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Cardioprotective Potential of the Ethanol and Water Extracts of Four Psilocybin Mushrooms on Angiotensin II-Induced Hypertrophy and Oxidative Stress on H9C2 Cardiomyocytes

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04 June 2023

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06 June 2023

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Abstract
Psilocybin-containing mushrooms, commonly known as magic mushrooms have antidepressant effect, however, their safety in cardiovascular diseases such as heart failure is not fully known and needs to be investigated. Cardiac hypertrophy is an independent risk factor for heart failure morbidity and mortality. Angiotensin II (Ang-II) plays a major role in the pathogenesis of cardiac hypertrophy. We investigated the cardiovascular safety of extracts of Panaeolus cyanescens, Psilocybe natalensis, Psilocybe cubensis, and Psilocybe cubensis leucistic A+ strain mushrooms, well-known psilocybin-containing mushrooms in the Panaeolus and Psilocybe genus on Ang II-induced hypertrophy oxidative stress. The four mushrooms were grown, dried and extracted with 70% ethanol, cold and hot water. Extracts were tested for cytotoxicity on H9C2 cardiomyoblast cells. The cardiomyocytes were induced with (10 µM) AngII and treated with the three extracts of the four mushrooms over 48 hours. Control cells were serum starved but neither AngII induced nor treated while AngII cells were serum starved and stimulated with AngII but not treated. Losartan, an inhibitor of AngII type 1 receptor was used as positive control. Effects of the extracts on actin-filament labelling and cell surface area, mitochondrial activity, reactive oxygen species (ROS) and atrial natriuretic peptide levels were determined. Stimulation with AngII lowered cell viability, increased the cell width measurements and intracellular ROS levels significantly compared to control cells. The results indicated that the ethanol and water extracts of the four psilocybin mushrooms did not exacerbate the angiotensin II-induced hypertrophy conditions, but the extracts had cardio-protective activity against angiotensin II-induced oxidative stress. The phytochemical analysis of the extracts confirmed detections of known compounds with antioxidant and anti-inflammatory effects in the water and ethanol extracts of these four psilocybin mushrooms.
Keywords: 
Subject: Medicine and Pharmacology  -   Cardiac and Cardiovascular Systems

1. Introduction

Depression is a burden to society and associated with chronic stress and aging [1]. Psilocybin-containing mushrooms have been used by different tribes to improve quality of life and for mind healing [2]. Many studies have also demonstrated the antidepressant effects of psilocybin (4-phospholoxy-N-N-dimethyltryptamine), the classic psychedelic agent occurring naturally in psilocybin-containing mushrooms [3,4]. Consequently, the use and awareness of psilocybin-containing mushrooms, commonly known as magic mushrooms is increasing. However, psilocybin and psilocybin mushrooms also lead to a temporary increase in heart rate and blood pressure which may pose as risk especially for users suffering from cardiovascular diseases [5]. Since depression is associated with aging people that are prone to cardiovascular disease such as hypertension and heart failure, investigating safety of the mushroom usage in these conditions is crucial.
Heart failure is an international public health problem of pandemic proportions and studies showed that about 64.3 million people globally are living with a heart failure condition [6,7]. Cardiomyocyte hypertrophy which is a major consequence of pressure and/or volume overload is considered a significant diagnostic component and plays a key role in the progression of heart failure [8]. Cell enlargement and apoptotic loss of cardiomyocytes are key pathological changes in cardiac hypertrophy [9,10]. Many factors are involved in the pathogenesis and regulation of cardiomyocyte hypertrophy including angiotensin II (AngII). Angiotensin II is a key factor of the renin-angiotensin system that induces cell hypertrophy, differentiation and apoptosis through activation of various intracellular signalling molecules including calcineurin, mitogen-activated protein kinase and many other factors [11].
Angiotensin II has two receptors, AngII type 1 (AT1R) which is known to mediate pro-hypertrophic effects of AngII and AngII type 2 (AT2R) that attenuates the AT1R activation-induced hypertrophy [12]. A fibroblast-derived factor is identified as a biochemical process used by AngII to stimulate direct cardiomyocyte hypertrophy which can be blocked by using bromodeoxyuridine, a fibroblast proliferation inhibitor [12]. Many studies also showed that AngII stimulated protein synthesis which could also be eliminated by losartan, the AT1R blocker, further indicating direct role of AngII in the production of some fibroblasts factor [12,13]. Furthermore, studies have also showed that the pro-hypertrophic effects of AngII are also mediated through mitochondrial and induced-cell death by activating NAD(P)H oxidase through ATR1 receptors leading to increased generation of reactive oxygen species (ROS) and oxidative stress [14,15]. Oxidative stress is the lack of balance state where production of ROS such as superoxide, hydrogen peroxide and hydroxyl radicals exceed the antioxidant defences [13].
Plasma levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), well-known as hall markers of heart failure, have been found to increase with the severity of heart failure [16]. Their main physiological effects are to suppress progression of heart failure by inducing several effects that includes inhibition of the renin-angiotensin-aldosterone and promoting vasodilation and natriuresis [16].
This study aimed at investigating for the first time the risks and/or safety of Panaeolus cyanescens, Psilocybe natalensis, Psilocybe cubensis and Psilocybe cubensis leucistic A+ strain mushrooms, well-known psilocybin-containing mushroom in the genus Panaeolus and Psilocybe, on AngII-induced hypertrophy using a rat H9C2 cardiomyoblast cells model which is well-known and a widely used in vitro cell model with accepted reliability in cardiovascular drug discovery [17].

2. Results

This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.

2.1. Cytotoxicity of the Extractsn

The cytotoxicity results of the extracts on H9C2 cardiomyocytes showed that the three extracts of the four magic mushrooms were not toxic when comparing their LC50 values with the positive control doxorubicin, a well-known toxic drug, Table 6.1.
Table 1. Cytotoxicity effects of the extracts over 48 hours on H9C2 cardiomyocytes.
Table 1. Cytotoxicity effects of the extracts over 48 hours on H9C2 cardiomyocytes.
Sample LC50 (µg/mL)
Pan cyanescens cold-water (PC) 66.1 ± 1.8
Pan cyanescens hot-water (PH) >100
Pan cyanescens 70% ethanol (PE) > 100
P. cubensis cold-water (GC) >100
P. cubensis hot-water (GH) >100
P. cubensis 70% ethanol (GE) >100
P. A+ strain cold-water (AC) >100
P. A+ strain hot-water (AH) >100
P. A+ strain 70% ethanol (AE) >100
P. natalensis cold-water (NC) >100
P. natalensis hot-water (NH) >100
P. natalensis 70% ethanol (NE) >100
Doxorubicin 0.2169 ± 0.037

