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Citation: Panossian, A.; Lemerond, T.;
Efferth, T. State-of-the-Art Review on
Botanical Hybrid Preparations in
Phytomedicine and Phytotherapy
Research: Background and
Perspectives. Pharmaceuticals 2024,17,
483. https://doi.org/10.3390/
ph17040483
Academic Editor: Daniela De Vita
Received: 12 March 2024
Revised: 28 March 2024
Accepted: 4 April 2024
Published: 10 April 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
pharmaceuticals
Review
State-of-the-Art Review on Botanical Hybrid Preparations in
Phytomedicine and Phytotherapy Research: Background
and Perspectives
Alexander Panossian 1, * , Terry Lemerond 2and Thomas Efferth 3,*
1Phytomed AB, Sjöstadsvägen 6A, Lgh 1004, 59344 Västervik, Sweden
2EuroPharma USA Inc., Green Bay, WI 54311, USA; terryl@europharmausa.com
3Department of Pharmaceutical Biology, Institute of Pharmaceutical and Biomedical Sciences,
Johannes Gutenberg University, 55099 Mainz, Germany
*Correspondence: ap@phytomed.se (A.P.); efferth@uni-mainz.de (T.E.)
Abstract: Background: Despite some evidence supporting the synergy concept, the commonly known
assumption that combinations of several herbs in one formulation can have better efficacy due to
additive or synergistic effects has yet to be unambiguously and explicitly studied. Study aim: The
study aimed to reveal the molecular interactions in situ of host cells in response to botanical hybrid
preparations (BHP) intervention and justify the benefits of implementing BHP in clinical practice. Results:
This prospective literature review provides the results of recent clinical and network pharmacology
studies of BHP of Rhodiola rosea L. (Arctic root) with other plants, including Withania somnifera (L.) Dunal
(ashwagandha), (Camellia sinensis (L.) Kuntze (green tea), Eleutherococcus senticosus (Rupr. and Maxim.)
Maxim. (eleuthero), Schisandra chinensis (Turcz.) Baill. (schisandra), Leuzea carthamoides (Willd.) DC.,
caffeine, Cordyceps militaris L., Ginkgo biloba L. (ginkgo), Actaea racemosa L. (black cohosh), Crocus sativus L.
(saffron), and L-carnosine. Conclusions: The most important finding from network pharmacology
studies of BHP was the evidence supporting the synergistic interaction of BHP ingredients, revealing
unexpected new pharmacological activities unique and specific to the new BHP. Some studies show
the superior efficacy of BHP compared to mono-drugs. At the same time, some a priori-designed
combinations can fail, presumably due to antagonistic interactions and crosstalk between molecular
targets within the molecular networks involved in the cellular and overall response of organisms to the
intervention. Network pharmacology studies help predict the results of studies aimed at discovering
new indications and unpredicted adverse events.
Keywords: network pharmacology; gene expression; botanical hybrid preparations;
Rhodiola rosea clinical trials; synergy
1. Introduction
The use of complex herbal formulations comprising fixed combinations of several
plant extracts has a long history in TCM, Kampo, Ayurveda, and other traditional medical
systems [
1
–
3
]. The potential health benefits of consuming combined nutrients or dietary
supplements have gained considerable attention due to their impact on overall well-being
due to the synergy concept, recently defined as nutrient synergy [
4
]. The term synergy
is differently defined in the various scientific disciplines and comes from the Attic Greek
word συνεργ´
ιαsynergia [1] from synergos,συνεργóς, meaning “working together”.
Combining two or more plants assumes that a hybrid botanical preparation (BHP)
is more active due to synergistic effects brought about by different mechanisms. Figure 1
illustrates an allegoric analogy with two or more kinds of hybrids from ancient mythology.
Pharmaceuticals 2024,17, 483. https://doi.org/10.3390/ph17040483 https://www.mdpi.com/journal/pharmaceuticals
Pharmaceuticals 2024,17, 483 2 of 27
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 2 of 30
Figure 1. Images of two, three, and more kinds of hybrids from ancient mythology.
https://en.wikipedia.org/wiki/List_of_hybrid_creatures_in_folklore, accessed on 3 April 2024.
• Lama an Assyrian protective hybrid deity with a human head, a bull’s body or lion’s
body, and wings symbolizing the synergistic interaction between these elements.
• The centaur Chiron, from Greek mythology, raised Achilles at the request of Achilles’
mother (reproduced from Peter-Paul Rubens in 1630–1635).
• Navagunjara—A Hindu creature with the head of a rooster, the neck of a peacock, the
back of a bull, a snake-headed tail, three legs of an elephant, tiger, and deer or horse,
with the fourth limb being a human hand holding a lotus.
• Nureonna—a creature with the head of a woman and the body of a snake (Japanese
mythology).
• Kotobuki—a Japanese Chimera with the head of a rat, the ears of a rabbit, the horns of
an ox, the comb of a rooster, the beard of a sheep, the neck of a Japanese dragon, the
mane of a horse, the back of a wild boar, the shoulders and belly of a South China
tiger, the arms of a monkey, the hindquarters of a dog, and the tail of a snake.
Based on the assumption of synergistic interaction of several components,
researchers propose that combinations of several active ingredients in one formulation
can have superior effectiveness and better efficacy due to their multiple effects on various
targets (Figure 2) [5–10].
The term botanical “Hybrid” preparation (BHP) is coined to describe the
biological/pharmacological activity (conditional pharmacological “signature”) of a fixed
herbal combination with a specific chemical composition (e.g., TLC of HPLC conditional
chemical “fingerprint”) (Figure 2) [7]. BHP is like a “newborn” unique biologically active
substance, a hybrid of the “parent” ingredients [7].
Similarly, phytochemical hybrid preparation (PHP) is used to determine the
pharmacological signature of a combination of phytochemicals comprising specific plant
species, e.g., R. crenulata (Hook. f. and Thomson) H. Ohba, R. sacra (Prain ex Raym.-
Hamet) S.H. Fu, R. kirilowii (Regel) Maxim., R. quadrifida (Pall.) Fisch. & C. A. Mey., and
R. dumulosa (Franch.) S. H. Fu, etc., characterized by specific chemical fingerprints (Figure
2).
