1. Introduction
Adaptogens are natural compounds or plant extracts that increase the adaptability, resilience, and survival of living organisms subject to stress [
1,
2,
3]. During the last decade, many molecular targets, networks, and signaling pathways that are regulated by adaptogens have been identified [
1,
4,
5,
6,
7,
8,
9,
10]. The current and potential uses of adaptogens include treatment of stress-induced fatigue, impaired cognitive function, mental illness, and behavioral- and aging-related disorders [
11,
12,
13,
14,
15,
16,
17,
18,
19,
20,
21,
22,
23,
24,
25,
26,
27,
28,
29,
30,
31,
32,
33].
Of particular interest are two adaptogenic plants,
Andrographis paniculata and
Withania somnifera, that have been extensively studied. However, there is limited evidence regarding the clinical efficacy of adaptogens as applied in treatment of cognitive impairment. A number of preclinical studies indicate synergistic interactions of various combinations of adaptogenic plants, e.g.,
A. paniculata with
W. somnifera (Adaptra
® Forte). Growing in Southeast Asia (mainly in China, Thailand, India, and Nepal),
A. paniculata (Burm. F. Nees*), (
Andrographidis Kraut: Ger.) is one of the mostly used medicinal plants in Asia for treatment of respiratory infections, such as the common cold, influenza with fever, sore throat, acute and chronic cough, sinusitis, bronchitis, and pharyngotonsillitis [
33,
34,
35]. The functional claims of
A. paniculata dietary supplements (for a consolidated list of Article 13 health claims of the European Food Safety Authority (EFSA), please refer to
http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_article13.htm) are related mainly to the categories “respiratory health”, “immune function”, and “body defenses against external agents” [
33,
34,
35]. Animal studies showed that andrographolide ameliorates the symptoms of experimental autoimmune encephalomyelitis [
36]. Furthermore, treatment with
Andrographis downregulates expression of
KIT,
IRF1,
CD24,
CASP1,
VCAM1,
NFRSF14, and
EPHA4 genes and upregulates expression
NRG1 and
NGF genes, suggesting predated inhibition of encephalitis [
37]. The main active ingredient, andrographolide, improved mediators of synaptic plasticity in the hippocampus and cerebral cortex and prevented neurodegeneration, suggesting potential use of andrographolide and
Andrographis as neuroprotective agents for the management of neurological disorders associated with memory impairment [
38].
Radix Withaniae consisting of the dried roots of
Withania somnifera (L.) Dunal. (Solanaceae) is widespread from the Mediterranean coast to India in semi-arid habitats. Radix Withaniae, known as ‘Ashwagandha’ in Sanskrit and as ‘Indian ginseng’ in Ayurveda [
25], is used both in Ayurveda and Unani traditional medicinal systems for the treatment of numerous diseases, including neurological and mental disorders. The anxiolytic- and antidepressant-like activities of the alcohol root extract and isolated glycowithanolides, sitoindosides, and withaferin A were demonstrated in animal models of depression and anxiety [
26,
27,
28,
29].
Withania somnifera root extract induced sedation, a reduction in locomotor activity, potentiation of thiopental-induced sleep, reduction of catecholamines and acetylcholine with an increase in histamine and serotonin in the brain tissue of mice, and a delay in semicarbazide- and pentylenetetrazole-induced seizures [
29,
30]. It is possible that GABAergic activity is the mechanism through which GABAergic signaling dysfunctions, such as in anxiety disorders and insomnia, are mitigated through treatment [
31].
Seven clinical studies described
Withania somnifera as a safe and effective adaptogen for treating stress-related neuropsychiatric conditions, such as anxiety and bipolar disorder [
18]. For example, in a double-blind, randomized placebo-controlled trial (RPCT), 64 adult subjects exposed to chronic stress took one capsule twice daily of either
W. somnifera extract (300 mg) or placebo for 60 days [
32]. Telephone interviews were used to assess safety and compliance on days 15, 30, and 45, and at endpoint on day 60. At the study endpoint, subjects taking
W. somnifera extract showed significantly greater reductions as measured on all stress scales versus placebo (
p < 0.0001), and a significantly greater reduction in serum cortisol levels versus placebo (
p = 0.0006). Adverse event rates were comparable in both groups.