2.2. Effects of the Extracts on AngII-Induced Hypertrophy in H9C2 Cardiomyocytes

Morphological analysis of the actin filaments of the cardiomyocytes using rhodamine phalloidin reagent showed that AngII increased the cell size of the stimulated cardiomyocytes F-actin over 48 hours, Figure 1. Treatment with 50 µg/mL of the three extracts of Pan cyanescens (PC, PH and PE) and P. cubensis (GC, GH and GE) and the positive control losartan (100 µM) reduced the F-actin sizes of the cells, Figure 1.
Cell surface area measurements performed using CellSens Dimension 1,12 software on the cells showed in Figure 2 that AngII increased the cell width measurements of the induced cells 1.4-fold significantly (p< 0.001) compared to the non-induced negative control cells. The positive control losartan reversed these AngII effects significantly (p< 0.001). The cold-water (PC), hot-water (PH) and ethanol (PE) extracts of Pan cyanescens and P. cubensis’s cold-water (GC), hot-water (GH) and ethanol (GE) mushroom extracts also all reversed the AngII effects significantly, Figure 2.
Florescence morphological studies showed a decreased F-actin sizes in the AngII-induced cells treated with the cold-water, hot-water and ethanol extracts of P. A+ strain (AC, AH and AE) and P. natalensis (NC, NH and NE) mushrooms over 48 hours, in comparison to the AngII-stimulated cells, Figure 3.
The treatment with the cold-water, hot-water and ethanol extracts of P. A+ strain (AC, AN and AE) and P. natalensis (NC, NH and AE) mushrooms reduced the AngII-induced cell size measurements significantly compared to the AngII-stimulated cells, Figure 4.

2.3. Effects of the Extracts on ANP Levels in AngII-Induced Cardiomyocytes

The AngII non-significantly increased the ANP levels of stimulated cells compared to the control cells. All the extracts of the four mushrooms lowered AngII-induced ANP concentration, however non-significantly, results not showed. Only the positive control, losartan reversed the AngII-induced ANP concentration significantly (p= 0.0093).

2.4. Effects of the Extracts on Mitochondrial Activity of AngII-Induced Cells

Angiotensin II stimulation reduced the mitochondrial activity indicated by lowering viability of cells significantly (p< 0.001) below 80% in comparison to the control cells, Figure 5. Positive controls, losartan and L-NAME increased the viability of AngII-induced cells in a concentration-dose manner and the effects were more pronounced with L-NAME treatment that restored viability of cells in line with control cells and above 100% cell viability with the 100 µM treatment.
The cold-water extract of Pan cyanescens increased viability of AngII-induced cells above LNAME the positive control and the control cells with the concentration investigated in the study(50 µg/mL and 25 µg/mL concentration), Figure 5. The hot-water and ethanol extracts of Pan cyanescens increased the viability same as the P. cubensis above 80% also in a dose dependant manner, Figure 5. P. A+ strain treatment increased the % viability of the cells above 80% with lowest concentration (25 µg/mL) investigated in the study while the higher 50 µg/mL concentration slightly increased the viability around and below 80% with the three extracts. The P. natalensis increased viability of cells above 80% in a concentration-dependent manner and to the same level as losartan with the 25 µg/mL concentration.

2.5. Effects of the Extracts on Intracellular ROS Levels in AngII-Induced Cardiomyocytes

Angiotensin II stimulation increased ROS levels significantly (p< 0.001) when compared with the non-induced but serum starved control cells, Figure 6. The cold-water, hot-water and ethanol extracts of all the extracts reduced the AngII stimulated ROS levels significantly similar to losartan (p< 0.001) when compared to AngII-induced cells.

2.6. Phytochemistry Analysis of the Water and Ethanol Extracts of Pan cyanescens, P. cubensis and A+ Strain Mushrooms

Figure 7 show the GCMS-MS chromatograms of the cold-water, hot-water and ethanol extracts of Pan cyanescens mushrooms respectively. The compounds with known anti0xidant and anti-inflammatory biological activities from the chromatograms of the three extracts are tabulated in Table 2 with their different peak numbers, compound name, molecular weight, formulas, similarity, area%, diemention time and height per extracts. Four compounds (n-hexadecanoic acid; 3-octanone, nonadane and tetradecane) with known natural antioxidant and anti-inflammatory activities similar to the ones extracted from P. natalensis identified in [21] were also present in the Pan cyanescens mushroom extracts. The compounds 9-Octadecenamide, (Z)- and n-hexadecanoic acid were found in all the extracts. Tetradecane was found in the hot-water extract and the ethanol extract. Nonadecane and decane were detected in the ethanol extract together with 9,12-Octadecadienoic acid (Z,Z)- and heineicosane. Dodecane, 1,1-dimethoxy- compound was detected in different peaks only with cold water together with hexanoic acid, methyl ester while dotriacontane compound and olean-12-ene-3,28-diol, (3á)- was detected only in the hot water extract of Pan cyanescens, Table 2.
Figure 8 show the GCMS-MS chromatograms of the cold-water, hot-water and ethanol extracts of P. cubensis mushrooms respectively. The compounds with known biological activities from the chromatograms of the three extracts are tabulated in Table 3 with their different peak numbers, compound name, molecular weight, formulas, similarity, area%, diemention time and height per extracts. Four compounds (n-hexadecanoic acid; 3-octanone, nonadane and tetradecane) with natural antioxidant and anti-inflammatory activities similar to the ones extracted from P. ntalensis identified in [21] were also present in these mushroom extracts, Table 3. The compound n-hexadecanoic acid was present in all the extracts while tetradecane compound was detected only in the ethanol extract together with hexadecane, 9,12-Octadecadienoic acid (Z,Z)-, nonadecane, heneicosane, oleic acid and decane. Compound 9-Octadecenamide, (Z)- was detected in different peaks in both ethanol and hot-water extracts. In the cold and hot-water extracts, dodecane, 1,1-dimethoxy- was detected in many different peaks and hexadecanoic acid, methyl ester; 3-octanone and dodecanoic acid, methyl ester compounds were also detected in the extract.
Figure 9 show the GCMS-MS chromatograms of the cold-water, hot-water and ethanol extracts of P. A+ strain mushrooms respectively while the compounds with know biological activities from the chromatograms of the three extracts are tabulated in Table 4 with their molecular weight, formulas and area% per extracts. The four compounds (n-hexadecanoic acid; 3-octanone, nonadane and tetradecane) with natural antioxidant and anti-inflammatory activities similar to the ones extracted identified in [21] were also present in the P. A+ strain mushroom extracts. The compound n-hexadecanoic acid was detected in all the extracts of P. A+ strain. Decane, tetradecane, 9,12-octadecadienoic acid (Z,Z)-, and eicosane compounds were detected in the ethanol extract. The compounds hexadecanoic acid, methyl ester, 3-Octanone and 9-octadecenamide, (Z)-, were detected in both cold and hot water extracts with dodecane, 1,1-dimethoxy detected in many different peaks in both water extracts of P. A+ strain mushroom while glycine was detected only in the hot-water extract.
In Table 5 below, there are other compounds that were extracted and not included in Nkadimeng et al 2020 [21] and are tabulated here. Compound heneicosane, 9,12-octadecadienoic acid (Z,Z)-, 5-eicosene, (E)- and decane were detected only in the ethanol extract. Dodecane, 1,1-dimethoxy- was detected in many different peaks in both hot and cold-water extracts while hexadecanoic acid, methyl ester and dodecanoic acid, methyl ester was only detected in the cold-water extracts of P. natalensis mushroom.

