alike.
In studying the occumences in clinical evidence, we discovered that the clinical
symptoms in children who consumed the ALVEO formula decreased by almost two thirds.
Discussion:
Even in the more advanced countries there are both historical and cultural reasons for the use
and application of alternative medicine rather than conventional treatments. It is important to
acknowledge that for half the world's population this type of treatment is actually the norm.
The use of the term "supplementary"- alternative medicine (complementary - altemative -
medicine - CAM) stems form the close relationship between the classical and alternative
approaches to treatment. This can include altemative physical procedures such as acupuncture
and acupressure as well as the application of herbal remedies as practiced by Indian, Japanese
and Chinese cultures and in procedures such as phytotherapy and so on (19,29).
An example would be in the treatment of allergies where a blend of various botanicals
was used. The formulation Pollen contains a mixture of 12 different pollens. The reported
effect of this product was a decrease in the frequency of allergy symptoms and a lower intake
of antibiotics (2). The use of various herbal homeopathic formulas was observed in a series of
clinical trials in individuals suffering from allergies, asthma, bronchitis as well as other health
conditions (7, 8, 10, 13, 15, lg,zg,and 30). Althoughthe discussion about the positive effecs

of homeopathic products on the market remains intense, it is necessary to take a truly
qualifred stance on the matter; the evidence for which can only be provided by scientific and
complex studies (1, 2, and l2). Presently there are no trial results that completely negate the
effects of phytotherapeutic products; nor are there any results that entirely support them (2).
We discovered that from the total 26 ingredients contained in the tested formula, at least 16
have an effect on the modulation of immunity response while others seem to function via
modulation of CNS (10, 30). In present day USA, herbal extracts and the formulas containing
herbal extmcts are considered to be medicines, food and even dietary supplements. The latter
are commonly refened to as DSHEA (4). Some herbal products are considered to be
adaptogens - products which have a normalizing (restorative) influence on the body and help
defend it against stressors (4, 10). We still require more research about the antioxidant
tendencies of herbal extracts, their effects on the regulation of enzymatic functions and their
precise influence on the entire immune system (1, 7, and 8). In our observations at the Health
Institute we have noticed that herbal extracts have positive effects on the health of children
who are already successfully responding to treatment. But even with some of our patients, we
realize that short-term application of these herbs (over the course of one month) is not enough
to positively influence the observed immunity parameters.
The safety of herbal formulations depends on their.orrrportdon; though the fact
rcmains that the effects of many herbal extracts are still not known. For example, there are
more than 100 different components in tea tree oil alone (29).
Preference should be given to those products, which have undergone a series of
laboratory and clinical tests, and most importantly, which have been approved by the
appropriate bodies in the respective country. Many widely available folk remedies may
actually turn out to be potentially harmful, Therefore, it is important to always notify the
public about the possible side-effects and risks involved with taking any particular formula
(29). Chamomile can for example trigger an allergic reaction in individuals who have been
sensibilized by the allergens of ragweed or chrysanthemum (29). Other herbs such as Garden
angelica and rue can highly increase one's photosensitivity.
Herbs and herbal products demonstrate significant antimicrobial activity. This activity
is mainly influenced by the phenolic and polyphenolic substances, which also act as strong
antiviral, antibacteiial and antifungalagents (7, l3); Terperroids also dernonstrde

antibacterial, antifungal, antiviral and protozoaic activity. Similarly it is with alkaloids,
lectins, polypeptides and other substances (7, 13, and l8). Some ingredients in ALVEO such
as licorice are important besause they induce interferon activity, increase the activity of
natural killer (or NK) cells and display antiviral activity including against HIV. Glyconhizin
has anti-inflanrmatory and anti-allergic properties (7, 8, 15, andlS). Some ingredients
contained in the formulation not only demonstrate significant antimicrobial activity but they
also have the ability to modulate the immune system. These traits have been confirmed in a
series of clinical trials, mainly in individuals inflicted with respiratory illnesses (2' 20).
Additional benefits of the formula are many. There is evidence of favorable impact on skin
infections, immune dysfunctions, and fatigue syndrome (29). The use of the formula as a
preventative stems from, not only its effects on the modulation of the immune system, but
also from its effects against stress and fatigue as well as many its other benefits (21,22).
In addition to observing the clinical behaviors or reactions of an individual to an
ALVEO regime, we also decided to measure the parameters of salivary immunity and IgA,
from which we further calculated the ration of Albumin to IgA. The reason for this was to
check if our results were corrdct. We evaluated the diagnoses of the children prior to the study
and after its completion. The collection of saliva was carried out id a standard manner and
always at the same time of day (23,25). There is little possibility of comparing the
immunological findings after the phytotherapy with those of other studies because of the lack
of literature available on this subject. Only isolated studies study the effects of ginseng on the
levels of IgA (9, 11). The measuring of IgA in saliva alone is fairly inaccurate because it is
way too easily affected by the presence of inflammation or damage to the mucous lining of
the oral cavity. This is why we decided to also measure the amounts of albumin in the saliva.
The presence of which signifies the presence of an inflammation, irritation or infection (3, 16,
17,20,23,25,28). We also cannot omit the effects of tonsillectomy on the amounts of both
observed components. as in our study on body fat (5, l2). Circadian and seasonal rhythmicity
can also significantly influence the findings of certain proteins in saliva (25). This is why it is
so important to strictly adhere to a collection schedule. The influence of physical strain on
salivary immunity is also worth a mention (5).
In closing, we can state that we observed the benefits of regular application of the
ALVEO formulation in children. The conclusions we reached suggest a limitation caused by
tbe short application p€riod We believe tbat a oourse of snrdy over the period of three montbs

