Botanical hybrid preparations (BHP) in phytomedicine and phytotherapy research: background and perspectives

1. Introduction

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 [1,2]. In Europe, Rosenrot 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.

During the last two decades, more than 1200 studies were conducted in Europe, America, and China, providing results of preclinical and clinical efficacy, safety, and quality of Rhodiola 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 [3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38].

Seventeen clinical studies were conducted on the fixed combinations of Rhodiola rosea with Green Tea [39,40,41,42,43], Eleutherococcus, and Schisandra [44,45,46,47,48,49], Eleutherococcus, Schisandra, and Leuzea [47,48], caffeine [50], Cordyceps [51,52,53,54], Gingko [55], Black Cohosh [56], saffron [57], L-carnosine [58], Eleutherococcus and Glycyrrhiza glabra [59].

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. Combining two or more plants assumes a hybrid botanical preparation (BHP) is more active due to their additive or potentiating effects. Figure 1 illustrates an allegoric analogy with two or more kinds of hybrids from ancient mythology.

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 multiple effects on various targets. The term "hybrid" botanical preparation is coined for biological/pharmacological activity (conditional pharmacological "signature") of a fixed herbal combination with specific chemical composition (e.g., TLC of HPLC conditional chemical "fingerprint") (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 between various regulatory systems of the host cells and organism.

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 botanicals.

This overview provides the results of recent clinical and network pharmacology studies of BHP of Rhodiola rosea with other plants. 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 and Other Plant Extracts

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 multiple effects on various targets. Hypothetically, combining two pharmacologically active extracts aims to achieve more benefits or higher effectiveness of their combination due to additive (1 + 1 = 2), potentiating (0 + 1 > 1), amplifying (1 + 1 > 2) or synergistic (0 + 0>0) interactions. However, antagonistic (1 + 1 < 2) and attenuating (1 + 1 < 2) interactions of the components of complex herbal preparations may also have a negative impact both on efficacy and overall toxicity (Panossian et al., 2018b). Recent studies show that the beneficial outcome is not evident until it is observed in experimental studies or clinical trials due to possible antagonistic (1 + 1 < 2) interactions of the components of complex herbal preparations, which may also have an impact on the overall toxicity [60,61].

These assumptions were supported in the series of in vitro studies where the effects of several plant extracts and their hybrid combinations on the number and composition of deregulated genes in brain cell cultures were analyzed [61,62,63,64]. The composition of genes deregulated by hybrid combinations of plant extract was quantitatively and qualitatively different from the composition of genes deregulated by each plant separately, suggesting that the impact of the hybrid combination on the target cells was qualitatively different from the effects of ingredients [63] (Figure 3). In other words, the phytochemicals of two of three plant extracts exhibit quite different pharmacological activities if combined. These findings are essential for understanding unpredictable results obtained in clinical studies of multi-component drugs and dietary supplements [39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59].

Figure 3 illustrates the essence hybridization of ingredients of BHP, 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).

Meanwhile, the total extract of Rhodiola-containing 120 phytochemicals (including salidroside) or 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) [63] (Figure 3). Among those deregulated by ADAPT-232 were 206 genes that were not deregulated by any ingredient of BHP ADAPT-232 due to the synergy effect (Figure 3). 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 [63].

Figure 4 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 in inflammation and neurodegeneration in neuroglia cells.

In a recent study [62], BHP of Rhodiola with Ashwagandha (Adaptra) positively regulated 22 of 57 genes, which are known to activate the development of neurons (Figure 5), suggesting that Adaptra is potentially helpful in learning and memory, stress, and depression, insomnia, and aging-related neurodegenerative diseases preventing of Alzheimer's and Parkinson's diseases, and recovery from brain injury and stroke. Notably, 25 genes were deregulated due to RR and WS synergistic interactions in the fixed combination Adaptra (Figure 5 and Table 1). This means that in combination, these two ingredients of Adaptra act synergistically. In other words, Adapta is superior to Rhodiola or Withania in activation of neurogenesis and, consequently, has potential effects mentioned above [62].

In this study, isolated brain cells were incubated independently with Adaptra, Rhodiola, and Withania. Gene expression degree was measured, expressed in fold changes compared to control, and analyzed using Ingenuity Pathway Analysis software (QIAGEN Bioinformatics). IPA performs different calculations based on the Ingenuity Knowledge Base, a large gathering of observations with approximately 5 Mio findings manually curated from the biomedical literature or integrated from third-party databases [62].