3. Discussion

Previous studies have demonstrated that AngII plays a key role in progression of heart failure by affecting cell growth, differentiation and apoptosis, induction of pro-inflammatory cytokines and many other factors in cardiomyocytes [14]. In the study, morphological F-actin size results and cell width measurements showed that the cells that were induced with AngII increased the cell surface area of the cells significantly compared to non-induced serum starved control cells in agreement with previous studies [19]. Angiotensin II stimulation decreased mitochondrial activity by lowering cell viability < 80% signifying cell death and also increased levels of ROS production significantly while ANP level was non-significantly increased in cardiomyocytes. The results showed that the positive control losartan, which is an AngII inhibitor via blockage of ATR1 receptors, significantly reduced the AngII-induced cell surface area measurements, ROS and ANP levels of the stimulated cells. Losartan also improved cell viability of AngII-induced cells in a dose-dependent manner similar to L-NAME. This study showed that L-NAME, which is a non-selective NOS inhibitor, prevented the AngII-induced cell death in greater percentage indicating involvement of NOS uncoupling in the AngII-induced injury and cell death in the study.
Our study demonstrated that the cold-water, hot-water and ethanol extracts of Pan cyanescens, P. cubensis, P. A+ strain and P. natalensis mushroom extracts alleviated the cell enlargement induced by AngII stimulation same as the positive control, losartan. Cell enlargement is one of the key indices of hypertrophy and by reducing it, the extracts demonstrated not just safety but potential protective effects as well in AngII-induced hypertrophy conditions. The concentrations of ANP, which is a maker in heart failure, was also found to be lower however non-significant in the three extract-treatments. Since the increase levels of ANP are known to be associated with the severity of heart failure condition, their decrease agreed with the significant decrease in cell size enlargement supporting alleviation of hypertrophy observed with the mushroom extracts’ treatments.
Other well-investigated pro-hypertrophic mechanisms of AngII are also known to be mediated via activation of NAD(P)H oxidase through ATR1 receptors and inducing mitochondrial and induced-cell death leading to increased ROS generation and more oxidative stress [14,15]. In our study, we measured intracellular ROS especially superoxide and hydroxyl radicals both of which are known to increase significantly with AngII stimulation [13]. Our study showed that the cold-water, hot-water and ethanol extracts of all the four magic mushrooms alleviated these AngII-induced intracellular ROS generation of treated cells significantly similar to Losartan. By reducing accumulation of AngII-induced ROS, the extracts of Pan cyanescens, P. cubensis, P A+ strain and P. natalensis mushrooms demonstrated safety and protective potentials of the extracts in AngII-induced oxidative stress conditions in vitro in cardiomyocytes in the concentration investigated. These results agreed with our previous finding in endothelin-induced ROS activity following treatment with Pan cyanescens, P. cubensis water extracts [20]. Furthermore, we have also demonstrated invitro anti-inflammatory potential of the four extracts on LPS-induced human macrophage cells [21].
In addition, the four mushroom extracts also protected against AngII-induced mitochondrial and cell death signified by increasing in % viability of cells above 80% in safe margins with the concentrations investigated 50 µg/mL for Pan cyanescens and P. cubensis, and 25 µg/mL for P. A+ strain and P. natalensis mushrooms in the study. However, the study also showed that P. A+ strain mushroom extracts may be toxic if higher than 50 µg/mL concentrations are used in an AngII pathological condition.
The cytotoxicity assay results on H9C2 cardiomyocytes also showed that the three extracts of the four magic mushrooms were not toxic when compared to the positive control doxorubicin. The extracts were safe in the order ethanol> hot-water> cold-water for the three magic mushrooms and the order hot-water> ethanol> cold-water for P. cubensis. Moreover, in accordance to the American National Cancer Institution guidelines, it is indicated that extracts that exhibit an LC50 ≤ 20 µg/mL over 48 hours treatment are considered toxic. As a result, the three extracts of the four mushrooms will be considered to be safe with LC50 presented and also with the 50 µg/mL and 25 µg/mL concentration which were investigated in the study. However, further investigations in vivo to confirm this safety is recommended.
The phytochemistry analysis of the water and ethanol extracts showed presence of known natural compounds with antioxidant and anti-inflammatory activities in support of these ant-oxidative stress effects observed in the study. The two compounds, 9-Octadecenamide, (Z)- which is a potent antioxidant with antimicrobial activities, and n-hexadecanoic acid which is the most common saturated fatty acid known to have anti-inflammatory and antioxidant activities were detected in all the water and ethanol extracts of the four magic mushrooms [22,23,24].
Decane, an alkaline hydrocarbon compound, was found to possess activities such as phosphatase and membrane permeability inhibitor which means it prevents damage and preserve integrity of the cellular membrane [25]. The compound was also reported as platelet aggregation inhibitor and platelet activating factor beta antagonist which are known to inhibit thrombus production by decreasing platelet agglutination with potential therapeutic agents in different diseases including cardiovascular [25]. This compound was detected only in all the ethanol extracts of the four psilocybin mushrooms.
Tetradecane is an alkaline hydrocarbon compound which was found to have anti-inflammatory effects and also a potential membrane integrity agonist, cardiovascular analeptic (which are central nervous system stimulants agents that increases alertness, heart rate, blood pressure, breathing and blood glucose level, mood and euphoria among others) and nicotinic alpha6beta3beta4alpha5 receptor antagonist [25,26]. This compound was detected in all the ethanol extracts of the four psilocybin mushrooms extracts.
The three compounds, nonadecane, an alkaline hydrocarbon lipid molecule and vey hydrophobic with antioxidant, antibacterial and antimalarial activities [26,27,28]; 9,12-Octadecadienoic acid (Z,Z)- compound which has been reported to have antioxidant activity, and heneicosane, an aliphatic hydrocarbon compound that is reported to complements C5a chemotactic receptor antagonist which play important roles in inflammation and cell killing process [29], and also to possess anti-eczema atopic activities, phobic disorders treatment and betaadrenergic receptor kinase inhibitor which are known to ameliorate cardiac dysfunction and improve survival especially in heart failure [25] were all detected only in the ethanol extracts of the three psilocybin mushrooms, P. natalensis, Pan cyanescens and P. cubensis and it was not detected in any of the P. A+ strain mushroom extracts.
Hexadecanoic acid, methyl ester which is known to have antioxidant, anti-inflammatory (by inhibiting cyclooxygenase-2 enzymes) activities and also a blood cholesterol decreasing effect [30] was detected in the water extracts of P. A+ strain and P. cubensis, and also in the cold-water extracts of Pan cyanescens and P. natalensis mushrooms. Compound 3-Octanone reported to have antioxidant and anti-inflammation activities was detected in the water extracts of P. cubensis, P. A+ strain and P. natalensis mushrooms and in the cold water of Pan cyanescens mushroom [18,31]. Dotriacontane reported to have antioxidant activities were detected in the ethanol extracts of P. cubensis and P. natalensis mushrooms and also in the hot-water extracts of Pan cyanescens mushroom extracts [32].
Hexadecane has been found to have antibacterial, cognition disorder treatment, antianginal, nicotinic alpha6beta3beta4alpha5 and nicotinic alpha2beta2 receptor antagonist, kidney function stimulant and 5-hydroxytryptamine uptake stimulant. This compound was detected in ethanol extracts of P. cubensis [25,33]. Oleic acid is a mono-unsaturated omega9-fatty acid known to enhance antioxidant activity,inhibit adrenoleukodystrophy, boost memory, a key factor accounting for the hypotensive effects of olive oil [34] and generally known to improve and protect against cardiovascular disease [35], 2021) was detected in ethanol extracts of P. cubensis. Glycine, which is known to improves the body’s ability to use nitric oxide and relief blood pressure [36] was detected only in the hot-water extract of P. A+ strain mushroom. Olean-12-ene-3,28-diol, (3á)-, which is known to have anti-inflammatory and protecting activities against induced experimental autoimmune or allergic encephalomyelitis [37] was detected in the hot water extracts of Pan cyanescens mushroom extract.
In summary, the study showed that AngII induced cell enlargement and ANP levels signifying hypertrophy in the stimulated cells. Angiotensin II stimulation also induced cell injury and death by decreasing cell viability and increasing ROS generation and NOS activity in the induced cells. Losartan, the positive control reversed these AngII-induced hypertrophy effects and also cell injury effects similar to L-NAME in agreement with previous studies. The cold-water, hot-water and ethanol extracts of Pan cyanescens, P. cubensis, P. A+ strain and P. natalensis mushrooms reversed the cell size enlargement significantly and non-significantly lowered ANP levels (indices of AngII-induced hypertrophy effects) and protected the cardiomyocytes significantly against the AngII-induced oxidative stress and cell death in a manner similar to losartan at the 50 µg/mL (used for Pan cyanescens and P. cubensis) and 25 µg/mL (used for the P. A+ strain and P. natalensis) in the study. These findings suggested potential presence of compounds with antioxidant abilities known to neutralize the free radicals and alleviate intracellular ROS accumulation in the four mushrooms. The phytochemical analysis of the extracts confirmed these effects by showing detection of known compounds with antioxidant and anti-inflammatory effects in the water and ethanol extracts of these four psilocybin mushrooms.

