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Introduction
Neurodegenerative disease, such as Alzheimer’s disease (AD), has become a concern, especially in developed countries with a high aging population. This disease has created social and health problems since it burdens the patient, family, and health system 1. Most people with Alzheimer's are 65 and older, but the early stage of AD can be found at younger ages. AD mainly affects brain function, which causes memory loss, diminished judgment capacity, language impairment, and behavioral changes 2. This disease is progressive and, over the years, can develop dementia (60-70% of dementia is caused by AD) 3. Multiple factors are involved in the pathogenesis of AD, including deficiency of neurotransmitter acetylcholine (ACh) and oxidative stress. The regulation of acetylcholine is controlled by acetylcholinesterase (AChE) 4. This enzyme catalyzes the breakdown of ACh into choline and acetate. Therefore, inhibiting this enzyme will help maintain the level of ACh in the brain. The use of cholinesterase inhibitors has shown positive effects in the improvement of cognitive function of AD patients 5. Studies have revealed that oxidative stress plays a significant role in the pathogenesis of neurodegenerative diseases, including AD. Oxidative stress causes the loss of neurons and AD progression to dementia 6-8. Oxidative stress also involves the accumulation of a toxic peptide, β-amyloid, in the brain of AD patients 8,9.
Pternandra galeata, locally known as Tiju, is a plant found on Borneo Island in Indonesia and Malaysia. It is a small to medium size tree with a height of up to 27 m. The leaves and flowers of P. galeata are the food source for Borneo’s monkeys 10. A Literature search showed a limited study of P. galeata regarding its metabolites and bioactivities. The methanolic extract of these plant leaves showed potential antiamoebic activity against Entamoeba hystolitica11. There is no report on the antioxidant and cholinesterase inhibitory activity of this plant. In our continuing study searching for potential medicinal plants and marine organisms to contend with neurodegenerative diseases, we screened several plants from Borneo for antioxidant and cholinesterase activity. The current research focuses on the antioxidant and cholinesterase activity of the ethanolic extract of the stem of P. galeata.
Material and methods
Reagents
The chemical used for cholinesterase assays were acetylcholinesterase from electric eel (AChE type VI-S), acetylthiocholine iodide (ATCI), horse-serum butyrylcholinesterase (BChE), butyrylthiocholine iodide (BTCI), 5,5′-dithiobis[2-nitrobenzoic acid] (DTNB), bovine serum albumin (BSA), tris buffer, and galantamine. The chemicals used for antioxidant assays were 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and potassium persulfate. Folin-Ciocalteu’s phenol reagent, sodium carbonate, and gallic acid were used to determine total phenolics. All reagents were purchased from Sigma-Aldrich.
Preparation of plant extract
The stems of Pternandra galeata (Korth.) Ridl. was obtained from Landau Village, Nanga Taman District, Sekadanau, West Kalimantan, Indonesia. The plant was identified by Purwodadi Botanic Garden, Indonesian Institute of Sciences, with identification letter number: B-262/IPH.06/KS.02/VII/2020. The stems of Pternandra galeata were cut into small pieces, then dried in the shade for seven days, and finally grounded. A hundred grams of the powdered stem was soaked with 600 mL ethanol for 24 hours; then, the filtrate was separated under vacuum. The residue was re-extracted using the same procedure three times. All collected filtrates were evaporated under vacuo to obtain 0.55 gram ethanolic extract.
Acetylcholinesterase inhibitory assay
The assay was performed based on the modified Ellman’s method 12-14. The extract was dissolved in 10% methanol at 1-500 µg/mL. Twenty-five microliters of sample solutions were mixed with 1.5 mM ATCI (25 µL), 3 mM DTNB (125 µL), Tris buffer (50 µL), and 0.22 U/mL AChE (25 µL) in a 96-well microplate. The formation of yellow colored product, 5-thio-2-nitrobenzoate, was monitored in a microplate reader (Thermo Scientific Multiskan FC) at 405 nm every 5 s for 2 mins. Experiments were carried out in triplicates. Galantamine was used as a standard, and 10% methanol was used as a control/blank. The percent of inhibition was then calculated as follows:
DPPH radical scavenging assay
The assay was conducted in triplicates based on modified protocols of Herald et al. and Lee et al.15,16. The DPPH solution (0.25 mM) was made in methanol. Different concentrations of extract (1.25 - 40 µg/mL) in methanol and standard gallic acid were incubated with DPPH solutions in 96 well plates and allowed to stand at room temperature in the dark for 30 minutes. The solutions were shaken for 30 s, and the absorbance was measured at 517 nm in a microplate reader. Gallic acid was used as a standard. The following formula calculated the percent DPPH scavenging effect:
where abs control is the absorbance of DPPH radical + methanol and abs sample is absorbance DPPH radical + extract/standard.