would be optimal. Furthermore, new studies show that the inclusion of new indicators in
saliva would be appropriate for the purpose of nutritional immunology (l l).
Literature:
l. Bellavite, P., Ortolani, R., Pontarollo, F,, Piasere, V., Benato, G., Conforti, A.:
Immunology and Homeopaty. 4. Clinical Studies-Part 1. eCAM, 3,2006,s.293-301.
2. Bellavite, P., Ortolani, R., Pontarollo, F., Piasere, V., Benato, G., Conforti, A.:
Immunology and Horneopaty.4.Clinical Studies-Part 2. eCAM,3,2006,s.397-409.
3. Brown, L.R.. Frome, W.J., Wheatcroft, M.G., Riggan, L.J., Bussell, N.E., Johnston, D'A':
The Effect of Skylab on the Chemical Composition of Saliva. J.Dent.Res. 56,1977,
s.l 137-l 143.
4. Bucci, L.R.: Selected herbals and human exercise performance. Am.J. clin. Nutr.,
72,200A, s.624s-636s.
S. Cieslak. TJ.,Frost,G., Klentrou P.: Effects of physical aktivity, body fat, and salivary
cortisol on mucosal immunity in children., J. Appl. Physiol., 95,2003, s. 2315 -2320'
6. Childers, N.K., Powel, W.D., Tong, G., Kirk, K., Wiatrak,8., Michalek, S.M.: Human
salivary immunoglobulin and antigen-specific antibody activity after tonsillectomy.,Oral
Microbiol. Immunol., 16,2001, s. 265 -269.
7. Cowan,M.M.: Plant Products as Antimicrobial Agents, Clin.mikrobiol. Rev.,12,1999,
s.546 - 582.
8. Craig, W.J.: Health-promoting properties of common herbs. Am. J. Clin. Nutr., 70, 1999,
s.491-499.
9. Engels, H.J., Fahlman, M.M., Wirth, J.C.: Effects of ginseng on secretory IgA'
performance, and recovery from interval exercise. Med.Sci Sports Exerc.,35,2003, s.690-
696,.
10. Emst, E.,: The Risk - Benefit Profile of Commonly Used Herbal Therapies: Ginkgo, St.
John's Wort, Ginseng, Echinacea" Saw Palmetto, and Kava., Arur. Intem. Med., 136,
L
t_
I
2002, s. 42 - 53.
I l. Feig, S.A., Speigbt, N., Hynete,E.,Magaziner, A.: Summary of American College for
Advancement in Medicine November 2004 Conference on Emerging Concepts in
Immunology. eCAM, 2.2005, s. l2l -124.
12. Gidden, F., Shenkin. A.: Laboratory support of the Clinical Nutrition Service.
Clin.Chem.Lab.Med. 38; 2000,s -693-'7 L 4.