This distinction emphasizes that any new fixed combination exhibits unique
biological characteristics and effects different from the ingredients’ natural characteristics.
That is due to their multitarget effects on various mediators, which interact within various
regulatory systems of the host cells and organism [7,8].
Modern technologies in biomedical research and bioinformatics provide potent tools
in phytotherapy research and implement a concept of systems biology and network
pharmacology, uncovering numerous molecular targets and new mechanisms of action of
botanical (Figure 3) [11]. In these studies, the experimental protocol included mRNA
microarray hybridization, ingenuity pathway analysis (IPA), and statistical analysis to
uncover the mechanism of action of purified compounds, the plant extract, and their
Figure 1. Images of two, three, and more kinds of hybrids from ancient mythology. https://en.
wikipedia.org/wiki/List_of_hybrid_creatures_in_folklore, accessed on 3 April 2024.
•
Lama an Assyrian protective hybrid deity with a human head, a bull’s body or lion’s
body, and wings symbolizing the synergistic interaction between these elements.
•
The centaur Chiron, from Greek mythology, raised Achilles at the request of Achilles’
mother (reproduced from Peter-Paul Rubens in 1630–1635).
•
Navagunjara—A Hindu creature with the head of a rooster, the neck of a peacock, the
back of a bull, a snake-headed tail, three legs of an elephant, tiger, and deer or horse,
with the fourth limb being a human hand holding a lotus.
•
Nureonna—a creature with the head of a woman and the body of a snake
(Japanese mythology).
•
Kotobuki—a Japanese Chimera with the head of a rat, the ears of a rabbit, the horns
of an ox, the comb of a rooster, the beard of a sheep, the neck of a Japanese dragon,
the mane of a horse, the back of a wild boar, the shoulders and belly of a South China
tiger, the arms of a monkey, the hindquarters of a dog, and the tail of a snake.
Based on the assumption of synergistic interaction of several components, researchers
propose that combinations of several active ingredients in one formulation can have su-
perior effectiveness and better efficacy due to their multiple effects on various targets
(Figure 2) [5–10].
The term botanical “Hybrid” preparation (BHP) is coined to describe the biologi-
cal/pharmacological activity (conditional pharmacological “signature”) of a fixed herbal
combination with a specific chemical composition (e.g., TLC of HPLC conditional chemical
“fingerprint”) (Figure 2) [
7
]. BHP is like a “newborn” unique biologically active substance,
a hybrid of the “parent” ingredients [7].
Similarly, phytochemical hybrid preparation (PHP) is used to determine the pharma-
cological signature of a combination of phytochemicals comprising specific plant species,
e.g., R. crenulata (Hook. f. and Thomson) H. Ohba, R. sacra (Prain ex Raym.-Hamet) S.H.
Fu, R. kirilowii (Regel) Maxim., R. quadrifida (Pall.) Fisch. & C. A. Mey., and R. dumulosa
(Franch.) S. H. Fu, etc., characterized by specific chemical fingerprints (Figure 2).
This distinction emphasizes that any new fixed combination exhibits unique biological
characteristics and effects different from the ingredients’ natural characteristics. That is due
to their multitarget effects on various mediators, which interact within various regulatory
systems of the host cells and organism [7,8].
Modern technologies in biomedical research and bioinformatics provide potent tools in
phytotherapy research and implement a concept of systems biology and network pharma-
cology, uncovering numerous molecular targets and new mechanisms of action of botanical
(Figure 3) [
11
]. In these studies, the experimental protocol included mRNA microarray
hybridization, ingenuity pathway analysis (IPA), and statistical analysis to uncover the
mechanism of action of purified compounds, the plant extract, and their combinations
comprising BHP. This was achieved by assessing their effects on gene expression in isolated
neuronal cells and their potential therapeutic action.
Pharmaceuticals 2024,17, 483 3 of 27
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 3 of 30
combinations comprising BHP. This was achieved by assessing their effects on gene
expression in isolated neuronal cells and their potential therapeutic action.
Figure 2. Schematic representation of quality, efficacy, and safety characteristics of herbal medicinal
products comprising the mixtures of fixed combinations of molecules from herbal extracts.
Reproducible qualitative and quantitative chemical composition by HPLC and TLC fingerprint
ensures the reproducible quality of a fixed combination. Reproducible efficacy and safety of a
botanical/herbal hybrid preparation (BHP) is characterized by pharmacological profile—
conditional signature, e.g., microarray dataset of deregulated genes in response to exposure of BHP
in a bioassay providing further information on the effect on physiological functions and diseases in
the form of heatmaps.
Figure 2. Schematic representation of quality, efficacy, and safety characteristics of herbal medicinal
products comprising the mixtures of fixed combinations of molecules from herbal extracts. Repro-
ducible qualitative and quantitative chemical composition by HPLC and TLC fingerprint ensures the
reproducible quality of a fixed combination. Reproducible efficacy and safety of a botanical/herbal
hybrid preparation (BHP) is characterized by pharmacological profile—conditional signature, e.g.,
microarray dataset of deregulated genes in response to exposure of BHP in a bioassay providing
further information on the effect on physiological functions and diseases in the form of heatmaps.
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 4 of 30
Figure 3. Molecular network-based cause–effect relationships concept. The current drug-discovery
platform links drugs and targets with signaling pathways, molecular networks, and organism
effects. Molecular targets of adaptogens, their networks, and signaling pathways are associated with
chronic inflammation, atherosclerosis, neurodegenerative cognitive impairment, metabolic
disorders, and cancer. Predicting the response of the human body to medication requires an
understanding of drug–effect relationships at the organism, organ, tissue, cellular, and molecular
levels based on integrative personal OMICS (DNA-genomics, RNA-transcriptomics, microbiomes,
proteomics, and metabolomics) profiling and their changes in health and disease, as well as after
pharmacological intervention.
Recently, the Herbal Medicinal Products Platform Austria (HMPPA) committee
chose Rhodiola rosea L. as a medicinal plant in 2023 in Austria [12]. What is behind this
choice of HMPPA?
