Results

Tannins and Terpenoids content of TT-TeMac™

The level of tannins and terpenoids in the extract were 168.33 ± 8.1 mgEqAT/g TT-TeMac™ and 345.66 ± 3.16 mgEqOA/g TT-TeMac™, respectively.

Characterization and identification of terpenoids and tannins of the TT-TeMac™ by LCMS

The results of the LC-MS analysis are presented in Fig. 2 (chromatogram) and Table 1 (mass spectra).

Fig. 1: Experimentation design.

Fig. 2: Chromatographic profile of TT-TeMac™.

Chromatogram of TT-TeMac™

LC analysis of the combined fraction of terpenoids and tannin showed the presence of peaks with a retention time of 0–20 min (Fig. 2).

Mass Spectra of compounds identified of TT-TeMac™

The MS allowed to identify seven compounds: Terminolic Acid, Sericic acid, arjunolic acid, Gallic acid, ellagic acid, 3-O methyl ellagic acid and 3,3’-di-O-methyl ellagic acid) (Table 1).

Target Identification

A total of 293 TT-TeMac™ target genes and 123 genes associated with cholinergic dysfunction were identified, and a total of 15 genes were identified as intersection genes (Fig. 3).

Fig. 3: Venn diagram showing potential targets between TT-TeMac™ and cholinergic dysfunction targets.

Fig. 4: Compound-target network of TT-TeMac™ against cholinergic dysfunction generated by Cytoscape software. The nodes represent the interacting target genes of cholinergic dysfunction and the triangle represents the node of TT-TeMac™. SERPINE1: Serpin Family E Member 1; NOS2: Nitric-oxide synthase inducible; ALB: Albumin; ACHE: Acetylcholinesterase; SLC6A3: Dopamine transporter; BCHE: ButyrylCholinesterase; CHRM2: Muscarinic acetylcholine receptor M2; IL6: Interleukin−6; TNF: Tumor necrosis factor; AKT1: RAC-alpha serine/threonine-protein kinase; SNCA: Alpha-synuclein; COMT: Catechol O-methyltransferase; ACE: Angiotensin-converting enzyme; AR: Androgen receptor; STAT3: Signal transducer and activator of transcription 3.

Fig. 5: (A) Protein-protein interaction networks are constructed using the STRING database. The top ten hub gene networks of TT-TeMac™ against cholinergic dysfunction are obtained by integrating three algorithms: (B) Maximum Clique Centrality (MCC), (C) Maximum Neighborhood Component (MNC), (D) Node Degree. (E) Venn diagram illustrates nine hub genes. (F) Target network of nine hub genes generated by Cytoscape software.

TT-TeMac™-target network constructions

The compound target network consists of 15 compound target nodes, one active node of TT-TeMac™, and 126 edges. In the network, the circles represent the target proteins of cholinergic dysfunction, while the triangle denotes TT-TeMac™. This clearly demonstrates the multi-target nature of TT-TeMac™ against cholinergic dysfunction associated with memory loss. Therefore, we not only observed the relationship between TT-TeMac™ and cholinergic dysfunction, but also discovered the potential pharmacological effects of TT-TeMac™ from this network.

Top ten hub gene networks of TT-TeMac™ against cholinergic dysfunction

The figure shows the protein-protein interaction networks constructed using the STRING database. The top ten hub gene networks of TT-TeMac™ against cholinergic dysfunction are obtained by integrating three algorithms (maximum clique centrality (MCC), maximum neighbour component (MNC), node degree). The Venn diagram illustrates nine hub genes and the target network of nine hub genes generated by Cytoscape software. The nine hub genes include ACHE, IL6, TNF, SNCA, AKT1, SERPINE1, STAT3, ACE, and ALB.