4. Materials and Methods

4.1. Ethical Clearances

The protocol for this study was approved by the University of Pretoria research committee with the number REC045-18. The project was also approved by the Medical Control Council (MCC) of the South African Health Department with a permit license POS 223/2019/2020 since psilocybin mushrooms are schedule 7 substances in South Africa.

4.2. Growing Mushrooms and Making Extracts

The spores print syringe of Panaeolus (Copelandia) cyanescens (Pan cyanescens), Psilocybe nataleases (P. natalensis), Psilocybe cubensis (P. cubensis), and Psilocybe cubensis leucistic A+ strain (P. A+ strain) mushrooms were verified by the Sporespot Company and sterile substrate and they were grown and extracted with 70% ethanol, cold and hot-boiling water as described in [18].

4.3. Culturing of Cells

The rat H9C2 cardiomyoblast cells were obtained from American Type Culture Collection (ATCC ® CRL-1446™ ) and maintained using Dolbecco Modified Eargle media (DMEM) (Pan, Separations Scientific) supplemented with 10% fetal bovine serum (FBS) (Gibco, Sigma Aldrich) and 1% of 100 IUnits/mL penicillin and 100 µg/L streptomycin (Pan, Celtics diagnostic) in 75 cm2 tissue culture treated flasks (NEST, Whitehead Scientific). The cells were grown in an incubator (HERAcell 150, Thermo Electron Corporations, USA) at 370C in 5% CO2 balanced air.

4.3.1. Cytotoxicity Determination of the Mushroom

The cytotoxicity of the extracts was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay described by Mosmann (1983) with modifications by Nkadimeng et al. (2020a). When grown to confluence the H9C2 cardiomyocytes were washed with pre-warmed phosphate buffer (PBS) (Sigma-aldrich), passaged with trypsin-EDTA (Biochom biotch) and neutralised with DMEM. The cells were centrifuged for 7 minutes and pellet resuspended with 1 mL of fresh medium. Thereafter cells were counted and seeded 1 × 104 cells (all the wells in column 2 to 12) in a 96 well tissue culture treated plates (NEST, Whitehead Scientific). After 24 hours cells had adhered fully, and medium was removed and replaced with fresh media 100 µl per well. Then the cells in column 2 to 10 were treated with the mushroom extracts (0.0075, 0.01, 0.025, 0.05, 0.075 and 0.1 µg/mL concentrations) and doxorubicin chloride (Pfizer Laboratories), a well-known toxic drug was used as a positive control (2, 4, 10 and 20 µM). Plates were incubated for 48 hours at 370C in 5% CO2 incubator. The wells of the 1st column without cells were used as blank while the wells of the last two columns 11 and 12 which were not treated were used as negative controls.
After 48 hours, medium was removed, and the cells were washed with 200 µl of pre-warmed PBS. Then 100 µl of fresh medium was added and 30 µl of MTT (Inqaba biotec, stock solution of 5 mg/ml in PBS) was added and the plates were incubated for 4 hours in dark at 370C in 5% CO2 incubator. After 4 hours, the media with MTT was removed and the formazon salts were dissolved with 50 µl of dimethyl sulfoxide (DMSO) (Sigma-aldrich) in dark. The plates were shaken for 1 minute and plates read using a microplate reader (Biotek, Synergy HT) at a wavelength of 570 nm and a reference wavelength of 630 nm. Untreated cells (negative control) were included. The treatment was performed in triplicates and the experiments were repeated three times. Viability of cells in percentages was calculated using the formula: % Viability= ((Sample Absorbance/control Absorbance) x 100). The results were expressed as lethal concentration (LC) LC50 value which is the concentration of the sample necessary to kill viability of cells by 50%.