Rhodiola rosea L. (Crassulaceae, syn. Sedum rhodiola DC., Sedum rosea (L.), Scop cop,
known as Roseroot, Rosenroot, Golden Root, Arctic Root, Orpin Rose, Rhodiole, and
Rougeâtre) has an extensive history as a treasured medicinal plant and has appeared in
the Materia Medica of several European countries [13,14]. In Europe, Rosenroot was
formally adopted in Sweden as a natural remedy (national legislation) from 1987 to 2008,
and since 2008, as a traditional herbal medicinal product (THMP) and registered as an
adaptogen in decreased performance, such as fatigue and weakness [15].
During the last two decades, more than 1200 studies, including 33 clinical and 910
pre-clinical studies, were conducted in Europe, America, and China, providing results of
preclinical and clinical efficacy, safety, and quality of R. rosea preparations in various
stress-induced disorders, including fatigue syndrome, cognitive deficiencies,
mild/moderate depression, anxiety, and burnout symptoms, as well as in healthy subjects
under stress (Figure 4) [15–48].
Figure 3. Molecular network-based cause–effect relationships concept. The current drug-discovery
platform links drugs and targets with signaling pathways, molecular networks, and organism effects.
Pharmaceuticals 2024,17, 483 4 of 27
Molecular targets of adaptogens, their networks, and signaling pathways are associated
with chronic inflammation, atherosclerosis, neurodegenerative cognitive impairment,
metabolic disorders, and cancer. Predicting the response of the human body to medi-
cation requires an understanding of drug–effect relationships at the organism, organ, tissue,
cellular, and molecular levels based on integrative personal OMICS (DNA-genomics, RNA-
transcriptomics, microbiomes, proteomics, and metabolomics) profiling and their changes
in health and disease, as well as after pharmacological intervention.
Recently, the Herbal Medicinal Products Platform Austria (HMPPA) committee chose
Rhodiola rosea L. as a medicinal plant in 2023 in Austria [
12
]. What is behind this
choice of HMPPA?
Rhodiola rosea L. (Crassulaceae, syn. Sedum rhodiola DC., Sedum rosea (L.), Scop cop,
known as Roseroot, Rosenroot, Golden Root, Arctic Root, Orpin Rose, Rhodiole, and
Rougeâtre) has an extensive history as a treasured medicinal plant and has appeared in the
Materia Medica of several European countries [
13
,
14
]. In Europe, Rosenroot was formally
adopted in Sweden as a natural remedy (national legislation) from 1987 to 2008, and since
2008, as a traditional herbal medicinal product (THMP) and registered as an adaptogen in
decreased performance, such as fatigue and weakness [15].
During the last two decades, more than 1200 studies, including 33 clinical and 910 pre-
clinical studies, were conducted in Europe, America, and China, providing results of
preclinical and clinical efficacy, safety, and quality of R. rosea preparations in various stress-
induced disorders, including fatigue syndrome, cognitive deficiencies, mild/moderate
depression, anxiety, and burnout symptoms, as well as in healthy subjects under stress
(Figure 4) [15–48].
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 5 of 30
Figure 4. The number of publications (1200 results) worldwide on R. rosea from 1963 to 2024; the
author’s chart extracted from Pubmed: rhodiola rosea—search results—PubMed (nih.gov, accessed
on 3 April 2024).
Eighteen clinical studies were conducted on the fixed combinations of R.rosea with
green tea [49–53], Eleutherococcus, and Schisandra [54–57], Eleutherococcus, Schisandra,
and Leuzea [56], caffeine [58,59], Cordyceps [60–63], Ginkgo [64], black cohosh [65],
Saffron [66], L-carnosine [67], Eleutherococcus, and Glycyrrhiza [68].
This narrative state-of-the-art review provides the results of recent clinical [49–68] and
network pharmacology studies of the BHP of R. rosea with other plants [5,6,8–10]. The
studies aim to reveal the molecular interactions in situ of host cells in response to the
intervention of BHP and justify the benefits of implementing BHP in clinical practice.
2. Synergy and Antagonism of Active Ingredients of Rhodiola rosea and Other
Plant Extracts
Concomitant treatment of disease by two herbal drugs aiming to achieve better effect
suggests their additive (1 + 1 = 2), amplifying (1 + 1 > 2), potentiation (0 + 1 > 1), or
synergistic (0 + 0 > 1) result. Hypothetically, their interaction can result in beneficial
antagonistic interactions (1 + 1 < 2, or 1 + 1 = 0), e.g., decreased toxicity and detoxifying
effects [3,6].
This hypothesis was verified in a set of in vitro studies, where the effects of several
BHP and their ingredients on the number of deregulated genes in brain cell cultures were
analyzed [5–10]. The composition of genes (signature) deregulated by BHP was
quantitatively and qualitatively different from the signature (composition of genes)
deregulated by each plant separately, suggesting that the impact of the BHP on the target
cells was qualitatively different from the effects of individual ingredients [5] (Figure 5).
This implies that the BHP exhibits quite different pharmacological activities when the
ingredients are combined. These findings are essential for understanding the
unpredictable results of clinical studies of multi-component drugs and dietary
supplements [49–68].
Figure 4. The number of publications (1200 results) worldwide on R. rosea from 1963 to 2024; the
author’s chart extracted from Pubmed: rhodiola rosea—search results—PubMed (nih.gov, accessed
on 3 April 2024).
Eighteen clinical studies were conducted on the fixed combinations of R. rosea with
green tea [
49
–
53
], Eleutherococcus, and Schisandra [
54
–
57
], Eleutherococcus, Schisandra,
and Leuzea [
56
], caffeine [
58
,
59
], Cordyceps [
60
–
63
], Ginkgo [
64
], black cohosh [
65
], Saf-
fron [66], L-carnosine [67], Eleutherococcus, and Glycyrrhiza [68].
This narrative state-of-the-art review provides the results of recent clinical [
49
–
68
]
and network pharmacology studies of the BHP of R. rosea with other plants [
5
,
6
,
8
–
10
].
The studies aim to reveal the molecular interactions in situ of host cells in response to the
intervention of BHP and justify the benefits of implementing BHP in clinical practice.