Details of protein targets in the PDB database

Table 2 shows the details of protein targets identified in the Protein Data Bank (PDB), along with their respective PDB IDs, resolution, and R values obtained by X-ray diffraction except SNCA (Electron Microscopy). Target proteins include Acetylcholinesterase (ACHE), Angiotensin Converting Enzyme (ACE), tumor necrosis factor (TNF), albumin (ALB), Signal transducer and activator of transcription 3 (STAT3), Plasminogen activator inhibitor−1 (SERPINE1), protein kinase Akt (AKT1), alpha-synuclein (SNCA) and Interleukin−6 (IL6). Resolution values range from 1.80 to 2.98 Å.

Targets name | PDB ID | Resolution (Å) | R-Value Free | R-Value Work
ACHE | 4EY4 | 2.16 | 0.22 | 0.18
ACE | 1O8A | 2.00 | 0.22 | 0.18
ABL | 1AO6 | 2.5 | 0.28 | 0.21
STAT3 | 6NJS | 2.70 | 0.25 | 0.20
SERPINE1 | 1LJ5 | 1.80 | 0.24 | 0.19
AKT1 | 6S9W | 2.30 | 0.26 | 0.21
SNCA | 6SSX | 2.98 | / | /
TNF | 2AZ5 | 2.10 | 0.27 | 0.22
IL6 | 1ALU | 1.90 | 0.27 | 0.21

Molecular Docking results

Table 3 presents the molecular docking scores of the identified TT-TeMac™ compounds with the major targets involved in cholinergic dysfunction. The highest MOE scoring function was applied to the tested compounds to evaluate their binding affinities. The compounds showed good binding affinity values ranging from −3.39 kcal/mol (gallic acid) to −7.4 kcal/mol (Arjunolic acid). The considerable RMSD value of < 2 indicates the high stability of these compounds in the binding site (SM 2).

Compounds | ACHE | ACE | ABL | STAT3 | SERPINE1 | AKT1 | SNCA | TNF | IL6
Terminolic Acid | −4.19 | −7.10 | −5.67 | −4.13 | −5.49 | −6.36 | −3.96 | −4.50 | −5.01
Sericic acid | −5.59 | −7.19 | −5.31 | −4.26 | −5.66 | −4.99 | −4.38 | −4.91 | −4.62
Arjunolic acid | −5.07 | −7.40 | −5.45 | −4.42 | −5.96 | −5.34 | −3.99 | −4.62 | −4.82
Gallic Acid | −4.74 | −4.64 | −4.56 | −4.32 | −4.37 | −4.81 | −3.39 | −3.81 | −3.98
Ellagic Acid | −4.20 | −5.64 | −6.15 | −5.25 | −5.02 | −6.29 | −4.05 | −4.20 | −4.62
3-O methyl ellagic acid | −3.65 | −5.5 | −6.60 | −5.43 | −5.40 | −6.62 | −4.10 | −4.18 | −4.83
3,3’-di-O-methyl ellagic acid | −5.21 | −5.73 | −6.43 | −5.57 | −5.63 | −6.80 | −4.30 | −4.79 | −4.89

Table 4 and SM1 show the profiles and different interactions of the compounds of interest with the targets. The compounds interacted with important residues of the target active pockets.

Compounds | AChE | ACE | ABL | STAT3 | SERPINE1 | AKT1 | SNCA | TNF | IL6
Terminolic Acid | Trp86, Asp74, Tyr341, Tyr124 | Arg124, Asn66, Ser335, Asn70 | Lys395, Cys448 | Cys259 | Asp222 | Gln59, Ala58 | Gln79 | Leu43 | Asp160, Arg104
Sericic acid | Tyr72, Gly120, Glu202 | His 410, Asn70 | Ala201, Asn298, Lys614, Lys395 | Glu238 | Glu242, Lys241 | Ser56 | Gly36, Leu38 | Trp28 | Arg104
Arjunolic acid | Val73, Gly448, Glu202, His447 | Asn70, Asp358 | Lys195 | Arg246, Val136 | Arg157 | Arg200 | Tyr39 | Arg32 | Glu42, Lys46
Gallic Acid | Phe338, Tyr124 | Asn66, Glu143 | His242 | Cys259 | Val274 | Thr211 | Gln79 | Arg32, Trp28 | Arg104
Ellagic Acid | Tyr124, Glu202 | Asn374, Asp377, Glu162 | Ala291, His242 | Cys259 | Leu272 | Lys268, Thr211, Trp80 | Lys80 | Gln25 | Arg104, Thr43
3-O-methyl ellagic acid | Tyr337, Trp86 | Tyr62, Ser355 | Ala291, Trp214 | Gln141, Asn257 | Val274 | Tyr211, Trp80 | Leu38 | Gln25 | Arg104, Thr43
3,3’-di-O-methyl ellagic acid | Glu202, Trp86 | Tyr62, Ser355 | Ser287 | Val137 | Phe358, Val274 | Gly294 | Lys80 | Asp45 | Thr43