Figure 6 shows 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 and their hybrid combination (RC-ES-WS) [61]. Values indicate the number of unique genes up- or downregulated by each extract alone and the number of deregulated genes overlapping multiple extracts. 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. Synergistic or antagonistic effects on canonical pathways, cellular functions, physiological processes, diseases, and gene expression associated with the pathway (e.g., dendritic cell maturation) can be observed by comparison of the effects of the hybrid substance RC-ES-WS with a lack of (or opposite) effects of individual extracts at a significance level of p < 0.05 (-log = 1.3) and a z-score > 2. Up-regulated genes are shown in red, while down-regulated genes are in green.

3. Clinical Studies in Human Subjects

3.1. HHP of Rhodiola with Green Tea (Mg-Teadiola^®) in Psychological and Social Stress

Rhodiola rosea and Green tea (Camelia sinensis) supplementation were known to improve subjective stress perception and mood responses to acute stress. Their combinations 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 conducted in France and the UK to assess stress-protective effects compared to the efficacy Rhodiola rosea and Green tea [39,40,41,42,43], Table 2. The authors hypothesized the efficacy of BHS Mg-Teadiola^® is superior to 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, social stress experimentally induced by The Trier Social Stress Test (TSST, speech, and mental mathematics tasks). Outcome measures were: (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) [39,40,41,42,43].

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. Effects were found both during the peak stress response and recovery. The salivary cortisol response was unaffected by treatment [39].

The BHS Mg-Teadiola^® treatment significantly increased EEG resting state theta – 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 [40].

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 [41].

In a placebo-controlled randomized clinical trial, the repeated dose effect of Mg-Teadiola^® tablets for four weeks was studied in two parallel groups of 100 moderately chronically stressed otherwise healthy volunteers (the Depression Anxiety Stress Scale-42 score: > 14) and two groups of 40 individuals after acute thermal stimulation. Outcome measures were: (i) - blood-oxygen-level-dependent signal, (ii) - stress, (iii) - depression, (iv) – anxiety, (v) - salivary cortisol, and (vi) – sleep [42].

Mg-Teadiola was effective in relieving stress on days 14 and 28 in chronic stress and may diminish pain perception, underlining its potential benefits for patients suffering from pain in whom comorbidities such as stress and sleep disorders are frequent [42]. Supplementation with Mg-Teadiola reduced stress on D28 in chronically stressed but otherwise healthy individuals and modulated the stress and pain cerebral matrices during stressful thermal stimulus [43].

Green tea contains catechins, tannins, phenolic acids, flavanol glycosides, the alkaloid caffeine, and amino acid L-theanine [66,67], which are known capable of a significant effect on the general state of mental alertness or arousal [68], activating adaptive cellular stress responses inducing the production of cytoprotective proteins and protecting neurons in an animal model of Parkinson’s disease, Huntington’s disease, Alzheimer disease, and ischemia–reperfusion injury [69,70]. 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 [67,69,70].

The shortcoming of studies: The authors declare the labeled amount of active ingredients but do not adhere to CONSORT regarding the quality of the product, which is not adequately characterized in respect to extraction solvents, dry herb: dry native extract ratio (DER), the content of active markers (caffeine, theanine, Mg²⁺, salidroside, rosavin, etc.), the analytical methods validated for selectivity, accuracy, and procession and providing TLC and HPLC fingerprints to ensure reproducible quality and reproducible pharmacological activity. The placebo and Mg-Teadiola were distinguishable by appearance. Reporting the masking procedure is not convincing enough to provide an adequate double-blind study design. The magnesium and caffeine content contained in Green tea was uncontrolled and not specified; there is no guarantee that the effect of Mg-Teadiola will provide consistently reproducible efficacy.

Green tea products are known as herbs that have significant variability depending on numerous factors [71].

There are concerns regarding the rationale for including green tea containing caffeine in the formulation of the safe adaptogenic hybrid supplement Mg-Teadiole; addiction to caffeine and other undesired effects of regular caffeine supplementation are well known. In this context, the formulation's rationale does not stand up to scrutiny.