ABTS Radical Scavenging Assay
The bioassay was performed based on the previous method of Lee et al. with some modifications (16). ABTS radical was generated by mixing 5 mL ABTS (7 mM) with 88 𝜇L potassium persulfate (140 nM) and allowed to stand for 16 hours in the dark at room temperature. Samples at a concentration range of 1.25 - 25 𝜇g/mL in methanol were treated with 100 𝜇L of ABTS in a 96-well microplate, followed by incubation for 6 mins in the dark condition at room temperature. The absorbance was read at 734 nm in a microplate reader. Gallic acid was used as a standard. Experiments were carried out in triplicates. The ABTS radical scavenging activity of the extract was calculated as above.
Quantification of total phenolic content
The total soluble phenolic compounds (TPC) in the extract were measured according to the method by Zhang et al. and Herald et al. with slight modification using gallic acid as a standard 15,17. Briefly, 25 µL solution of extract (1000 µg/mL) or gallic acid (25 - 500 µg/mL) was mixed with water (75 µL) and Folin & Ciocalteu’s phenol reagent (25 µL) in a 96-well microplate. The mixtures were allowed to stand for 6 minutes at room temperature, followed by adding 100 µL Na2CO3 solution (75 g/L). The mixtures were then incubated for 90 minutes in the dark at room temperature. The absorbance was recorded at 765 nm, and the TPC was calculated based on the gallic acid standard calibration curve. The TPC of the extract was expressed as gallic acid equivalent (GAE) in milligrams per gram of dry extract.
Results
Acetylcholinesterase inhibitory activity
The ethanolic extract of P. galeata was screened against the AChE enzyme using Ellman’s method. In this assay, the enzyme AChE will hydrolyze the substrate acetylthiocholine Iodide (ATCI), resulting in the product thiocholine, which will react with the Ellman’s reagent (DTNB) to form a yellow-colored compound 5-thio-2-nitrobenzoate that can be monitored at 405 nm. The presence of the AChE inhibitor will prevent the hydrolysis of ATCI so that the yellow-colored product will not be formed 12. Various concentrations of P. galeata extract were prepared to evaluate the dose-response mode and the fifty percent inhibitory activity (IC50) of the extract. The result presented in Table 1 showed that the extract inhibited the AChE enzyme with an IC50 value of 74.62 µg/mL. Figure 1 showed that the extract inhibited AChE in a dose-dependent manner.
Antioxidant Activity
The antioxidant property of P. galeata was examined using DPPH and ABTS assays. In the DPPH assay, the antioxidant provides a hydrogen atom that reacts with the stable radical DPPH to form a yellow-colored non-radical diphenylpicrylhydrazine 18,19. The principle of ABTS assay is similar to that of DPPH assay, in which the antioxidant acts as a hydrogen donor to form a non-radical ABTS. The reduction of a dark-bluish color of ABTS radical can be monitored by a spectrophotometer 20. The results presented in Table 1 and Figure 2 suggested that the extract of P. galeata exhibited concentration-dependent radical scavenging activities in both DPPH and ABTS assays with IC50 values of 20.21 and 7.68 µg/mL, respectively.
Table 1 Radical scavenging effect and AChE inhibitory activities of P. galeata extract and standards
| Samples | IC50 (µg/mL) | ||
| AChE | DPPH radical scavenging | ABTS radical scavenging | |
| P. galeata extract | 74.62 ± 0.89 | 20.21 ± 0.08 | 7.68 ± 0.09 |
| Galantamine | 0.63 ± 0.05 | - | - |
| Gallic acid | - | 2.76 ± 0.02 | 0.97 ± 0.03 |
aEach value is the average of three analyses ± standard deviation
Total phenolic content
The total phenolic content of P. galeata extract was ascertained by the method of Folin-Ciocalteu and calculated as gallic acid equivalent (GAE). The total phenolic content of P. galeata extract was 164.71 ± 3.33 mg GAE/g of dry extract (Table 2).