TT-TeMac™ prevents non-spatial recognition learning and memory in the NOR test

Fig. 6 (A, B and C) presents the object recognition indices that provide information on the learning and memory of non-spatial recognition in rats. We noted a significant reduction in short-term IR in the SCO + H2O group compared to the Normal Control group (respectively 0.56 ± 0.01; 0.78 ± 0.014 p < 0.0001) (Fig. 6 A). The presence of TT-TeMac™ in the SCO + TT-TeMac™ group significantly increased this IR compared to the SCO + H2O group (respectively 0.67 ± 0.04; 0.56 ± 0.01 p < 0.05) (Fig. 6 A). Scopolamine injection in the SCO + H2O2 group resulted in a reduction of long-term IR compared to the normal control group (respectively 0.53 ± 0.02; 0.72 ± 0.02 p < 0.01) (Fig. 6 B). Co-treatment with TT-TeMac™ (100 mg/kg bw) in the SCO + TT-TeMac™ group showed an increase in this IR compared to the SCO + H2O group (0.59 ± 0.03; 0.53 ± 0.02 p > 0.05) (Fig. 6 B). No difference was noted between the SCO + Done group and the SCO + TT-TeMac™ group on this IR (short and long term).

Fig. 6: TT-TeMac™ prevents scopolamine-induced short- and long-term nonspatial learning and memory impairments in rats. (a) Short Term Memory IR (3 h after familiarization). (B) long term Memory IR (24 h after familiarization). (C) Representative tracks of rats moving during the test. Normal Control (n = 5): Rats treated with distilled water; SCO + H2O (n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + distilled water; SCO + TT-TeMacTM(n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + 100 mg/kg body weight/day TT-TeMacTM; SCO + Done (n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + 5 mg/kg body weight/day donepezil. One-way ANOVA followed by Tukey’s post-hoc: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 for each group.

TT-TeMac™ prevents spatial memory deficits in MWM test

Fig. 7 (A, B and C) shows the learning and memory parameters during MWM. Scopolamine induced learning impairments in the SCO + H2O group compared to the normal control group, characterized by a change in the learning profile (Fig. 7A); however, treatment with TT-TeMac™ (100 mg/kg bw) in the SCO + TT-TeMac™ group maintained the learning profile. On the last day of testing, we assessed memory parameters. It appears that scopolamine (1 mg/kg bw) treatment in the SCO + H2O group significantly increased the latency time (s) to reach the target quadrant (respectively 23.31 ± 2.38; 14.56 ± 1.49 p < 0.05) and reduced (respectively 3 ± 0.7; 5 ± 0.7 p < 0.0001) the number of entries into the target quadrant compared to the normal control group. In contrast, TT-TeMac™-treated rats in the SCO + TT-TeMac™ group showed improvements in spatial memory, characterized by a reduction (respectively 15.1 ± 0.77; 23.31 ± 2.38 p < 0.01) in the latency to reach the target (s) quadrant and an increase (respectively 4.2 ± 0.83; 3 ± 0.7 p > 0.05) in the number of entries into the target quadrant compared to the SCO + H2O group. TT-TeMac™ was comparable to that of donepezil (Fig. 7 B).