3.2. HHP of Rhodiola SHR-5 with Schisandra and Eleutherococcus (ADAPT-232/Chisan^®) for Relief of Mental and Physical Fatigue both in Healthy Subjects and in Patients with Pneumonia and COVID-19

ADAP232/Chisan is the combination of extracts of Rhodiola rosea L., roots (SHR-5) Eleutherococcus senticosus (Rupr. et Maxim) Harms, roots (SHE-3) and Schisandra chinensis (Turcz) Baill., fruits, containing 0.5% schizandrin, 0.47% salidroside, 0.59% rosavin, 0.11% eleutherosides B and 0.19% E. ADAPT 232® capsules and Chisan^® oral solution have been used to enhance mental and physical capacities in case of tiredness or during convalescence as a natural remedy in Sweden since 1979 and as an herbal medicinal product in Denmark since 2002. In 2008, it was approved as a traditional herbal medicinal product in 2008 in Sweden as an adaptogen in case of decreased performance, such as fatigue and sensation of weakness.

Early studies of ADAPT-232 showed improved cognitive functions and endurance under the stress of cosmonauts. It has been used in space and by cosmonauts during the longest flight in space in the 1990s. It is noteworthy that typically, only carefully verified and validated for their efficacy and safety aids were allowed for use in space. ADAPT-232 showed a good anti-fatigue effect, significantly increasing accuracy and precision in psychometric tests, particularly in complicated tests, decreasing the number of errors, and improving short memory in fatigue under space-like conditions. Significant improvements in concentration, oculomotor coordination, and short-term memory of cosmonauts have been demonstrated in the computerized Monotonic 3 test for attention and two complicated short-term memory tests [72].

Three randomized, placebo-controlled, double-blind clinical trials (RCT) were conducted on healthy subjects, and two RCT studies were conducted to assess the anti-fatigue effect of ADAPT232/Chisan during recovery of patients with infectious respiratory diseases [44,45,46,47,48,49]. The efficacy of a single dose of ADAPT-232 on cognitive functions in humans has been demonstrated in the participants who experienced stressful cognitive tasks, namely, the Stroop Colour-Word test followed by the d2 test of attention (d2), before and two hours after treatment. The results of the d2 test (attention deficit) are shown on the graph below [45], Figure 5a. ADAP232/Chisan increases tolerance to mental fatigue in healthy subjects [45]. ADAP232 significantly improved attention and increased speed and accuracy during stressful cognitive tasks compared to placebo [45]. ADAP232 significantly decreased the number of mistakes in complicated psychometric tests [44,45,47,48] and improved patients' quality of life and recovery from acute nonspecific pneumonia [44], Figure 5b. ADAPT232/Chisan has beneficial effects on stress-induced fatigue [45] in patients during their recovery from pneumonia [44] Figure 5b.

ADAP232 and the BHP containing Rhodiola, Eleutherococcus, Schisandra, and Leuzea (Rhaponticum cartamoides L.) roots extracts, significantly decreased inattention, impulsivity, and the perception of stress, reducing fatigue and increasing the anabolic index (testosterone/cortisol ratio) (Figure 5 c-f) in 200 elite athletes and improving their physical performance and the recovery rate of athletes after heavy physical and emotional load and increased adaptation to physical and emotional stress [47,48].

Recently, the effects of ADAPT-232 on the recovery of patients with Long-COVID symptoms (fatigue, headache, respiratory insufficiency, cognitive performance, mood disorders, loss of smell, taste, and hair, sweatiness, cough, pain in joints, muscles, and chest) was studied [49]. One hundred patients with confirmed positive SARS-CoV-2 test who were discharged from the intensive care unit and experienced at least three of nine Long-COVID symptoms in the 30 days before randomization were included in the study. Chisan^®/ADAPT-232 decreased the duration of fatigue and pain for one and two days, respectively, in 50% of patients (Figure 5 h,i). The number of patients with lack of fatigue and pain symptoms was significantly less in the Chisan^® treatment group than in the placebo group on days 9 (39% vs. 57%, pain relief, p = 0.0019) and 11 (28% vs. 43%, relief of fatigue, * p = 0.0157). Significant relief of severity of all Long-COVID symptoms over the time of treatment and the follow-up period was observed in both groups of patients, notably decreasing the level of anxiety and depression from mild and moderate to normal, as well as increasing cognitive performance in patients in the d2 test for attention and increasing their physical activity and workout (daily walk time). However, the significant difference between placebo and Chisan^® treatment was observed only with a workout (daily walk time) (Figure 5 g). A clinical assessment of blood markers of the inflammatory response (C-reactive protein) and blood coagulation (D-dimer) did not reveal any significant difference over time between treatment groups except significantly lower IL-6 in the Chisan^® treatment group. Furthermore, a substantial difference between the placebo and Chisan^® treatment was observed for creatinine: Chisan^® significantly decreased blood creatinine compared to the placebo, suggesting preventing renal failure progression in Long-COVID [49].