2. Synergy and Antagonism of Active Ingredients of Rhodiola rosea and Other
Plant Extracts
Concomitant treatment of disease by two herbal drugs aiming to achieve better effect
suggests their additive (1 + 1 = 2), amplifying (1 + 1 > 2), potentiation (0 + 1 > 1), or synergistic
(0 + 0 > 1) result. Hypothetically, their interaction can result in beneficial antagonistic interac-
tions (1 + 1 < 2, or 1 + 1 = 0), e.g., decreased toxicity and detoxifying effects [3,6].
This hypothesis was verified in a set of
in vitro
studies, where the effects of several
BHP and their ingredients on the number of deregulated genes in brain cell cultures were
analyzed [
5
–
10
]. The composition of genes (signature) deregulated by BHP was quantita-
Pharmaceuticals 2024,17, 483 5 of 27
tively and qualitatively different from the signature (composition of genes) deregulated
by each plant separately, suggesting that the impact of the BHP on the target cells was
qualitatively different from the effects of individual ingredients [
5
] (Figure 5). This implies
that the BHP exhibits quite different pharmacological activities when the ingredients are
combined. These findings are essential for understanding the unpredictable results of
clinical studies of multi-component drugs and dietary supplements [49–68].
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 5 of 30
Figure 4. The number of publications (1200 results) worldwide on R. rosea from 1963 to 2024; the
author’s chart extracted from Pubmed: rhodiola rosea—search results—PubMed (nih.gov, accessed
on 3 April 2024).
Eighteen clinical studies were conducted on the fixed combinations of R.rosea with
green tea [49–53], Eleutherococcus, and Schisandra [54–57], Eleutherococcus, Schisandra,
and Leuzea [56], caffeine [58,59], Cordyceps [60–63], Ginkgo [64], black cohosh [65],
Saffron [66], L-carnosine [67], Eleutherococcus, and Glycyrrhiza [68].
This narrative state-of-the-art review provides the results of recent clinical [49–68] and
network pharmacology studies of the BHP of R. rosea with other plants [5,6,8–10]. The
studies aim to reveal the molecular interactions in situ of host cells in response to the
intervention of BHP and justify the benefits of implementing BHP in clinical practice.
2. Synergy and Antagonism of Active Ingredients of Rhodiola rosea and Other
Plant Extracts
Concomitant treatment of disease by two herbal drugs aiming to achieve better effect
suggests their additive (1 + 1 = 2), amplifying (1 + 1 > 2), potentiation (0 + 1 > 1), or
synergistic (0 + 0 > 1) result. Hypothetically, their interaction can result in beneficial
antagonistic interactions (1 + 1 < 2, or 1 + 1 = 0), e.g., decreased toxicity and detoxifying
effects [3,6].
This hypothesis was verified in a set of in vitro studies, where the effects of several
BHP and their ingredients on the number of deregulated genes in brain cell cultures were
analyzed [5–10]. The composition of genes (signature) deregulated by BHP was
quantitatively and qualitatively different from the signature (composition of genes)
deregulated by each plant separately, suggesting that the impact of the BHP on the target
cells was qualitatively different from the effects of individual ingredients [5] (Figure 5).
This implies that the BHP exhibits quite different pharmacological activities when the
ingredients are combined. These findings are essential for understanding the
unpredictable results of clinical studies of multi-component drugs and dietary
supplements [49–68].
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 6 of 30
Figure 5. The upper panel shows a Venn diagram with Greek, Latin, and Cyrillic letter symbols,
where overlapped sections contain the same symbols. Eleven symbols are the same in all alphabets.
Similarly, Rhodiola, Eleutherococcus, and Schisandra separately deregulated 140 out of 1340 genes in
experiments with neuroglia cells; however, only 96 out of 640 genes were deregulated by the hybrid
combination (ADAPT) of these three plant extracts. External sections of the Venn diagram show the
number of deregulated genes specific to distinct extracts (antagonistic interactions in blue, yellow,
and green) and synergy-derived 206 deregulated genes characteristic to ADAPT-232 (in red). The
lower panel shows the number of compounds in extracts and 3D-HPLC fingerprints of Rhodiola,
Eleutherococcus Schisandra, and their combination ADAPT-232, and Venn diagrams showing
intersections of deregulated genes in neuroglia cells after exposure to these herbal extracts. Authors’
drawings adapted from freely accessible publications [5].
Figures 5 and 6 illustrate the essence of hybridization of ingredients of BHP or PHP,
their synergy, and the antagonism in these experiments and interpretations. The point is
the biological activity of a single compound, e.g., salidroside, an active compound of
Rhodiola rosea extract, interacts with many proteins in brain cells, deregulating 640 (!) genes
in neuroglia cells, associated with various physiological processes and effects in stress and
aging-induced disorders (e.g., neurodegeneration). This is illustrated in Figures 5 and 6
[9].
Figure 5. The upper panel shows a Venn diagram with Greek, Latin, and Cyrillic letter symbols,
where overlapped sections contain the same symbols. Eleven symbols are the same in all alphabets.
Similarly, Rhodiola,Eleutherococcus, and Schisandra separately deregulated 140 out of 1340 genes
in experiments with neuroglia cells; however, only 96 out of 640 genes were deregulated by the
hybrid combination (ADAPT) of these three plant extracts. External sections of the Venn diagram
show the number of deregulated genes specific to distinct extracts (antagonistic interactions in blue,
yellow, and green) and synergy-derived 206 deregulated genes characteristic to ADAPT-232 (in
red). The lower panel shows the number of compounds in extracts and 3D-HPLC fingerprints of
Rhodiola, Eleutherococcus Schisandra, and their combination ADAPT-232, and Venn diagrams showing
intersections of deregulated genes in neuroglia cells after exposure to these herbal extracts. Authors’
drawings adapted from freely accessible publications [5].