Fig. 7: TT-TeMac™ prevents scopolamine-induced spatial learning and memory impairment in rats. (a) Latency to reach the platform during training days (day 1 to day 4). (B) Latency on the final test day (day 5). (C) Number of entries in the target quadrant. (D) Representative tracks of mice swimming per group during the final test. Normal Control (n = 5): Rats treated with distilled water; SCO + H2O (n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + distilled water; SCO + TT-TeMacTM(n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + 100 mg/kg body weight/day TT-TeMacTM; SCO + Done (n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + 5 mg/kg body weight/day donepezil. One-way ANOVA followed by Tukey’s post-hoc: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 for each group.

TT-TeMac™ protects the loss of neuron on hippocampal microarchitecture rat’s brain

TT-TeMac™ protects the loss of neuron on CA1 hippocampal microarchitecture rat’s brain

Fig. 8a show the effect of TT-TeMac™ (100 mg/kg bw) in CA1 of the hippocampus in rats treated with scopolamine (1 mg/kg bw). Histological analysis with H&E revealed altered structural integrity, with leukocyte infiltration in the CA1 region of the hippocampus in SCO + H2O rats compared to the normal control. Administration of TT-TeMac™ in SCO + TT-TeMac™ protected the CA1 region of the hippocampus in these rats from this alteration (absence of leukocyte infiltration). However, the structural morphology of the CA1 region of the hippocampus was similar to that of the SCO + Done group. The use of CV showed that scopolamine caused neuronal apoptosis, thus reducing neuronal density in the CA1 area of the hippocampus of rats in the SCO + H2O group compared to rats in the normal control group. The group of rats receiving TT-TeMac™ (SCO + TT-TeMac™) prevented this neuronal apoptosis, thus reducing neuronal loss.

Fig. 8a: TT-TeMac™ protects loss of neurons in the CA1 of hippocampus of rats. HE = Hematoxylin-eosin; CV = Cresyl violet; Ne = Neuron; Ile = Leukocyte infiltration; Na = Apoptotic neuron; CNi = Nissl body; CA1: Cornu Ammonis 1. Normal Control (n = 5): Rats treated with distilled water; SCO + H2O (n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + distilled water; SCO + TT-TeMacTM(n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + 100 mg/kg body weight/day TT-TeMac™; SCO + Done (n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + 5 mg/kg body weight/day donepezil.

TT-TeMac™ protects the loss of neuron on CA2 hippocampal microarchitecture rat’s brain

Fig. 8b show the effect of TT-TeMac™ in CA2 of the hippocampus in rats treated with scopolamine (1 mg/kg bw). Observation of the CA2 region of the hippocampus in SCO + H2O rats, after H&E staining, revealed cell loss compared to the control group. Treatment with TT-TeMac™ reduced this loss. However, the structural morphology of the CA2 region of the hippocampus in SCO + TT-TeMac™ rats was closer to that of the control group than to that of the SCO + Done group. CV-specific staining highlighted hypotrophy and pyknosis in neurons of SCO + H2O rats, compared to the control group. Treatment with TT-TeMac™ (SCO+TT-TeMac™) prevented this hypotrophy and pyknosis, thus reducing neuronal loss.

Fig. 8b: TT-TeMac™ protects loss of neurons in the CA 2 of hippocampus of rats. HE = Hematoxylin-eosin; CV = Cresyl violet;; Pn = Neuronal loss; Hc = Cell hypotrophy; Py = Pycnosi; CA2: Cornu Ammonis 2;Normal Control (n = 5): Rats treated with distilled water; SCO + H2O (n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + distilled water; SCO + TT-TeMacTM(n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + 100 mg/kg body weight/day TT-TeMac™; SCO + Done (n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + 5 mg/kg body weight/day donepezil.