The anxiolytic effects of five different preparations of
W. somnifera were observed in five clinical studies using a total of 157 patients [
18]. Three of these studies were double-blind RCPTs with quality scores of 2–3 out of 5. The limitations of these studies include insufficient details regarding preparation of the extract, treatment administration, and randomization procedures. The best study (quality score 4), was an eight-week double-blind RCT which used a water extract and 250–500 mg/day
Withania (Sensoril
®) in testing 60 subjects with bipolar disorder (DSM-IV). Compared with the control, subjects taking
W. somnifera had significantly better performance on three cognitive tasks: digit span backward (
p = 0.035), flanker neutral response time (
p = 0.033), and social cognition response of the Penn Emotional Acuity Test (
p = 0.045). Other cognitive tests were not significantly different between groups. Adverse events were similar in both groups. These encouraging results need to be confirmed in clinical trials of standardized products.
The present study was initiated to test a combination of Andrographis paniculata and Withania somnifera (the dietary supplement Adaptra® Forte) in elderly subjects suffering from mild cognitive impairment. We hoped that the two preparations would interact positively with each other and provide adaptogenic efficacy within a clinical setting.
The primary aim of the study was to determine whether Adaptra® Forte is an effective and safe treatment for impaired cognitive functions, memory, learning ability, sleep disorders, electrical activity of the brain (at six electroencephalogram (EEG) brain frequencies δ, θ, α1, α2, β1, and β2 in 17 different brain regions), and whether Adaptra® Forte is superior to the placebo after single and repeated administration for four weeks. The secondary aim of the study was to assess the durability of the effect over a two-week-long, treatment-free follow-up period. This pilot study was designed to provide preliminary data to power a future large-scale study of Adaptra® Forte efficacy for cognitive impairment.
3. Discussion
We aimed to compare the effects of Adaptra® Forte with a placebo using three different types of evidence: quantitative EEG recording in the presence of different challenges, psychometric testing, and questionnaires. Whereas no significant effects of Adaptra® Forte were recognized with respect to the mood state using the POMS questionnaire, the sleep questionnaire showed statistically significant improvements (p < 0.05) with respect to sleep quality, refreshment, and occurrence of psychosomatic symptoms when the difference between the last experimental day was compared with the first experimental day. With respect to cognitive testing, our comparison of the difference between the first and last experimental days showed a statistically significant improvement (p < 0.05) according to the d2-attention-Test compared to baseline in the case of Adaptra® Forte. Likewise, the difference in the baseline value between the first and last day was significantly better (p < 0.05) with respect to performance in the memory test for the Adaptra® group. The results of cognitive testing could be confirmed in the future using a larger number of participants in a future study.
Quantitative EEG data revealed statistically significant effects in comparison with the placebo under different recording conditions. Typically, brain wave patterns reveal balances and imbalances within the brain, e.g., overactivity in certain brain sections may be correlated with the pharmacological effects of various drugs under various conditions [
39].
During relaxation, increases in spectral δ and θ power emerged 2 h after intake of Adaptra® Forte in numerous brain regions as early as the first experimental day. Spectral changes were more pronounced during the eyes closed recording condition than during the eyes open condition. After four weeks of repetitive intake of Adaptra® Forte, spectral changes were even more pronounced in comparison with the first day.
With respect to spectral EEG changes during cognitive testing, only slight changes were observed 2 h after intake of Adaptra® Forte (data not shown). Possibly due to the general increase in δ and θ spectral power during relaxation, an attenuation was observed for the Adaptra® Forte in comparison to the placebo group, especially for the d2-attention-Test. Baseline values one day after continuous repetitive intake for four weeks showed a statistically significant focal increase in δ/θ power during performance of this test.