Discussion
Alzheimer’s disease is one of the most prevalent neurodegenerative diseases that mainly occurs in older adults. Cognitive decline is an apparent symptom of this disease, caused by the deficit of neurotransmitter acetylcholine, particularly affecting cholinergic neurons in the basal forebrain. Another factor involved in the etiology of AD is oxidative stress. It is associated with the mechanism of Aβ-induced cytotoxicity of cholinergic neurons. Currently, AChE inhibitors, such as galantamine, rivastigmine, and donepezil, have been proven to be the most effective therapy to improve AD patients’ memory and cognitive function 21. In addition, antioxidants have shown benefits in improving cognitive function and behavioral deficits in AD animal models 22. Therefore, it is suggested that natural antioxidants with potent cholinesterase inhibitory activity can be a better drug for treating AD 23,24. In the current study, the stem of Pternandra galeata was extracted with ethanol. The extract was then subjected to in vitro antioxidant and acetylcholinesterase inhibitory assays.
Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are enzymes that are related to the level of acetylcholine (ACh) in the brain 25. However, AChE is reported as the major cholinesterase enzyme in the brain that catalyzes the hydrolysis of acetylcholine and shows higher specificity toward acetylcholine. Therefore, AChE enzyme has been an attractive target for AD therapy 26. In our study, P. galeata extract showed moderate inhibition against the AChE enzyme in a dose-dependent manner. Currently, we have not found a report from the genus Pternandra as a cholinesterase inhibitor. However, a plant from the same family Melastomataceae, Miconia sp., has been reported to have moderate AChE inhibitory activity 27. A triterpene, sumaresinolic acid, isolated from Miconia stenostachya showed AChE inhibitory activity 28,29.
Oxidative stress has been reported to play an essential role in AD pathogenesis. Therefore, preventing or reducing oxidative damage by using antioxidants for AD and another therapy may provide better results 24. Plant materials, such as fruits, vegetables, herbs, and spices, have been known as the source of natural antioxidants 30. The current study examined the antioxidant property of P. galeata by DPPH and ABTS methods. The DPPH and ABTS free radicals are stable free radicals widely used to estimate the radical scavenging activity of antioxidants. The results showed that P. galeata gave strong radical scavenging activities in both DPPH and ABTS assays. This is due to the different reactivity of compounds in the P. galeata extract to DPPH and ABTS radicals. The reaction of DPPH radicals depends on the steric accessibility of compounds to them. Small molecules generally react better with DPPH radicals compared to large molecules. ABTS radical can be used to measure the antioxidant capacity of both hydrophilic and lipophilic compounds 31,32. The extract showed a lower IC50 value in the ABTS assay than in the DPPH assay. There has been no report of P. galeata as an antioxidant. However, a plant from the same genus, P. azurea exhibited radical scavenging activity against DPPH, ABTS, and nitric oxide (NO) 33.
There has been evidence of the correlation of phenolic compounds with antioxidant and cholinesterase inhibitory activities. The presence of hydroxyl groups in the phenolic compounds is believed to play a significant role in AChE inhibitory activity. The multiple hydroxyl groups in the phenolic compounds can enhance the inhibitory action of AChE due to their stronger binding capacity 34,35. Several phenolic compounds have been reported to show inhibition against AChE and BChE in both in vitro and in vivo studies, such as quercetin, resveratrol, curcumin, gallocatechin, as well as cinnamic acid and its derivatives 35. Phenolic compounds are also well known to be essential in the antioxidant activities of medicinal plants. The antioxidant potential of the phenolics is predominantly due to the redox capability so that it can absorb and neutralize free radicals, decompose peroxide, and quench singlet or triplet oxygen 36. The number and arrangement of the hydroxyl groups in phenolics are believed to be closely related to their antioxidant capacity.
Conclusion
The stem extract of P. galeata exhibited antioxidant and acetylcholinesterase inhibitory activities. The presence of phenolic compounds in the extract may be responsible for the antioxidant and AChE activities. Further study is needed to investigate the chemistry of the phenolic compounds in the extract.