TT-TeMac™ protects the loss of neuron on CA3 hippocampal microarchitecture rat’s brain

Fig. 8c show the effect of TT-TeMac™ in CA3 of the hippocampus in rats treated with scopolamine. Observation of the CA3 region of the hippocampus in rats from the SCO + H2O group, after H&E staining, revealed altered morphology and cell loss compared to the control group. Treatment with TT-TeMac™ reduced this loss. However, the structural morphology of the CA3 region of the hippocampus in rats from the SCO + TT-TeMac™ group is closer to that of the control group than to that of the SCO + Done group. CV-specific staining revealed the presence of pyknosis in neurons of SCO + H2O rats, compared to the control group. Treatment with TT-TeMac™ (SCO + TT-TeMac™) prevented this hypotrophy and pyknosis.

Fig. 8c: TT-TeMac™ protects loss of neurons in the CA3 of hippocampus of rats. HE = Hematoxylin-eosin; CV = Cresyl violet; Pn = Neuronal loss; Hc = Cell hypotrophy; Py = Pycnosis; CA3: Cornu Ammonis 3; Normal Control (n = 5): Rats treated with distilled water; SCO + H2O (n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + distilled water; SCO + TT-TeMacTM(n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + 100 mg/kg body weight/day TT-TeMac™; SCO + Done (n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + 5 mg/kg body weight/day donepezil.

TT-TeMac™ protects the loss of neuron on GD hippocampal microarchitecture rat’s brain

Fig. 8d show the effect of TT-TeMac™ in GD of the hippocampus in rats treated with scopolamine. Observation of the hippocampal GD region in rats from the SCO + H2O group, using H&E staining, showed a morphological alteration with a marked opening of the hippocampal sulcus (or hippocampal fissure) and cellular loss, compared to the control group. Treatment with TT-TeMac™ reduced this loss. However, the structural morphology of the CA3 area of the hippocampus in rats from the SCO + TT-TeMac™ group is closer to that of the normal group than to that of the SCO + Done group.

Fig. 8d: TT-TeMac™ protects loss of neurons in the DG of hippocampus of rats. HE = Hematoxylin-eosin; CV = Cresyl violet; Pn = Neuronal loss; GD = Dentate gyrus; Normal Control (n = 5): Rats treated with distilled water; SCO + H2O (n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + distilled water; SCO + TT-TeMacTM(n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + 100 mg/kg body weight/day TT-TeMac™; SCO + Done (n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + 5 mg/kg body weight/day donepezil.

TT-TeMac™ reduces cholinesterase activity in the brain

Acetylcholinesterase activity was significantly increased in the SCO + H2O group compared to the normal control group (respectively 0.00339 ± 4.96E−05; 0.000259 ± 0.0002, p < 0.0001) (Fig. 9). In addition, administration of TT-TeMac™ at 100 mg/kg body weight/day in the SCO + TT-TeMac™ group resulted in a significant reduction in this enzyme activity compared to the SCO + H2O group (respectively 0.00064 ± 0.0001; 0.00339 ± 4.96E−05, p < 0.001).

Fig. 9: TT-TeMac™ counteracts the increase in brain cholinesterase activities. (a): Acetylcholinesterase (AchE), (b): Butyrylcholinesterase (BuchE). Normal Control (n = 5): Rats treated with distilled water; SCO + H2O (n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + distilled water; SCO + TT-TeMacTM(n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + 100 mg/kg body weight/day TT-TeMacTM; SCO + Done (n = 5): Rats treated with 1 mg/kg body weight/day scopolamine + 5 mg/kg body weight/day donepezil. One-way ANOVA followed by Tukey’s post-hoc: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 for each group.

In the same direction, the activity of butyrylcholinesterase was significantly higher in the SCO + H2O group than in the normal control group (respectively 0.0046 ± 0.0004; 0.00072 ± 0.00004, p < 0.001). Concomitant administration of TT-TeMac™ at 100 mg/kg body weight/day in the SCO + TT-TeMac™ group resulted in a significant reduction in the activity of this enzyme compared to the SCO + H2O group (respectively 0.00061 ± 0.00018; 0.00072 ± 0.00004, p < 0.001). An inhibitory effect of TT-TeMac™ similar to that of the reference drug, donepezil, was observed (0.00061 ± 0.00018; 0.0005 ± 5.75E−05 respectively, p > 0.05) (Fig. 9).