Figures 5and 6illustrate the essence of hybridization of ingredients of BHP or PHP,
their synergy, and the antagonism in these experiments and interpretations. The point
is the biological activity of a single compound, e.g., salidroside, an active compound of
Rhodiola rosea extract, interacts with many proteins in brain cells, deregulating 640 (!) genes
in neuroglia cells, associated with various physiological processes and effects in stress and
aging-induced disorders (e.g., neurodegeneration). This is illustrated in Figures 5and 6[
9
].
Pharmaceuticals 2024,17, 483 6 of 27
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 7 of 30
Figure 6. The impact of Rhodiola and its active compounds on gene expression in human brain cells
suggests potential effects on canonical intracellular signaling pathways, molecular, cellular, and
physiological functions, and diseases; updated from the authors’ freely accessible publication [9]
and authors’ drawings.
Meanwhile, the total extract of Rhodiola-containing 120 phytochemicals (including
salidroside) or the BHP of Rhodiola, Schisandra, and Eleutherococcus extracts (ADAPT-232)
containing 207 phytochemicals deregulate almost the same number of genes (Schizandra—
625 genes, Rhodiola—631 genes, Eleutherococcus—669 genes, ADAPT232—678 genes) [6]
(Figure 7). Among those deregulated by ADAPT-232, there were 206 genes that were not
deregulated by any ingredient of BHP ADAPT-232 due to the synergy effect (Figure 7).
The synergy-derived biological effect is characteristic of the BHP (ADAPT-232), which has
a distinct pharmacological profile (signature) and typical chemical composition
(fingerprint), which are different from Rhodiola rosea extracts [5].
Gene expression profiling was conducted on the human neuroglial cell line, T98G,
after treatment with either Rhodiola SHR-5 extract or several of its constituents separately,
including salidroside, triandrin, and tyrosol. Rhodiola SHR-5 and individual constituents
had similar effects on G-protein-coupled receptor (GPCR)-mediated signal transduction
through cAMP, phospholipase C, and phosphatidylinositol signaling pathways (Figure
6).
Figure 6. The impact of Rhodiola and its active compounds on gene expression in human brain cells
suggests potential effects on canonical intracellular signaling pathways, molecular, cellular, and
physiological functions, and diseases; updated from the authors’ freely accessible publication [
9
] and
authors’ drawings.
Meanwhile, the total extract of Rhodiola-containing 120 phytochemicals (including
salidroside) or the BHP of Rhodiola, Schisandra, and Eleutherococcus extracts (ADAPT-232)
containing 207 phytochemicals deregulate almost the same number of genes (Schizandra—
625 genes, Rhodiola—631 genes, Eleutherococcus—669 genes, ADAPT232—678 genes) [
6
]
(Figure 7). Among those deregulated by ADAPT-232, there were 206 genes that were not
deregulated by any ingredient of BHP ADAPT-232 due to the synergy effect (Figure 7). The
synergy-derived biological effect is characteristic of the BHP (ADAPT-232), which has a
distinct pharmacological profile (signature) and typical chemical composition (fingerprint),
which are different from Rhodiola rosea extracts [5].
Gene expression profiling was conducted on the human neuroglial cell line, T98G,
after treatment with either Rhodiola SHR-5 extract or several of its constituents separately,
including salidroside, triandrin, and tyrosol. Rhodiola SHR-5 and individual constituents
had similar effects on G-protein-coupled receptor (GPCR)-mediated signal transduction
through cAMP, phospholipase C, and phosphatidylinositol signaling pathways (Figure 6).
Pharmaceuticals 2024,17, 483 7 of 27
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 8 of 30
The interpretations of microarray data and gene expression changes were conducted
using Ingenuity Pathways Analysis (IPA) software (QIAGEN Bioinformatics, Aarhus C,
Denmark), which is based on a continuously updated database (the Ingenuity Knowledge
Base), gathering research observations with more than 8.1 million findings manually
curated from biomedical literature and integrated from 45 third-party databases. The IPA
network contains more than 40,000 nodes representing human genes, molecules, and
biological functions, linked by 1,480,000 edges representing experimentally observed
cause–effect relationships (either inhibiting or activating) associated with gene
expression, transcription, molecular metabolism, and receptor binding. Network edges
are also linked to activating or inhibiting effects. The IPA core analysis of transcriptomic
datasets provides information about the impact of test samples on canonical signaling and
metabolic pathways, diseases, and molecular and cellular functions that are activated or
inhibited in experiments.
Two statistical methods of analysis of gene expression datasets are used in the IPA:
(i) the gene-set-enrichment method, where differentially expressed genes are intersected
with sets of genes that are associated with a particular pathway or biological function,
providing a so-called “enrichment” score (Fisher’s exact test p-value). This score measures
the overlap of the observed and predicted regulated gene sets. (ii) The method based on
cause–effect relationships related to the direction of effects reported in the literature,
which provides the so-called z-score measuring the match of observed and predicted
up/down-regulation [10,11]. The predicted effects are based on gene expression changes
in the experimental samples relative to the control; z-score > 2, −log p-value > 1.3.
Figure 7 shows the synergy, potentiation, and antagonistic effects of hybridization of
a combination of Rhodiola with Withania, Withania with melatonin, and Curcuma longa with
Boswellia on eicosanoids signaling pathways, which play an important role in
inflammation and neurodegeneration in neuroglia cells.
Figure 7. The synergy, potentiation, and antagonistic effects of hybridization of a combination of
Rhodiola with Withania, Withania with melatonin, and Curcuma longa with Boswellia on eicosanoids
signaling pathways, which has an essential role in inflammation and neurodegeneration assessed
in isolated neuroglia cells. The authors’ drawings were adapted from freely accessible publications
[65].
In a recent study [8], BHP of Rhodiola with Withania (Adaptra) positively regulated 22
of 57 genes, which are known to activate the development of neurons (Figure 8),
suggesting that Adaptra is potentially helpful in learning and memory, stress, and
Figure 7. The synergy, potentiation, and antagonistic effects of hybridization of a combination of
Rhodiola with Withania,Withania with melatonin, and Curcuma longa with Boswellia on eicosanoids
signaling pathways, which has an essential role in inflammation and neurodegeneration assessed in
isolated neuroglia cells. The authors’ drawings were adapted from freely accessible publications [
65
].