Figure 7. (a)-Single dose effects of ADAPT-232 on cognitive functions. All participants were submitted to stressful cognitive tasks (Stroop CW and d2 tests) four times over three consecutive days (Day 1: in the morning when subjects were not tired; Day 2: in the morning when subjects were not tired and in the afternoon when subjects were tired; Day 3: in the afternoon, two hours after treatment with ADAPT or placebo, when subjects were tired). Repeated testing in the mornings of days 1 and 2 allowed to exclude the effect of learning. The degree of tiredness in the afternoon of day 2 was assessed by comparing these results with those obtained in the morning of the same day. A significant difference (p < 0.05) between the two groups was observed with respect to TN-E score (the main parameter in the d2 test), where ADAPT-232 increases speed and accuracy of mental performance, improving attention, in comparison to placebo. Thus, the cognitive performance (attention measured as TN-E score) increased for 43 units in the ADAPT-232 group, while the increase was for eight units in the placebo group.

Figure 7. (a)-Single dose effects of ADAPT-232 on cognitive functions. All participants were submitted to stressful cognitive tasks (Stroop CW and d2 tests) four times over three consecutive days (Day 1: in the morning when subjects were not tired; Day 2: in the morning when subjects were not tired and in the afternoon when subjects were tired; Day 3: in the afternoon, two hours after treatment with ADAPT or placebo, when subjects were tired). Repeated testing in the mornings of days 1 and 2 allowed to exclude the effect of learning. The degree of tiredness in the afternoon of day 2 was assessed by comparing these results with those obtained in the morning of the same day. A significant difference (p < 0.05) between the two groups was observed with respect to TN-E score (the main parameter in the d2 test), where ADAPT-232 increases speed and accuracy of mental performance, improving attention, in comparison to placebo. Thus, the cognitive performance (attention measured as TN-E score) increased for 43 units in the ADAPT-232 group, while the increase was for eight units in the placebo group.

3.3. HHP of Rhodiola With Caffeine for Improvement of Muscle Strength and Muscular Endurance

Alkaloid caffeine is a central nervous system (CNS) stimulant widely consumed in coffee and tea, promoting athletic capacities, including anaerobic exercise capacity, strength, and muscular and aerobic endurance. From 1984 to 2004, the World Anti-Doping Agency (WADA) banned caffeine because it enhances athletic performance in sports such as running, swimming, and cycling, but in 2004, lifted the ban, removing caffeine from the list of the Prohibited List, despite growing evidence that it is a sports booster. The athletes usually receive 3–6 mg/kg doses of caffeine to enhance resistance exercise performance acutely. However, a dose above 9 mg/kg often fails to cause a positive impact on various performance improvements and may also lead to side effects, such as tachycardia, headache, and anxiety. Long-term caffeine intake leads to an upregulation of adenosine receptor expression, resulting in a progressive reduction of the stimulatory effect on adenosine receptors and caffeine resistance in the human body that has gradually occurred after 28 days of long-term caffeine intake [73]. Furthermore, regular use of caffeine-contained beverages may develop a physical, emotional, and psychological dependence, and one may experience a caffeine withdrawal syndrome characterized by headache, anxiety, irritability, low energy levels, dizziness or light-headedness, mental fogginess, negative mood after abrupt cessation of caffeine intake. Unlike other psychoactive drugs, it is legal, cheap, and not regulated in almost all parts of the world [74].

In a recent study, the stimulating effects of caffeine, Rhodiola, and their fixed combination were compared in human subjects and rats, suggesting the superimposed effects of the two supplements due to their different application mechanisms. The impact of supplementation of caffeine (3 mg/kg/daily), Rhodiola, (2.4 g/daily), and their fixed combination for 28 days on muscle strength and muscular endurance physical performance of resistance exercise-untrained volunteers and resistance exercise-trained subjects were assessed in randomized, double-blind, crossover clinical trial including four groups of resistance exercise of trained subjects and two groups of resistance exercise-trained volunteers.