4.3.2. Cell Culture for Treatment

The cells were cultured according to the method of [19] with modification. Briefly as soon as cells reached 70% confluence they were passaged, counted and 1 × 106 cells seeded and grown on glass cover slips in 6 well plates (NEST, Whitehead scientific). After 24 hours medium was removed, the cells in the 6 well plates were washed with 1 mL serum free DMEM and deprived of serum for 18 hours by adding 2 mL of serum-free DMEM. After 18 hours sera-free media was removed, and the cells were treated AngII (10 µM) (Sigma-aldrich) and incubated for 45 min before treated with the three extracts (50 μg/mL) of Pan cyanescens and P. cubensis and the three extracts (25 µg/mL) of P. A+ strain and P. natalensis, and positive control 100 µM Nω-nitro-L-arginine methyl ester (L-NAME, Sigma-aldrich) over 48 hours in 1% FBS media. Losartan100 µM (Sigma-aldrich), another positive control which is the selective AT1R inhibitor was induced 45 minutes prior to stimulation with AngII and treated over 48 hours in media supplemented with 1% FBS and 1% penicillin-streptomycin. This medium was used to prepare and dilute all the treatment and drugs. Stock concentrations of chemicals were prepared in sterile pure water. AngII cells were induced with AngII but not treated, while the control cells were serum starved but neither induced with AngII nor treated.

Mitochondrial Activity

To test for mitochondrial activity, 1 × 104 cells were seeded in 96 well plates (NEST, Whitehead scientific), deprived of serum the same way as above by removing old medium after 24 hours and adding 100 µl of serum-free DMEM over 18 hours prior to inducing the cells with 10 µM AngII for 45 minutes. Then cells were treated with the three extracts and positive controls over 48 hours in the presence of 1% FBS DMEM as above. AngII cells were cells that were induced with AngII but not treated and control cells were only serum starved but not stimulated with AngII nor treated. Mitochondrial activity was measured using the Resazurin assay kit AR002 (R & D, Whitehead scientific) according to the manufacture manual. The viability of cells in percentages was calculated using the formula: % Viability= ((Sample Absorbance/control Absorbance) x 100). The experiments were performed in triplicate and repeated in three different times.

Actin Filament Labelling and Surface Area Measurements

After 48 hours of treatment, the cells on coverslips in the 6 well plates were prepared for rhodamine phalloidin reagent fluorescence staining using ab235138 Rhodamine Phalloidin Reagent (Biocom Africa) for labeling, identifying and quantifying actin filaments (F-actin) in the cardiomyocyte cells according to the manual. Briefly the cells on coverslip were washed with PBS. Then cells were fixed with 3.5 % formaldehyde fixation in PBS at room temperature for 20 minutes. Formaldehyde solution was aspirated carefully, and cells washed with PBS. Then 0.1%Triton X-100 in PBS was added to the cells to increase permeability for 5 minutes and then washed with PBS. Then 1X phalloidin conjugate working solution was added into each well of fixed cells and incubated for 90 minutes. After 90 minutes, excess phalloidin conjugate was removed and mounting media added to preserve fluorescence and sealed. The cells were then observed using a fluorescents microscope (Olympus BX63) fitted with filter at Ex/Em=546/575 nm. Morphological images were taken using 50 µm lense and the cells were analysed for cell size width measurements using CellSens Dimension 1,12 software. The surface area of cells from each group (60-80 cells/group) were determined and compared with the AngII-induced cells. Negative control was neither treated nor induced with AngII. The results showed represented analysis from three independent experiments.

ANP Concentration Measurements

After 48 hours the effects of the extracts on levels of ANP were determined and quantified using the rat Atrial natriuretic peptide (ANP) ELISA kit (E-EL-R0017, Elabscience, Biocom Africa) following the same protocol as above using the instructor manual on the cell culture medium. The absorbance of samples and controls were inversely proportional to the concentrations of ANP in the media.

Intracellular ROS Measurements

To measure the reactive oxygen species (ROS) generated by the cells induced with AngII and treated with the extracts, the cells were seeded in 96 wells plates and deprived with serum for 18 hours. Thereafter the cells were induced with AngII over 2 hours before treated with 50 µg/mL for Pan cyanecsens and P. cubensis and 25 µg/mL for P. natalensis and P. A+ strain mushroom extracts and 100 µM losartan for 1 hour. Fluorometric Intracellular ROS assay kit (Green Fluorescence) MAK 143 (Sigma-Adrich) was used according to manual instructions to detect intracellular ROS (especially superoxide and hydroxyl radicals) in live cells with a green fluorescence intensity at λex= 485/20,520/25 nm on AngII-induced cardiomyocytes. The experiment was done in duplicate and repeated three times.

4.3.3. Phytochemical Determination of the Ethanol and Water Extracts

Phytochemical determination of extracts was performed using the gas chromatography-mass spectrometry (GCMS-MS) by the LC-MS (Synapt) facility at the Chemistry Department, University of Pretoria. The water and ethanol extracts of Pan cyanescens, P. cubensis and A+ strain mushrooms were dissolved in methanol (1mg/mL). Chromatograms and presence of compounds in the three extracts were produced. Phytochemistry analysis of P. natalensis water and ethanol mushroom extracts were done and the chromatograms and results are published in Nkadimeng et al 2020 [18].

4.3.4. Statistical Analysis

Results are expressed as mean ± standard deviations and statistically significant values were compared using one-way ANOVA analysis of variance using an interactive statistical program (Sigmastat, SPSS version 26, USA) and pairwise multiple comparison procedures using Holm-Sidak method according to [21]. Normality test was done using Shapiro-Wilk and equal variance test using Brown-Forsythe. The p-value of ≤ 0.050 was considered statistically significant. The cold-water, hot-water and ethanol extracts are symbolised in the chapter for Pan cyanescens as PC, PH and PE respectively; for P. cubensis as GC, GH and GE; for P. A+ strain as AC, AH and AE while P. natalensis is symbolised as NC, NH and NE respectively and losartan is symbolised with LOS.

5. Conclusions

In conclusion, 70% ethanol, hot-water and cold-water extracts of Panaeolus cyanescens, Psilocybe nataleases, Psilocybe cubensis, and Psilocybe cubensis leucistic A+ strain mushroom did not exacerbate the AngII-induced hypertrophy and the study revealed for the first time the potential cardio-protective effects of the four mushroom extracts against AngII-induces oxidative stress and hypertrophy in the concentration investigated in the study. Further investigations to support these findings in vivo and also to examine the underlying mechanisms in vivo and in vitro are recommended.

Author Contributions

Conceptualization, S.M.N.; Methodology, S.M.N.; writing—original draft preparation, S.M.N.; writing—review and editing S.M.N., C.M.L.S., J.N.E. All authors have read and agreed to the published version of the manuscript.

Funding

The study project (REC045-18) was funded by the Health and Welfare Sector Education and Training Authority (HWSETA) and MJ Medtech grants to S.M. Nkadimeng. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Data Availability Statement

Data for this research will be obtained from the corresponding author upon request.