Drug-independent evidence from human EEG recordings [
40] suggests that θ band activity increases when subjective alertness decreases in a given individual, even with moderate levels of sleepiness experienced during the daytime. Strong preclinical evidence for a calming action related to θ activity comes from the administration of drugs biochemically acting on norepinephrine α2 autoreceptors, like clonidine or medetomidine in rats [
41]. This effect on θ spectral power was reproduced in humans using dexmedetomidine, which acts as an agonist on α2-adrenergic autoreceptors [
42]. The general increases in δ and θ power are therefore indicative of a calming action of Adaptra
® Forte without inducing sedation.
Similar increases in δ and θ spectral power were observed in humans in the presence of theanine, a compound contained in tea [
43]. Theanine is well known for its calming properties. Increases in spectral θ activity were reported for the synthetic drug buspirone [
44]. Buspirone is prescribed for relieving anxiety and has been referred to as anxioselective. Thus, the observed general increase in θ activity in the presence of Adaptra
® Forte might be indicative of anxiolytic effectiveness. The focal increase in δ/θ spectral power during performance of a mental challenge indicates a positive effect of repetitive dosing of Adaptra
® Forte on concentration, since focal (opposite to general) enhancement of θ activity is related to changes in the attention system [
45].
According to discriminant analysis, the efficacy of Adaptra
® Forte is projected not too far from and with a similar color to Lasea
®, a herbal drug for relief of anxiety. Hence, Adaptra
® Forte might be used with a similar indication as Lasea
®—namely, to treat anxious states. This result is in line with reports in the literature. One of the constituents of Adaptra
® Forte,
Withania somnifera, is reported to be effective in the treatment of anxiety. According to [
46], five clinical studies concluded that
W. somnifera intervention resulted in greater score improvements (significantly, in most cases) than placebo in outcomes on anxiety or stress scales. Broad evidence from preclinical studies indicates a possible anxiolytic and/or anti-depressive activity of constituents from
W. somnifera [
26,
47]. Regarding stress, some evidence in the literature indicates the capability of
Andrographis paniculata, the second constituent of Adaptra
® Forte, to be effective in rats [
48]. Thus, the combination of extracts from
W. somnifera and
A. paniculata seem to supplement each other, providing stress-coping and anxiolytic properties. Finally, a trend in improving cognitive functioning emerged during longer duration of treatment.
In conclusion, spectral changes in the quantitative EEG induced by Adaptra® Forte seem to indicate calming as well as anxiolytic properties, possibly also resulting in patients better handling stress. Improvements with respect to mental challenges, despite the calming action, were only observed when comparing the difference between the first and last experimental day. The significance of the results could be improved in a future study by increasing the number of subjects. The results are in line with the adaptogenic properties that have previously been characterized for Withania and Andrographis preparations in the literature.
4. Materials and Methods
4.1. Participant Eligibility
The study was conducted in the Contact Research Organisation CRO NeuroCode AG (Wetzlar, Germany) with regulatory approval of an independent Ethical Committee at Landesärztekammer Hessen, im Vogelsgesang 3, D-60488 Frankfurt, Germany (Approval Date: 12 July, 2018; protocol number: PP_1717_EuroPharma_FINAL V2_15 May 2018; PP_1717_EuroPharma_FINAL V3_28 August 2018). The study was performed in accordance with the current version of the declaration of Helsinki (52nd WMA General Assembly, Edinburgh, Scotland, October 2000). The trial was conducted in agreement with the International Conference on Harmonization (ICH) guidelines on Good Clinical Practice (GCP). This trial is registered at
ClinicalTrials.gov (Identifier: NCT03780621;
https://www.clinicaltrials.gov/ct2/show/NCT03780621). All volunteers provided informed written consent to partake in the study. The investigator explained the aims, procedures, potential risks, anticipated benefits of the study, and provided a subject’s information sheet to each volunteer. The participants signed the consent form to state that the information had been explained and that they understood. They received a copy of the form and the original copy was maintained in a confidential file in the investigator’s records.
4.2. Selection of the Study Population
4.2.1. Screening and Recruitment
Sixteen elderly volunteers of both genders aged 60 to 75 years were assessed for eligibility and enrolled in the study. Participants were eligible for participation in this trial in the period of December 2018 to May 2019. Patients taking dietary supplements that may affect cognitive functions were asked to terminate that treatment at least two weeks prior to beginning the study.