BHP Rhodiola-caffeine improved the rat model's forelimb grip strength, erythropoietin, dopamine, and oxygen consumption rate. BHP Rhodiola-caffeine significantly increased the bench press one-repetition maximum, deep squat 1RM, maximum voluntary isometric contraction, and maximum repetitions of 60% 1RM bench press in resistance exercise-untrained volunteers. BHP Rhodiola-caffeine improved resistance exercise performance significantly by increasing the bench press one-repetition maximum, deep squat 1RM, maximum voluntary isometric contraction, and maximum repetitions of 60% 1RM bench press for both resistance exercise-untrained and -trained volunteers. The authors concluded that the stimulating effect of BHP Rhodiola-caffeine was superior to Rhodiola and caffeine separately, presumably due to their different mechanisms of action [73].

3.4. HHP of Rhodiola with Cordyceps for Boost Exercise Performance

The aim to improve the physical performance of Rhodiola by combining with Cordyceps has not been achieved in all three studies; the efficacy of HHP of Rhodiola rosea with Cordyceps was inactive compared to placebo [51,52,54]. The fixed combination of Rhodiola rosea with Cordyceps has shown no ergogenic effect on oxygen consumption, cycling time, muscle strength, oxygen uptake, and muscle performance during maximal graded tests following 14 days of supplementation. Meanwhile, both Rhodiola rosea and Cordyceps alone have demonstrated increased muscle fatigue resistance and strength benefits in active men; Cordyceps increased aerobic capacity, and Rhodiola increased performance and time to exhaustion. The combination of Rhodiola with Cordyceps negatively affects the efficacy of intervention due to an unknown antagonistic interaction, probably due to the impact of purine alkaloid Cordycepin [75]. Its chemical structure resembles the nucleoside adenosine, which is involved in adenosine triphosphate (ATP) metabolism, providing energy in many processes, including muscle contraction.

3.5. HHP of Rhodiola with Ginkgo for Improvement of Cognitive Function

Ginkgo biloba flavones and polyphenols have a potential neuroprotective effect in Alzheimer's dementia by inhibiting amyloid fibril accumulation [76,77]. G. biloba delays age-induced cognitive decline and may have more delicate and subtle therapeutic effects on the speed of cognitive alterations [78]. G. biloba is known to improve cognitive impairment and memory, increase cerebral blood circulation in the elderly with dementia syndrome and Alzheimer’s disease, and relieve headaches and migraine [77,78]. Several human clinical trials provide preliminary positive evidence of the antidepressant effects and anxiolytic activity of G. biloba [79,80].

The effects of BHP of Rhodiola with Ginkgo on the cognitive functions of 112 healthy subjects were recruited in a double-blind, randomized, placebo-controlled four parallel groups trial. The efficacy of G. biloba, R. rosea extracts, and their fixed combination was assessed in psychomotor vigilance task (PVT) and short-term working memory accuracy tests as compared to placebo effects; critical flicker-fusion frequency, PVT, and computerized N-back test assessed the central cognitive effect. The short-term working memory accuracy and sustained attention were measured in the psychomotor vigilance task, which provided a numerical measure of sleepiness by counting the number of lapses in the attention of the tested subject. G. biloba or R. rosea improve PVT and exhibit low to moderate working memory accuracy, whereas BHP exhibits superior effects on PVT, short-term working memory accuracy, and critical fusion versus flicker compared to G. biloba or R. rosea if used alone. The author concluded that combining R. rosea with G. biloba led to a more significant effect on cognitive performance than either G. biloba or R. rosea if used alone [55].