Acknowledgments

We very much appreciate the support of Mr L Morland who assisted with the growing of mushrooms, and Ms LE Moagi for assistance with statistics. We higly appreciate and acknowledge the LC-MS Synapt Facility (Department of Chemistry, University of Pretoria) for chromatography and mass spectrometry services provided by Dr M Wooding and Dr Y Naude.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Morphological effects of Pan cyanescens (PC, PH and PE) and P. cubensis (GC, GH and GE) mushroom extracts (50 µg/mL) and positive control losartan (LOS) (100 µM), non-induced negative control (CTR) on the actin filaments of AngII-induced (ANG) H9C2 cardiomyocytes (50 µm) over 48 hours using a fluorescence filter at Ex/Em= 546/575 nm.
Figure 1. Morphological effects of Pan cyanescens (PC, PH and PE) and P. cubensis (GC, GH and GE) mushroom extracts (50 µg/mL) and positive control losartan (LOS) (100 µM), non-induced negative control (CTR) on the actin filaments of AngII-induced (ANG) H9C2 cardiomyocytes (50 µm) over 48 hours using a fluorescence filter at Ex/Em= 546/575 nm.
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Figure 2. The effects of Pan cyanescens (cold-water PC, hot-water PH and ethanol PE) and P. cubensis (cold-water GC, hot-water GH and ethanol GE) mushroom extracts (50 µg/mL) and positive control Losartan (100 µM) on the cell width measurements on AngII-induced hypertrophy on H9C2 cardiomyocytes over 48 hours. Control: non-induced negative control. (*: significant).
Figure 2. The effects of Pan cyanescens (cold-water PC, hot-water PH and ethanol PE) and P. cubensis (cold-water GC, hot-water GH and ethanol GE) mushroom extracts (50 µg/mL) and positive control Losartan (100 µM) on the cell width measurements on AngII-induced hypertrophy on H9C2 cardiomyocytes over 48 hours. Control: non-induced negative control. (*: significant).
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Figure 3. Morphological effects of P. A+ strain (AC, AH and AE) and P. natalensis (NC, NH and NE) mushroom extracts (25 µg/mL) and positive control losartan (LOS) (100 µM) on the actin filaments of AngII-induced H9C2 cardiomyocytes (50 µm) over 48 hours using a fluorescence filter at Ex/Em= 546/575 nm. CTR: non-induced negative control cells.
Figure 3. Morphological effects of P. A+ strain (AC, AH and AE) and P. natalensis (NC, NH and NE) mushroom extracts (25 µg/mL) and positive control losartan (LOS) (100 µM) on the actin filaments of AngII-induced H9C2 cardiomyocytes (50 µm) over 48 hours using a fluorescence filter at Ex/Em= 546/575 nm. CTR: non-induced negative control cells.
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Figure 4. The effects of P. A+ strain (cold-water AC, hot-water AH and ethanol AE) and P. natalensis (cold-water NC, hot-water NH and ethanol NE) mushroom extracts (25 µg/mL) and positive control Losartan (100 µM) on the cell width measurements on AngII-induced hypertrophy on H9C2 cardiomyocytes over 48 hours. Control: non-induced negative control. (*: significant).
Figure 4. The effects of P. A+ strain (cold-water AC, hot-water AH and ethanol AE) and P. natalensis (cold-water NC, hot-water NH and ethanol NE) mushroom extracts (25 µg/mL) and positive control Losartan (100 µM) on the cell width measurements on AngII-induced hypertrophy on H9C2 cardiomyocytes over 48 hours. Control: non-induced negative control. (*: significant).
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Figure 5. Effects of the extracts (25 and 50 µg/mL) of Pan cyanescens (cold-water PC, hot-water PH and ethanol PE), P. cubensis (cold-water GC, hot-water GH and ethanol GE), P. A+ strain (cold-water AC, hot-water AH and ethanol AE) and P. natalensis (cold-water NC, hot-water NH and ethanol NE) mushroom and positive controls; losartan and LNAME (50, 100 µM) on the mitochondrial activity of AngII-induced hypertrophy over 48 hours. Control: non-induced negative control. (*: significant).
Figure 5. Effects of the extracts (25 and 50 µg/mL) of Pan cyanescens (cold-water PC, hot-water PH and ethanol PE), P. cubensis (cold-water GC, hot-water GH and ethanol GE), P. A+ strain (cold-water AC, hot-water AH and ethanol AE) and P. natalensis (cold-water NC, hot-water NH and ethanol NE) mushroom and positive controls; losartan and LNAME (50, 100 µM) on the mitochondrial activity of AngII-induced hypertrophy over 48 hours. Control: non-induced negative control. (*: significant).
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Figure 6. Effects of 1-hour treatment with 50 µg/mL extracts of Pan cyanescens (cold-water PC, hot-water PH and ethanol PE), P. cubensis (cold-water GC, hot-water GH and ethanol GE), and 25 µg/mL of P. A+ strain (cold-water AC, hot-water AH and ethanol AE) and P. natalensis (cold-water NC, hot-water NH and ethanol NE) and the positive control Losartan (100 µM) treatments on fluorometric intracellular ROS (superoxide and hydroxyl radicals) production measured using a green fluorescence intensity at λex= 485/20,520/25 nm on AngII-induced cardiomyocytes. Control: non-induced negative control. (*: significant).
Figure 6. Effects of 1-hour treatment with 50 µg/mL extracts of Pan cyanescens (cold-water PC, hot-water PH and ethanol PE), P. cubensis (cold-water GC, hot-water GH and ethanol GE), and 25 µg/mL of P. A+ strain (cold-water AC, hot-water AH and ethanol AE) and P. natalensis (cold-water NC, hot-water NH and ethanol NE) and the positive control Losartan (100 µM) treatments on fluorometric intracellular ROS (superoxide and hydroxyl radicals) production measured using a green fluorescence intensity at λex= 485/20,520/25 nm on AngII-induced cardiomyocytes. Control: non-induced negative control. (*: significant).
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Figure 7. The GCMS-MS chromatogram of cold-water (PC), hot-water (PH) and 70% ethanol (PE) extracts of Pan cyanescens mushroom.
Figure 7. The GCMS-MS chromatogram of cold-water (PC), hot-water (PH) and 70% ethanol (PE) extracts of Pan cyanescens mushroom.
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Figure 8. The GCMS-MS chromatogram of cold-water (GC), hot-water (GH) and 70% ethanol (GE) extracts of P. cubensis mushroom.
Figure 8. The GCMS-MS chromatogram of cold-water (GC), hot-water (GH) and 70% ethanol (GE) extracts of P. cubensis mushroom.
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Figure 9. The GCMS-MS chromatogram of cold-water (AC), hot-water (AH) and 70% ethanol (AE) extracts of P. A+ strain mushroom.
Figure 9. The GCMS-MS chromatogram of cold-water (AC), hot-water (AH) and 70% ethanol (AE) extracts of P. A+ strain mushroom.
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Table 2. The compounds with anti-inflammatory and antioxidant effect identified in Pan cyanescens cold-water (PC), hot-water (PH) and 70% ethanol (PE) mushroom extracts.
Table 2. The compounds with anti-inflammatory and antioxidant effect identified in Pan cyanescens cold-water (PC), hot-water (PH) and 70% ethanol (PE) mushroom extracts.
PC Peak # Name Weight Formula CAS Similarity Area % 1st Dimension Time (s) Height
4 Hexanoic acid, methyl ester 130 C7H14O2 106-70-7 927 2,0459 385,6 614562
6 3-Octanone 128 C8H16O 106-68-3 794 2,9534 453,6 647828
7 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 705 2,8617 455 639963
22 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 778 0,66502 785,8 265590
26 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 816 0,79635 886 265712