4.2.2. Inclusion Criteria
The target population included elderly subjects experiencing mild cognitive impairment but who were otherwise healthy. The inclusion criteria were as follows:
Male and female volunteers suffering from cognitive deficits.
Questionnaire-DemTect. “DemTect” (for pre-selection of subjects) score values of 8–12 were regarded as conclusive.
Age between 60 and 75 years inclusive.
Subjects should be right-handed.
Subject must be capable of providing informed consent.
Acceptance of written consent to participate in the study after instruction in written and oral form (informed consent).
4.2.3. Exclusion Criteria
Subjects were excluded having the following characteristics:
Clinically relevant acute or chronic diseases as determined by case history or clinical examination.
Clinically relevant allergic symptoms.
Intake of clinically relevant medication in the last two weeks prior to screening (SC) as well as during the active study period based on notification by the subject or their case history.
Intake of medication with primarily central nervous action (e.g., psychopharmaceuticals or centrally acting antihypertensives, antiepileptics, antidepressants).
Known intolerance/hypersensitivity (allergy) to plant-derived extracts or any other of the ingredients of the investigational product (anamnestic survey).
Presence of a rare genetic disease, such as fructose intolerance, glucose–galactose malabsorption, or sucrose–isomaltase deficiency (anamnestic).
Intake of unusual quantities or abuse of coffee (more than 4 cups a day), tea (more than 4 cups a day), or tobacco (more than 20 cigarettes per day).
Detection of alcohol at the time of the initial examination (day SC) or on study days A, B, C, D, and FU (positive alcohol test) or by case history.
Smoking in the study center on study days A, B, C, D, and FU.
Results of the DemTect Questionnaire score being <8 or >12.
Participation in another clinical trial within the last 60 days.
Poor compliance.
Cancellation of informed consent.
4.2.4. Withdrawal of Subjects
There were no withdrawals. The participants were free to leave the study at any time without explanations. They were advised that it would have no undesirable consequences.
4.3. Study Design
This was a randomized, double-blind, placebo-controlled, two-armed crossover trial of the fixed combination of
Andrographis paniculata and
Withania somnifera (Adaptra
® Forte, Europharma) compared with placebo on electric activity of brain (EAB) and cognitive functions of elderly subjects with cognitive deficits. The effects of Adaptra
® Forte and placebo capsules, taken for 4 weeks, were studied (
Figure 7).
4.4. Intervention and Comparator
The dietary supplement used in this trial was a 550 mg Adaptra® Forte capsule containing 400 mg of A. paniculata herb dry native extract (ratio of herbal substance to genuine herbal preparation 15–23:1, extraction solvent using 70% ethanol) standardized for the content of andrographolides (40 mg), and 150 mg of Withania somnifera roots and dry leaf extract Shoden® (Arjuna Natural Pvt. Ltd, Kerala, India), (ratio of herbal substance to genuine herbal preparation 40:1, extraction solvent using 70% ethanol), standardized for the content of withanolide glycosides (50 mg).
The visually identical placebo capsules each contained 550 mg microcrystalline cellulose, corn starch, magnesium stearate, and brown sugar. The label included the drug name, study code, and storage conditions. Reference samples were retained and stored at Europharma USA (Green Bay, WI, USA).
Doses and Treatment Regimens
Participants received a package containing either Adaptra
® Forte capsules or placebo. They were instructed to take one capsule in the morning and one capsule at the evening with water for weeks. After four weeks of washout period, all participants started to take another test preparation for the next four-week period (
Figure 7).