The interpretations of microarray data and gene expression changes were conducted
using Ingenuity Pathways Analysis (IPA) software (QIAGEN Bioinformatics, Aarhus C,
Denmark), which is based on a continuously updated database (the Ingenuity Knowledge
Base), gathering research observations with more than 8.1 million findings manually cu-
rated from biomedical literature and integrated from 45 third-party databases. The IPA
network contains more than 40,000 nodes representing human genes, molecules, and biolog-
ical functions, linked by 1,480,000 edges representing experimentally observed cause–effect
relationships (either inhibiting or activating) associated with gene expression, transcription,
molecular metabolism, and receptor binding. Network edges are also linked to activating
or inhibiting effects. The IPA core analysis of transcriptomic datasets provides information
about the impact of test samples on canonical signaling and metabolic pathways, diseases,
and molecular and cellular functions that are activated or inhibited in experiments.
Two statistical methods of analysis of gene expression datasets are used in the IPA:
(i) the gene-set-enrichment method, where differentially expressed genes are intersected
with sets of genes that are associated with a particular pathway or biological function,
providing a so-called “enrichment” score (Fisher’s exact test p-value). This score measures
the overlap of the observed and predicted regulated gene sets. (ii) The method based
on cause–effect relationships related to the direction of effects reported in the literature,
which provides the so-called z-score measuring the match of observed and predicted
up/down-regulation [
10
,
11
]. The predicted effects are based on gene expression changes in
the experimental samples relative to the control; z-score > 2, −log p-value > 1.3.
Figure 7shows the synergy, potentiation, and antagonistic effects of hybridization of a
combination of Rhodiola with Withania,Withania with melatonin, and Curcuma longa with
Boswellia on eicosanoids signaling pathways, which play an important role in inflammation
and neurodegeneration in neuroglia cells.
In a recent study [
8
], BHP of Rhodiola with Withania (Adaptra) positively regulated
22 of 57 genes, which are known to activate the development of neurons (Figure 8), sug-
gesting that Adaptra is potentially helpful in learning and memory, stress, and depression,
insomnia, and aging-related neurodegenerative diseases, preventing Alzheimer’s and
Parkinson’s diseases, and aiding recovery from brain injury and stroke.
Pharmaceuticals 2024,17, 483 8 of 27
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 9 of 30
depression, insomnia, and aging-related neurodegenerative diseases, preventing
Alzheimer’s and Parkinson’s diseases, and aiding recovery from brain injury and stroke.
Figure 8. The effects of RR-WS (Adaptra) on gene expression in human T98G neuroglia cells and
the predicted activation of the development of neurons. The authors’ drawings were adapted from
freely accessible publications [8]. The synergy effects (red arrows) of hybridization of Rhodiola with
Withania on neurogenesis signaling pathways in isolated neuroglia cells. The intensity of green and
red squares indicates fold-changes compared to control, where green means down- and red means
up-regulation. Synergistic or antagonistic effects on gene expression were observed by comparing
the impact of the BHP Adaptra combination of RR-WS (sample A1) with the lack of impact of
individual extracts of RR (R. rosea), WS (Withania somnifera), and WSL Withania somnifera low dose,
corresponding to samples A2, A3, and A7, at a significance level of p < 0.05 (−log = 1.3) and a z-score
Figure 8. The effects of RR-WS (Adaptra) on gene expression in human T98G neuroglia cells and the
predicted activation of the development of neurons. The authors’ drawings were adapted from freely
accessible publications [
8
]. The synergy effects (red arrows) of hybridization of Rhodiola with Withania
on neurogenesis signaling pathways in isolated neuroglia cells. The intensity of green and red squares
indicates fold-changes compared to control, where green means down- and red means up-regulation.
Synergistic or antagonistic effects on gene expression were observed by comparing the impact of
the BHP Adaptra combination of RR-WS (sample A1) with the lack of impact of individual extracts
of RR (R. rosea), WS (Withania somnifera), and WSL Withania somnifera low dose, corresponding to
samples A2, A3, and A7, at a significance level of p< 0.05 (
−
log = 1.3) and a z-score > 2. The symbolic
interpretation of synergy and antagonism by the image of hybrid creature from Greek mythology:
ichthyocentaurs with a human head, a horse’s body-derived fish-tail due to their synergistic and
antagonistic (e.g., lack of human legs) interactions.
Pharmaceuticals 2024,17, 483 9 of 27
Notably, 25 genes were deregulated due to RR and WS synergistic interactions in
the fixed combination Adaptra (Figure 8and Table 1). This means that in combination,
these two ingredients of Adaptra act synergistically. In other words, Adaptra is superior to
Rhodiola or Withania in the activation of neurogenesis and, consequently, has the potential
effects mentioned above [8].
Figure 9shows the Venn diagrams of deregulated genes induced by the treatment of
neuroglial cells with Withania somnifera (WS), Rhaponticum cartamoides L. (RC), and Eleutherococcus
senticosus (RS) root extracts, as well as their hybrid combination (RC-ES-WS) [6].
Table 1. Effect of Rhodiola rosea (RR), Withania somnifera (WS), and their combination RR-WS (BHP
Adaptra) on genes involved in the regulation of neuronal development *.