3.6. BHP of Rhodiola with Black Cohosh for Relief of Aging-Related Menopausal Symptoms

Ageing-related decline in the production of sex hormones is associated with many menopause symptoms, including hot flashes, sweating, sleeplessness, nervousness, irritability, depressive state, palpitation, joint pain, libido changes, vaginal dryness, etc. Long-term hormonal replacement therapy has been shown to increase the risk of developing breast cancer. In this context, there is a high demand for relatively safe aids that can relieve or prevent menopausal symptoms, as more than 1.2 billion women globally will experience menopause syndrome by the year 2030. The recent study aimed to assess the efficacy and safety of a new herbal preparation (Menopause Relief EP^®), the hybrid combination of Actaea racemosa L. (Black Cohosh, BC) and Rhodiola rosea L. (RR) root extracts, compared with the most effective dose of BC extract in women with menopausal complaints [56]. A total of 220 women (mean age 52 years) were randomly assigned to receive two capsules of either BC (6.5 mg), BC500 (500 mg), Menopause Relief EP ^® (206,5), or placebo once per day for 12 weeks. The efficacy endpoints were relief of menopausal symptoms, including hot flushes, sweating, heart discomfort, sleep problems, joint and muscular discomfort, depressive mood, irritability, anxiety, physical and mental exhaustion, sexual problems, bladder problems, vaginal dryness and measured using the Kupperman Menopausal Index (KMI), Menopause Relief Score (MRS), and menopause Utian Quality of Life (UQOL) index. The symptom relief effects of Menopause Relief EP^® were significantly superior in all tests to the effects of BC and placebo after their repeated administration for 6 and 12 weeks. Menopause Relief EP^® significantly improved the UQOL index in patients, compared to BC, BC500, and placebo, mainly due to the beneficial effects on the emotional and physical health domains (Figure 8). It was concluded that the effect of BBHP is more effective of the mono-drug in relief of menopausal symptoms, particularly psychological symptoms.

3.7. HHP of Rhodiola with Saffron in Mild and Moderate Depression

Findings from clinical trials conducted to date indicate that saffron supplementation can improve symptoms of depression in patients with MDD. Saffron was found to be as effective as treatment with conventional antidepressants, such as imipramine and fluoxetine, in randomized, double-blind clinical trials [81]. Supplementation of a BHP of Rhodiola with saffron extracts in a daily dose of 308 mg Rhodiola and 30 mg Crocus for six weeks significantly decreased anxiety and depression scores compared to the baseline in 45 patients with mild-moderate depression. The results of this observational study are encouraging, and serious adverse effects were not recorded. However, a double-blind, randomized study with a positive comparator and placebo control design is needed to confirm these results [57].

3.8. BHP of Rhodiola with L-carnosine in Aging Skin

Carnosine (β-alanine-L-histidine) is an endogenous water-soluble dipeptide distributed mainly in skeletal muscles depending on age (decreases with age), gender (lower in females), and food (lower in a vegetarian diet) and playing an essential role in aging-related neurodegeneration, Diabetes mellitus, cardiovascular diseases, cancer, and other disorders. The meta-analysis of clinical trial findings indisputably demonstrated a significant beneficial effect of L-carnosine only for diabetes mellitus and cognitive impairment [82]. The pathophysiology of sensitive skin consists of an inflammatory reaction resulting from the abnormal penetration of potentially irritating substances in the skin, which occurs due to skin barrier dysfunction and changes in the production of local neuromodulators. A double-blind comparative study was conducted on 124 volunteers with sensitive skin who received either a formulation containing 1% of Rhodiola-L-carnosine BHP or a placebo applied twice a day for 28 consecutive days [58]. The reduction of transepidermal water loss, positive perceptions of improvements in skin dryness and skin comfort sensation, and reduction of discomfort sensation after the stinging test were measured. Rhodiola-L-carnosine BHP treatment produced in vivo protective effects in skin barrier function and positive subjective response of sensitive skin volunteers. The authors suggested that the protective effect of Rhodiola-L-carnosine BHP in skin barrier function and the positive reaction produced in human subjects with sensitive skin is due to an increase in opioid peptides release, an inhibitory effect on neuropeptides production, and modulation of cytokines production by keratinocytes under ultraviolet stress observed in vitro testing [58].

4. Conclusions

The most important finding from BHP's network pharmacology studies was the evidence supporting the synergistic interaction of its ingredients, revealing unexpected new pharmacological activities unique to and specific to the new BHP.

The results of some studies show the superior efficacy of BHP, such as the combinations of Rhodiola with Black Cohosh, Rhodiola with Ashwagandha, Rhodiola with Green tea, Rhodiola with Ginkgo, Rhodiola with Eleutherococcus and Schisandra, compared with the mono drugs. On the contrary, some a priori designed combinations, such as Rhodiola with cordyceps, were inactive in several clinical studies, presumably due to antagonistic interactions and crosstalk between molecular targets within molecular networks involved in the cellular and overall response of organisms on the intervention.

Network pharmacology studies help predict the results of studies to discover new indications and unpredicted adverse events. Predictions based exclusively on in silico analysis require experimental validation in animals and clinical studies.