4.5. Allocation, Study Procedures, and Follow-Up
4.5.1. Randomization, Blinding, and Allocation Concealment
Study preparations were randomly labeled by a qualified pharmacist (QP) and the random sequence of treatment codes was retained at the manufacturing site until the study was completed. The randomization code was kept secret from the investigators, and the code was only revealed after termination of the study. In this way, the investigators were blinded to the study medication and placebo control, thus ensuring a double-blind design. Study preparations were delivered to the study site pre-labeled and coded according to the random sequence. Participants were sequentially enrolled by the principal investigator (PI) at the study site. Participants’ allocation sequence (The Participants List), identifying the subjects and the study supplement packages (Treatment Code No.) were generated and maintained by the PI, who assigned each patient to a Treatment Code No. (from 1 to 16) and filled the patient name in the Case Report Form (CRF) and on the label of the package. The Participants List was used in statistical analysis at the end of the study together with the random sequence received from the QP. Information concerning the allocation of participants was kept in sequentially numbered and sealed envelopes that were stored by the chief executive officer (CEO) of the contract research organization.
4.5.2. Evaluation of Compliance
The products under investigation were taken (days A, B, C, and D) under the supervision of the investigator and the time of application was documented (days A, B, C, and D). Therefore, compliance for all subjects on these experimental days was 100%. Each subject documented the medication intake daily with the date and the time of the application over the four weeks. From day A, day B (Phase A—4 weeks) and day C and day D (Phase B—4 weeks). The compliance was calculated by counting the remaining tablets for each subject. Overall, it was 99.53% (placebo = 99.72%; Adaptra® Forte = 99.34%). The study monitor checked overall compliance with the study protocol upon their visits and the remaining capsules were counted at the end of the study. All unused capsules (two capsules in each package) were returned to the sponsor.
4.5.3. Efficacy Measurement
At all visits, the patients were sitting alone in a quiet separate room with dimmed light in a comfortable chair. Baseline recording for 6 min under the condition of eyes open (Eo) was followed by 4 min eyes closed (Ec), 5 min d2-Test, 5 min ME-test, and 5 min CPT.
Test conditions were standardized and validated. EEG data were recorded twice: before (baseline) and 120 min (2 h) after ingesting the medication. Between the measurements, subjects spent their time in the facility’s recreation room. All experiments occurred at the same time each day (starting at 07:00).
Phase A, Screening and Training
On visit 1, the screening visit, participants were informed about the details of the pending study. All volunteers were checked for eligibility and signed written informed consent, including obligations that they would not take medicine or dietary supplements that may have potential effects on cognitive functions and would not consume more than one cup of coffee daily (in the morning) during the course of the study. After providing written informed consent and passing routine medical examination, eligible patients were included in the trial.
Phase A, Treatment and Assessment
On visit 2, participants passed all tests for cognitive functions and stress in the morning before intake of the trial medication (baseline). Then, the PI randomly assigned participants to the treatments and gave them the test compound. Participants orally took the Phase I treatment and repeated all tests 2 h after intake of trial medication. The treatment lasted 28 consecutive days. Participants took two capsules per day (one in the morning and one in the evening). Phase A treatment was followed by a washout period of 28 days.
Phase B, Treatment and Assessment
At visit 3, participants passed all tests for cognitive functions and stress in the morning before intake of trial medication (baseline). Then, participants orally took Phase II treatment and repeated all tests 2 h after intake of trial medication. Treatment lasted 28 consecutive days. Participants took two capsules per day (1 in the morning and 1 in the evening). Phase II treatment was followed by a washout period of 2 weeks. The study was then considered completed.
4.6. Efficacy and Safety Evaluation
4.6.1. Efficacy Primary Outcome
The efficacy primary outcome measures were responses of electric brain activity as spectral power in 17 different brain regions within six specially defined frequency ranges (δ, θ, α1, α2, β1, and β2) during the test of cognitive performance: d2-Test, ME-Test, and CPT with financial reward. The EEG was recorded bipolarly from 17 surface electrodes according to the International 10–20 system with Cz as physical reference electrode (computer-aided topographical electroencephalometry: CATEEM®, MEWICON CATEEM-Tec GmbH, D94089 Altreichenau, Germany) using an electro-cap. For a detailed description of the procedure, please refer to references.