13. Glatthaar-saalmuller,8., Sacher, F., Esperester, A': Antiviral Activity of an extract
derived from roots of Eleutherococcus senticosus. Antiviral Res., 50,2001, s.223'228' ,
14. Gleeson, M., Pyne, D.B.: Exercise effects on mucosal imunity. Immunol'Cell Biol', 78,
2000, s.536-544.
15. Haddad, P.S., ArAt,G.A., Groom, S., Bolvin, M.: Natural Health Products, Modulation of
Immune Function and Prevention of Chronic Disease. eCAM,2'2005,s.513-520'
16. Kaufman, E, Lamster, I.B.: The Diagnostic Application of Saliva - a review'
Crit.Rev.Oral.Biol.Med."
1 3,2002, s.197'212'
17. Kugler, J., Hess, M., Haake, D.: Secretion of salivary immunoglobulin A in relation of
age, salivary flow" mood state, secretion of albumin, cortisol' and catecholamines in
saliva., J.clin.Immunol., 12,1992,s. I 573'
18. O'Hara,M., Kiefer, D., Farrel,K, Kemper, K.: A review of l2 Commonly Used medicinal
Herbs. Arch.Fam.Med., 7,1998,s.523-536'
19. Passalacqua" G., BOuqUet, P.J.. Carlsen, K.H., Kemp, J., LoCkey, R.F., Niggemann' B"
Pawankar,R., Price, D., Bouquet, J.: ARIA update: I-Systematic review of complementary
and alternative medicine for rhinitis and asthma. J.Allergy Clin.Immunol', 117, 2006'
s.1054-1062.
20. Pilette. C., Quadrhiri. Y., Godding, V., Vaerman,J'-P', Sibille, Y': Lung mucosal imunity:
immunoglobulin - A revised., Eur.Respir.J., I 8.2001,s.571 -588.
21. Predy, G.N., Goel,V., Lovlin, R., Donner, A., stitt, L., Basu, T.K.: Efficasy of an extract
of North American ginseng containing poly-furasonyl-saccharides for preventing upper
respiratory tract infections: a randomised conrolled trial. CMAJ ,173,2005, s.1043-1048.
22. Reid, V.I., Gleeson, M., Williams, N., Clancy, R.I.: Clinical investigation of athlets with
persistent fatigue and/or recurrent infections. Br.J.Sports Med.' 38, 2004, s-42'45'
23. Richter, J., Pelech. L.: Immunological findings in groups of children after compensatory
measures. Toxicology Letters. 88, 1996, s'165-168'
24. Richter, J., Kr6l, V., zukov, I., subn, P., Rahm, J.: circadian changes of the SIgA,
lysozyme, albumin and copper kontent in SAliva., Czechol'Med', 3,1980
' s'249-254'
25. Riedler,J., Hofstotter, H., Huttengger,I., Golser, A-: Secretory immunoglobulin A in
childhood: does the saliva value reflects the bronchial value?, Klin.Padiatr.,205,
1993,s.92-98.;
26. Rytschkova, M.A., Tuev, A.V., Krasnoshtejn, A:8., Airapetova" N'S', Verichova, L'A', et
al.: Treatrrent in speleoclimatic chamber made from potassium-magnesium salts of

7
verchnekamskoye deposit. Recomanded methods. Moscow-1994...., Health and Medical
industry ministry of Russian Federation,1994.
27. Schaad, U.8., Mutterlein, R., Goffrn, H.: lmmunostimulation With OM-85 in Children
With Recurrent Infections of the Upper Respiritory Tract: A Double-Blind, Placebo-
Controlled Multicenter Study. Chest, 122, 2002, s.2042-2049.
28. Schenkels, L., Veerman, 8., Nieuv Amerongen" A.V.: Biochemical Composition of
Human Saliva in Relation to Other Mucosal Fluids. Crit.Rev.Oral Biol.Med.,6, 1995,
s.1 67- 1 75.
29. Wilson, K., Busseo J.W.. Gilchrist, A., Vohr4 S., Boon, H., Mills, E.: Chracteristic of
Pediatric and Adolescent Patients Attending a Naturopathic College Clinic in Canada.
Pediatrics, I15, 2005. s.338-343.
30. Woolf, A.D.: Herbal Remedies and Children: Do They Work? Are They Harmful?
Pediatrics, I12. 2003, s.240-246.
Appendix:
Albumin in saliva of children before (1) and after (2)
application of Alveo and placebo
140
o 130
E tzo
@
5 110
E 100
E90
c
E80
Ero

-\
i SlgA in saliva of children befor€ (1) and afier (2)
I application of Alveo and placebo
i
o
25t)
fr zoo
o
b rso
CD
E roo
8so
0
i SlgAl I SlgA2 , SlgAl SlgA2
Controlgrdp
Case grouP
it
Josef Richter, MD, PhD, \Dpt of Immunology, Regional Public Helth Instute, Usti nad
Labem, Czech republic.
Josef.richter@ zuusti.cz
(W fi'"'e-L{'u'