30 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 820 0,36008 980,6 132773
35 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 843 0,4545 1070,3 185705
38 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 815 0,46437 1155,4 155284
42 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 794 0,93345 1236,3 196900
43 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 800 0,56178 1241,5 176011
47 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 781 1,0495 1313,4 297088
51 n-Hexadecanoic acid 256 C16H32O2 57-10-3 899 2,891 1369 754734
52 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 840 0,65301 1386,9 155364
53 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 774 0,66727 1392,4 200963
58 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 798 0,22375 1524,4 90547
62 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 908 1,1739 1621,3 345374
63 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 816 0,81034 1624,9 215839
64 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 733 0,51097 1628,1 131186
66 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 770 0,35671 1716,6 102006
68 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 791 0,20132 1774,1 81447
PH Peak # Name Weight Formula CAS Similarity Area % 1st Dimension Time (s) Height
13 Tetradecane 198 C14H30 629-59-4 884 4,0398 920,1 731151
19 n-Hexadecanoic acid 256 C16H32O2 57-10-3 868 1,9932 1369,2 270343
22 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 921 2,6151 1621,3 395816
23 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 829 0,98533 1625 141739
24 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 877 1,1489 1628,4 130670
26 Dotriacontane 450 C32H66 544-85-4 885 5,8278 1673,2 221028
32 Olean-12-ene-3,28-diol, (3á)- 442 C30H50O2 545-48-2 613 19,938 1900,8 766581
PE Peak # Name Weight Formula CAS Similarity Area % 1st Dimension Time (s) Height
5 Decane 142 C10H22 124-18-5 846 8,8254 605,8 1051849
10 Tetradecane 198 C14H30 629-59-4 880 18,263 919,9 2801754
14 Nonadecane 268 C19H40 629-92-5 893 10,729 1101,5 1772907
15 Nonadecane 268 C19H40 629-92-5 904 5,3724 1265 980759
17 n-Hexadecanoic acid 256 C16H32O2 57-10-3 880 2,671 1369,1 353542
19 9,12-Octadecadienoic acid (Z,Z)- 280 C18H32O2 60-33-3 910 3,4485 1485,3 489829
22 Heneicosane 296 C21H44 629-94-7 899 1,3394 1548,6 251573
23 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 924 2,509 1621,2 466852
Table 3. The compounds identified in P. cubensis cold-water (GC), hot-water (GH) and 70% ethanol (GE) mushroom extracts.
Table 3. The compounds identified in P. cubensis cold-water (GC), hot-water (GH) and 70% ethanol (GE) mushroom extracts.
GC Peak # Name Weight Formula CAS Similarity Area % 1st Dimension Time (s) Height
3 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 739 6,3304 343,2 2096696
6 Hexanoic acid, methyl ester 130 C7H14O2 106-70-7 928 2,8062 385,1 955717
9 3-Octanone 128 C8H16O 106-68-3 830 2,7283 452,8 762082
27 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 779 0,71327 785,7 314589
31 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 813 0,69149 885,8 276576
35 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 855 0,35927 980,5 151860
38 Dodecanoic acid, methyl ester 214 C13H26O2 111-82-0 900 0,70445 1019,3 279675
41 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 807 0,44597 1070,2 197083
48 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 809 0,78095 1236,2 194599
53 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 736 1,2754 1313,2 346174
55 Hexadecanoic acid, methyl ester 270 C17H34O2 112-39-0 910 3,7206 1346,9 1438966
57 n-Hexadecanoic acid 256 C16H32O2 57-10-3 876 1,7469 1368,7 479084
58 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 837 0,81401 1386,7 214202
59 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 761 0,89417 1392,1 245098
65 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 851 0,21253 1524,3 93108
71 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 729 0,20497 1716,4 85138
GH Peak # Name Weight Formula CAS Similarity Area % 1st Dimension Time (s) Height
3 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 718 5,7808 343,8 2236529
11 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 739 3,5171 454,8 833100
20 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 745 0,93596 569 403943
28 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 759 0,65184 785,8 327037
32 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 827 0,73365 885,8 331466
37 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 833 0,44286 980,5 180459
40 Dodecanoic acid, methyl ester 214 C13H26O2 111-82-0 862 0,76451 1019,3 332902
46 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 813 0,47338 1155,3 187669
48 Hexadecanoic acid, methyl ester 270 C17H34O2 112-39-0 829 1,4125 1191,3 568445
50 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 802 0,88819 1236,2 252623
55 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 784 1,2221 1313,2 405746
56 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 789 0,80275 1318,5 238850
59 n-Hexadecanoic acid 256 C16H32O2 57-10-3 891 3,1125 1368,8 1020178
60 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 827 0,72977 1386,7 208562
61 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 772 0,67475 1392,2 260361
69 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 845 0,23403 1524,2 119865
74 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 901 1,5525 1620,9 476077
75 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 828 0,94615 1624,4 327188
76 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 768 0,71306 1627,7 212987
78 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 839 0,41231 1716,3 134307
80 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 772 0,22056 1773,7 103514
81 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 735 0,27314 1829,2 96189
GE Peak # Name Weight Formula CAS Similarity Area % 1st Dimension Time (s) Height
1 Decane 142 C10H22 124-18-5 897 0,44944 490,3 252016
3 Decane 142 C10H22 124-18-5 842 0,98073 605,7 379188
8 Tetradecane 198 C14H30 629-59-4 864 4,5504 716,3 2342975
14 Tetradecane 198 C14H30 629-59-4 873 12,505 919,9 7151813
20 Hexadecane 226 C16H34 544-76-3 914 11,926 1101,7 7087174
24 Nonadecane 268 C19H40 629-92-5 919 6,5048 1265 3973633
26 n-Hexadecanoic acid 256 C16H32O2 57-10-3 916 3,2652 1368,9 1698554
29 Heneicosane 296 C21H44 629-94-7 901 2,7681 1413,3 1765288
30 9,12-Octadecadienoic acid (Z,Z)- 280 C18H32O2 60-33-3 920 7,7412 1485,4 3942406
31 Oleic Acid 282 C18H34O2 112-80-1 901 1,7768 1490,7 498823
32 Dodecanamide 199 C12H25NO 1120-16-7 822 0,1794 1506,4 175567
36 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 920 2,013 1620,9 1001169
37 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 875 1,1399 1627,8 415638
42 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 827 0,33757 1745 161336
Table 4. The compounds identified in P. A+ strain cold-water (AC), hot-water (AH) and 70% ethanol (AE) mushroom extracts.
Table 4. The compounds identified in P. A+ strain cold-water (AC), hot-water (AH) and 70% ethanol (AE) mushroom extracts.
AC Peak # Name Weight Formula CAS Similarity Area % 1st Dimension Time (s) Height
3 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 732 7,2746 343,1 1201850
5 Hexanoic acid, methyl ester 130 C7H14O2 106-70-7 928 3,7608 385 524718
7 3-Octanone 128 C8H16O 106-68-3 852 3,7989 452,8 422676
8 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 725 3,7989 454,5 422676