The signals of all 99 electrode positions (17 real and 82 virtual) were subject to fast Fourier transformation (FFT) based on 4 s sweeps of data epochs (Hanning window). Data were analyzed from 1.25 to 35 Hz using CATEEM
® software). In this software, the resulting frequency spectra are divided into six frequency bands: δ (1.25–4.50 Hz), θ (4.75–6.75 Hz), α1 (7.00–9.50 Hz), α2 (9.75–12.50 Hz), β1 (12.75–18.50 Hz), and β2 (18.75–35.00 Hz) [
49]. This frequency analysis is based on absolute spectral power values and calculated as source density [
50,
51,
52,
53].
4.6.2. Efficacy Secondary Outcomes
The efficacy secondary outcome measures were:
The accuracy of processing the test for attention, concentration, and visual scanning speed, which is expressed as an error rate (ER) score (E%), is defined as the ratio of errors made to the total number of correct responses during 4 min and 40 s
“Accuracy score” obtained in ME-Test
Concentration performance score in CPT
Profile of Mood States (POMS) using a validated POMS questionnaire [
54,
55,
56]. The questionnaire contains 65 words/statements that describe people’s feelings. The test requires the patient to indicate for each word or statement how they have been feeling in the past week including today. Scores are: S1 = dejection (Niedergeschlagenheit); S2 = sullenness (Missmut); S3 = fatigue (Müdigkeit), S4 = thirst for action (Tatendrang).
Quality of sleep score obtained by SF-B/R (Schlaffragebogen) questionnaire (a subjective assessment of sleep quality), used for quantitative and qualitative description and evaluation of sleep behavior and sleep experience. The SF-B/R comprises 31 questions and refers to the last two weeks [
57,
58,
59]. The following subscores were used for the evaluation of the sleep questionnaire SF-B/R: SQ = sleep quality (Schlafqualität); GES = feeling refreshed after sleep/feeling of being recovered after sleep (Gefühl des Erholtseins nach dem Schlaf); PSYA = psychic well-balanced feeling in the evening (psychische Ausgeglichenheit vor dem Schlafenlegen); PSYE = psychic exhausted feeling in the evening (psychisches Erschöpftsein vor dem Schlafenlegen); PSS = psychosomatic problems during sleep (psychosomatische Symptome in der Schlafphase); TRME = dream recall (Traumerinnerung); SWR = sleep–wake regulation (Schlaf-Wach-Regulation).
4.7. Safety Outcomes
Safety outcome measures included incidence and severity of adverse events (AEs). The subjects were informed to report any AE occurring during the study to the PI or the PI’s personnel. Open-ended standardized AE questions were asked by the PI (or designee) during each contact with the subject. AEs observed or reported by a subject and/or staff member was recorded in the CRF. The following variables were recorded for each AE: description of the event, onset (date and time), resolution (date and time), maximum intensity, action taken, outcome, causality (yes or no), and whether or not it constitutes an serious AE. Safety outcome measures were assessed from the date of randomization until the end of the treatment.
4.8. Sample Size Considerations
We enrolled 16 patients (16 per treatment condition). Sample size was not determined during this pilot study. Estimation of the number of subjects to be included into the study was performed by considering data from earlier experimental results obtained under a similar experimental design.
4.9. Statistical Analysis
EEG data from the first recording session before intake of the capsules are reported as absolute numbers (µV2). For mathematical comparison to the results obtained for other preparations or drugs under comparable conditions, the linear discriminant analysis according to Fischer was used. Results from the first three discriminant functions were projected into space (x, y, and z coordinates), whereas results from the fourth to sixth discriminant functions were coded into red, green, and blue, respectively, followed by an additive color mixture (RGB-mode). To statistically document the different electric reactions of the brain to various cognitive loads, data from each challenge were documented as absolute spectral power (µV2). Adaptra® Forte capsules versus placebo were compared by evaluation of the baseline recording of the last day in comparison to the baseline values of the first day (effect of single and repetitive dosing). Data from the first recording (baseline) were set as 100% and electrophysiological changes produced by placebo or Adaptra® Forte capsules are reported as percentage changes. Since this study was an exploratory study with a small number of participants and EEG data were not normally distributed, the non-parametric sign test was chosen for comparing between the effects of placebo and Adaptra® Forte. Exploratory statistics provided p-values.