Gene Symbol Entrez Gene Name Literature
Findings Prediction ** Gene Expression,
Fold Change
RR-WS RR WS
ADGRF1 adhesion G protein-coupled receptor F1 Affects (4) Affected 2.29 2.28
ADGRL1 *** adhesion G protein-coupled receptor L1 Increases (2) Increased 6.93
APOE apolipoprotein E Affects (13) Affected −2.84
BICDL1 BICD family like cargo adaptor 1 Affects (2) Affected −3.98 −2.34
CACNA2D2 calcium voltage-gated channel auxiliary
subunit α2δ2Affects (2) Affected 3.76 6.93
CDK5R1 cyclin-dependent kinase 5 regulatory
subunit 1 Increases (4) Increased 2.33
CDKL3 cyclin-dependent kinase like 3 Increases (3) Increased 4.82
CHRNA3 cholinergic receptor nicotinic α3 subunit Affects (2) Affected −3.09 −2.45
CHRNA7 cholinergic receptor nicotinic α7 subunit Increases (1) Decreased −3.74
CHRNB2 cholinergic receptor nicotinic β2 subunit Increases (8) Increased 2.45 −5.20
CHRNE
cholinergic receptor nicotinic epsilon subunit
Increases (1) Decreased −2.65 −2.59
COLQ collagen-like tail subunit
of acetylcholinesterase Affects (2) Affected −2.65 −6.30 −2.69
CRIP1 cysteine rich protein 1 Increases (1) Increased 2.41 3.01
ELFN1 extracellular leucine-rich repeat and
fibronectin type III domain-containing 1 Affects (1) Affected −5.31
FGF5 fibroblast growth factor 5 Increases (1) Increased 3.52 4.23
FOXO6 forkhead box O6 Increases (3) Decreased −7.93 −2.10 −3.89
GAS7 growth arrest specific 7 Increases (3) Decreased −2.85 −2.26
GFI1 growth factor independent 1
transcriptional repressor Affects (1) Affected −2.65 −2.59
GHSR growth hormone secretagogue receptor Affects (3) Affected 3.15
GRIN3A glutamate ionotropic receptor NMDA type
subunit 3A Decreases (4) Increased −3.33
HAP1 huntingtin-associated protein 1 Affects (1) Affected −2.21
ITGB2 integrin subunit β2 Increases (1) Increased 2.45 3.05 2.66
LRRC7 leucine-rich repeat containing 7 Affects (1) Affected −2.66 −2.11
LRRK2 leucine-rich repeat kinase 2 Affects (4) Affected 2.26
MAGI2 membrane-associated guanylate kinase, Affects (10) Affected 2.01 3.01 2.18
MBP myelin basic protein Increases (1) Increased 3.48
mir-10 microRNA 100 Increases (1) Increased 2.89 3.53
MYH7B myosin heavy chain 7B Affects (1) Affected 3.01 5.70 3.08
MYO16 myosin XVI Affects (1) Affected −3.32
NEFH neurofilament heavy Decreases (18) Decreased 3.02
NKX2-1 NK2 homeobox 1 Affects (4) Affected −2.38
NTF4 neurotrophin 4 Increases (5) Decreased −2.61
PAK3 p21 (RAC1) activated kinase 3 Affects (4) Affected 2.86 2.36
PARD6A par-6 family cell polarity regulator αDecreases (2) Increased −2.84 −2.39
PCDHB8 protocadherin β8 Affects (1) Affected −3.97 −3.89
PLXNA4 plexin A4 Increases (5) Increased 2.25 9.49 10.97
POU3F2 POU class 3 homeobox 2 Affects (4) Affected −2.65
PPP1R9A
protein phosphatase 1 regulatory subunit 9A
Affects (6) Affected 4.51 2.85 5.38
PRKCZ protein kinase C ζDecreases (2) Increased −2.23
PROX1 prospero homeobox 1 Increases (1) Increased 3.76 2.85
PTPRD protein tyrosine phosphatase, receptor
type D Increases (3) Decreased −4.32 −3.42 −2.11
RAB33A RAB33A, member RAS oncogene family Increases (1) Increased 2.81 −3.11
RAPGEF4 Rap guanine nucleotide exchange factor 4 Increases (2) Increased 6.31 11.27 3.92
RELN reelin Increases (9) Increased 3.01
ROR2 receptor tyrosine kinase-like orphan
receptor 2 Increases (5) Increased 3.01
RYR2 ryanodine receptor 2 Increases (2) Increased 3.75 2.85 3.07
SERPINF1 serpin family F member 1 Increases (1) Increased 3.04 2.88
SH3GL2 SH3 domain containing GRB2-like 2,
endophilin A1 Affects (2) Affected 3.02 2.16
SYN2 synapsin II Affects (3) Affected 2.41 2.56
TENM4 teneurin transmembrane protein 4 Increases (3) Increased 2.25
Pharmaceuticals 2024,17, 483 10 of 27
Table 1. Cont.
Gene Symbol Entrez Gene Name Literature
Findings Prediction **
Gene Expression,
Fold Change
RR-WS RR WS
TLX2 T cell leukemia homeobox 2 Decreases (2) Increased −2.64 2.39 −2.59
TNIK TRAF2 and NCK interacting kinase Affects (1) Affected −3.53 −3.60
UCN urocortin Affects (1) Affected 2.35 2.38
UGT8 UDP glycosyltransferase 8 Affects (2) Affected −2.21
UNC13A unc-13 homolog A Affects (2) Affected 2.25
WNT7B Wnt family member 7B Affects (2) Affected −3.54
ZNF423 zinc finger protein 423 Affects (4) Affected −2.66
* Development of neurons predicted to be increased (z-score
−
2.87). Overlap p-value 7.29
×
10
−3
. ** Prediction
is based on measurement direction and literature data: 22 of 57 genes deregulated by RR-WS have measurement
direction consistent with an increase in the development of neurons. *** Twenty-five genes (in bold text) deregulated
due to synergistic interactions of RR and WS in the fixed combination Adaptra are highlighted in red text.
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 12 of 30
Figure 9. (a) Venn diagrams of deregulated genes caused by treatment of neuroglial cells with
Rhaponticum cartamoides L. (RC), Eleutherococcus senticosus (RS), and Withania somnifera (WS) root
extracts, as well as their hybrid combination (RC-ES-WS). Values show the number of unique genes
up- or downregulated by each extract alone and the number of deregulated genes overlapping
extracts. (b,c) Heatmaps of canonical pathways, cellular functions, physiological processes, and
diseases activated (brown) and inhibited (blue) by treatment of neuroglial cells with WS, RC, ES,
and the hybrid combination RC-ES-WS. (d) Heatmap of gene expression showing synergistic or
antagonistic effects of the ingredients of BHP RC-ES-WS on dendritic cell maturation pathway.