24 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 742 0,77714 785,7 152661
27 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 805 1,1092 885,8 160107
30 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 841 0,47523 980,5 80179
35 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 850 0,5429 1070,1 98825
38 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 810 1,0146 1155,1 128129
39 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 748 0,71579 1160,2 83648
41 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 829 1,066 1236,1 112539
45 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 754 1,2357 1313,2 144052
46 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 732 0,83455 1318,6 97752
47 Hexadecanoic acid, methyl ester 270 C17H34O2 112-39-0 910 4,4828 1347 720709
49 n-Hexadecanoic acid 256 C16H32O2 57-10-3 884 2,0361 1369,1 181013
50 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 802 0,68996 1386,7 85828
60 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 895 1,3478 1621 146411
61 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 764 0,62809 1624,5 93200
63 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 859 0,36481 1716,4 56531
AH Peak # Name Weight Formula CAS Similarity Area % 1st Dimension Time (s) Height
3 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 729 7,5091 343,1 1023514
6 Hexanoic acid, methyl ester 130 C7H14O2 106-70-7 942 3,4369 384,8 481718
8 3-Octanone 128 C8H16O 106-68-3 822 0,94495 452,6
9 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 711 4,0912 454,3 366395
26 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 783 0,82861 785,6 133376
30 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 817 1,059 885,7 146601
33 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 854 0,51922 980,3 80840
38 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 827 0,58444 1070,1 106177
41 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 817 0,60789 1155,2 76344
43 Hexadecanoic acid, methyl ester 270 C17H34O2 112-39-0 851 1,3131 1191,4 202951
44 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 828 1,0656 1236,1 99142
45 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 779 0,30158 1241,2 66062
48 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 773 1,0275 1313,2 116536
50 Hexadecanoic acid, methyl ester 270 C17H34O2 112-39-0 910 3,9708 1347,1 619698
52 n-Hexadecanoic acid 256 C16H32O2 57-10-3 894 2,3541 1368,8 236893
53 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 831 0,54835 1386,7 69077
54 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 740 0,54021 1392,2 75964
59 Glycine 75 C2H5NO2 56-40-6 754 1,0692 1530,1 87122
63 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 864 0,62408 1621,2 97425
65 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 808 0,58739 1716,4 61884
AE Peak # Name Weight Formula CAS Similarity Area % 1st Dimension Time (s) Height
10 Decane 142 C10H22 124-18-5 933 20,924 497,5 783673
31 Tetradecane 198 C14H30 629-59-4 888 3,381 925,5 289073
39 n-Hexadecanoic acid 256 C16H32O2 57-10-3 911 3,0487 1375,1 418335
37 Hexadecanoic acid, methyl ester 270 C17H34O2 112-39-0 851 0,54568 1353 74443
42 9,12-Octadecadienoic acid (Z,Z)- 280 C18H32O2 60-33-3 914 2,1293 1491,4 204219
35 Eicosane 282 C20H42 112-95-8 896 1,4654 1107,3 168172
Table 5. Other compounds identified in P. natalensis cold-water, hot-water and 70% ethanol mushroom extracts. (Chromatograms published in [18]).
Table 5. Other compounds identified in P. natalensis cold-water, hot-water and 70% ethanol mushroom extracts. (Chromatograms published in [18]).
NH Peak # Name Weight Formula CAS Similarity Area % 1st Dimension Time (s) Height
7 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 715 3,2977 454,6 567775
23 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 817 1,1041 885,9 210887
26 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 830 0,42934 980,6 105250
28 Dodecanoic acid, methyl ester 214 C13H26O2 111-82-0 907 1,1472 1019,6 224678
30 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 843 0,63486 1070,3 134265
33 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 810 0,86841 1155,4 139945
36 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 806 0,92241 1236,2 152342
37 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 821 0,4793 1241,4 120478
40 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 750 1,677 1313,3 251622
45 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 809 1,1774 1386,7 152379
46 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 777 0,97402 1392,3 175193
50 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 795 0,26298 1524,4 67498
55 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 860 1,1976 1621,4 205738
57 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 771 0,27229 1716,4 72493
59 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 731 0,47328 1773,9 89699
NC Peak # Name Weight Formula CAS Similarity Area % 1st Dimension Time (s) Height
13 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 733 3,6742 454,9 751534
31 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 779 0,68667 785,7 315435
33 Decanoic acid, methyl ester 186 C11H22O2 110-42-9 890 0,63556 827,8 263610
35 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 831 0,74448 885,8 274267
40 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 837 0,43806 980,5 161533
43 Dodecanoic acid, methyl ester 214 C13H26O2 111-82-0 913 0,67789 1019,4 277970
46 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 797 0,44929 1070,1 184949
49 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 789 0,58295 1155,3 183945
53 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 783 0,87504 1236,2 203315
58 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 751 0,94274 1313,2 320486
59 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 809 0,79948 1318,7 210365
63 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 820 0,75158 1386,7 170332
64 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 759 0,71847 1392,2 224832
65 Hexadecanoic acid, methyl ester 270 C17H34O2 112-39-0 844 0,87915 1419,5 273632
70 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 836 0,24407 1524,3 103354
73 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 758 0,50675 1594,8 141017
75 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 889 0,73559 1621,2 243511
76 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 713 0,68859 1624,7 202379
78 Dodecane, 1,1-dimethoxy- 230 C14H30O2 14620-52-1 776 0,52186 1656,7 149748
NE Peak # Name Weight Formula CAS Similarity Area % 1st Dimension Time (s) Height
2 Decane 142 C10H22 124-18-5 874 1,1285 490 365956
17 5-Eicosene, (E)- 280 C20H40 74685-30-6 904 1,1568 1257,2 551903
23 5-Eicosene, (E)- 280 C20H40 74685-30-6 904 0,76517 1406,5 329793
24 Heneicosane 296 C21H44 629-94-7 901 3,1921 1413,2 1359751
25 9,12-Octadecadienoic acid (Z,Z)- 280 C18H32O2 60-33-3 884 1,6761 1485,1 574310
27 Oleic Acid 282 C18H34O2 112-80-1 828 1,6454 1508,6 534460
28 Dotriacontane 450 C32H66 544-85-4 921 1,3086 1548,6 624893
29 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 927 2,1573 1621 787743
30 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 857 0,99448 1624,7 316129
31 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 847 1,0811 1627,8 304295
35 9-Octadecenamide, (Z)- 281 C18H35NO 301-02-0 827 0,26441 1745 100584
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