Upregulated genes are shown in red, while downregulated genes are highlighted in green. (e)
Figure 9. (a) Venn diagrams of deregulated genes caused by treatment of neuroglial cells with
Rhaponticum cartamoides L. (RC), Eleutherococcus senticosus (RS), and Withania somnifera (WS) root
Pharmaceuticals 2024,17, 483 11 of 27
extracts, as well as their hybrid combination (RC-ES-WS). Values show the number of
unique genes up- or downregulated by each extract alone and the number of deregulated
genes overlapping extracts. (b,c) Heatmaps of canonical pathways, cellular functions,
physiological processes, and diseases activated (brown) and inhibited (blue) by treatment
of neuroglial cells with WS, RC, ES, and the hybrid combination RC-ES-WS. (d) Heatmap
of gene expression showing synergistic or antagonistic effects of the ingredients of BHP
RC-ES-WS on dendritic cell maturation pathway. Upregulated genes are shown in red,
while downregulated genes are highlighted in green. (e) Effects on CFH Canonical Pathway.
Authors’ drawings adapted from freely accessible publications [6].
3. Clinical Studies in Human Subjects
3.1. BHP of Rhodiola with Green Tea (Mg-Teadiola®) in Psychological and Social Stress
Green tea contains catechins, tannins, phenolic acids, flavanol glycosides, the alkaloid
caffeine, and the amino acid L-theanine [
69
,
70
], which are known to be capable of signif-
icant effects on the general state of mental alertness or arousal [
71
], activating adaptive
cellular stress responses, inducing the production of cytoprotective proteins, and protecting
neurons in animal models of Parkinson’s disease, Huntington’s disease, Alzheimer disease,
and ischemia–reperfusion injury [
72
,
73
]. Green tea components, such as epigallocatechin
gallate (EGCG), flavonoids kaempferol, and genistein activate protective mechanisms,
including antioxidant and detoxifying enzymes via activation of Nrf2 signaling pathway,
and upstream PKC, PI3K, and MAPKs modulation [70,72,73].
Both Rhodiola rosea and green tea (Camelia sinensis) supplementation were known to
improve subjective stress perception and mood responses to acute stress. Their combi-
nations with magnesium, vitamins B6, B9, B12, and L-theanine in a BHS Mg-Teadiola
®
was developed by Sanofi-Aventis Group, France, and studied in two clinical trials con-
ducted in France and the UK to assess stress-protective effects compared to the efficacy of
Rhodiola rosea and green tea [
49
–
53
], as shown in Table 2. The authors hypothesized that the
efficacy of BHS Mg-Teadiola
®
is superior to that of the ingredients and/or placebo. In a
DB-R-PC-PG clinical trial (NCT03262376), the single dose effect of Mg-Teadiola
®
tablets,
Rhodiola, and green tea extracts was studied in four parallel groups of 100 moderately
stressed, otherwise healthy volunteers (DASS score: 13–25) after acute psychological and
social stress experimentally induced by The Trier Social Stress Test (TSST, speech, and
mental mathematics tasks). The outcome measures were as follows: (i) spectral theta
brain activity associated with cognitive task performance, (ii) subjective stress (stress and
arousal), (iii) mood (profile of mood states), (iv) salivary cortisol, and (v) cardiovascular
parameters (BP, HRV) [49–53].
BHS Mg-Teadiola
®
significantly alleviated subjective stress and mood responses to
acute stress; analyses supported the superiority of the BHS Mg-Teadiola vs. placebo and
the ingredients—Rhodiola and green tea. The BHS Mg-Teadiola
®
significantly attenuated
subjective stress, tension, and total mood disturbance ratings after acute stress exposure.
These effects were found both during the peak stress response and recovery. The salivary
cortisol response was unaffected by treatment [
49
]. The BHS Mg-Teadiola
®
treatment
significantly increased EEG resting state theta activity—considered indicative of a relaxed,
alert state, attenuated subjective stress, anxiety, and mood disturbance, and heightened
emotional and autonomic arousal; Mg-Teadiola may enhance coping capacity and offer
protection from the harmful effects of stress exposure [50].
The BHS Mg-Teadiola
®
increased spectral theta brain activity during the execution of
two attentional tasks, suggesting a potential to increase attentional capacity under stress
conditions [51].
Pharmaceuticals 2024,17, 483 12 of 27
Table 2. Clinical studies of BHP of Rhodiola with other plants.
Reference/
Year
BHP Name,
Ingredients Condition Population (n)/
Country Dosage and Active Markers
Daily Dose
and Duration of
Treatment
Study Design *
and
Comparator
Result and
Outcomes
Dye et al., 2020
[49]
Mg-Teadiola®:
Rhodiola rosea L. +
Camelia chinensis [L.]
Kuntze +
Mg + vitamins B6, B9,
B12+L-theanine
Acute social
stress
100 (25 + 25 + 25 + 25)
Healthy, moderately
stressed
(DASS score: 13–25)
125 mg of IC
dry extracts
of Camellia sinensis L. leaf
containing
50 mg L-theanine, and 222 mg of
IC Rhodiola rosea L. root extract
(corresponding to
1887 mg plant),
and Mg (150 mg elemental) +
vitamins B6 (0.7 mg), B9 (0.1
mg), B12 (0.00125 mg)
One tablet of Mg- Teadiola®
contains 150 mg of Mg, 0.7 mg
of vitamin B6, 0.1 mg of vitamin
B9, and 1.25 g of vitamin B12,
and 222 mg of Rhodiola rosea
rhizome dry extract, as well as
125 mg of green tea extract,
including 50 mg of L-theanine
Single dose
One tablet
DB-R-PC-PG,
Placebo
Capsules
Tablets
Subjective stress (stress and arousal),
Mood (profile of mood states) TSST
Boyle et al.,
2021 [50]Mg-Teadiola®Acute social
stress
25+25+25+25
Healthy, moderately
stressed
(DASS score: 13–25)
DB-R-PC-PG,
Placebo
Capsules
tablets
TSST
Spectral theta brain activity associated
with cognitive task performance
Salivary cortisol, cardiovascular
parameters (BP, HRV)
Boyle et al.,
2022 [51]Mg-Teadiola®Acute social
stress
25+25+25+25
Healthy, moderately
stressed
(DASS score: 13–25)
DB-R-PC-PG,
Placebo
Capsules
tablets
TSST
Spectral theta