Antioxidant Potential of Colebrookea oppositifolia Sm. Extracts: An In Vitro Screening Study ^†

Abstract

Alzheimer’s disease is a degenerative neurological condition mostly affecting memory and cognitive abilities in older people. This study aimed to determine how acteoside, a major plant phytoconstituent, protects against neuronal death in the hippocampus region, activates the cholinergic system, and acts as an antioxidant to help people with Alzheimer’s-type dementia. Early research on the extraction process and subsequent in vitro testing revealed that the plant extract had potent antioxidant qualities. Initial assessment highlighted the yield percentage, which was 14.10% using the Soxhlet method. In order to explore this plant’s possible medical uses, further in vivo studies are required.

1. Introduction

Alzheimer’s disease (AD), the most common kind of dementia, develops gradually and leads to the impairment of behavior, mood, and cognitive function. More than 36 million people worldwide are affected by this age-related neurodegenerative illness. Furthermore, it has been estimated that this number would surpass 115 million by 2050 [1]. However, the degenerative process that causes AD starts well before typical symptoms appear, so there is a widespread sentiment that therapies must start as soon as the disease is diagnosed in order to have the most beneficial effect on the illness. None of the currently available therapies prevent or cure the illness, even though they can be beneficial by the time AD is diagnosed [2,3]. In order to increase our awareness of the condition and develop better techniques for early identification when neuronal protection is feasible, it is crucial to switch the diagnostic emphasis towards the detection of more preclinical AD. Adult-onset dementia is thought to be mostly brought on by neurodegeneration. Alzheimer’s disease, Lewy bodies, vascular dementia, frontotemporal lobar degeneration, and Parkinson’s disease are the most common degenerative dementias among elderly people [4,5]. On the other hand, non-neurodegenerative mild cognitive impairment can be brought on by vitamin deficiency, hypothyroidism, chronic alcohol abuse, cognitive impairment from chemical therapy, normal pressure hydrocephalus, viral infections like human immunodeficiency virus, subdural hematomas, brain tumors, traumatic brain injury (TBI), and psychiatric conditions like anxiety, as well as profound depression. Preclinical AD is defined by a lengthy period during which cognitive performance is unaffected but neuropathological deficits in AD begin to accumulate [6,7]. Biomarkers for preclinical AD have gained a lot of attention from both doctors and researchers as instruments that aid in the detection and diagnosis of the illness. The condition often begins with increasing memory loss; however, sometimes the predominant symptoms are behavioral, visuospatial, or linguistic. After Alzheimer’s disease symptoms appear, survivors typically live for 10 to 12 years. The “preclinical” stage of Alzheimer’s disease, which starts 20 years before symptoms appear, is defined by the gradual buildup of protein amyloid plaques (also called neuritic plaques or amyloid plaques), neurofibrillary tangles (NFTs), and amyloid beta (Aβ) [8,9]. The pathophysiology of Alzheimer’s disease is complex and involves various molecular, cellular, and structural alterations in the brain. This discussion provides an overview of the key aspects of the pathophysiology of Alzheimer’s disease [3]. Despite the availability of pharmaceutical interventions, the potential neuroprotective and therapeutic benefits of plants and their phytoconstituents have drawn attention to their use in the treatment of Alzheimer’s disease. Numerous plants and their phytoconstituents have potent anti-Alzheimer’s and antioxidant properties. One of the main causes of AD pathogenesis is oxidative stress, which results in the buildup of free radicals and the death of neurons. Acorus calamus [8], Curcumin [10], Ginkgo biloba [11], Bacopa monnieri [12], Panax Ginseng [13], grapes and berries [14], Ashwagandha [15], Rhodiola Rosea [16], and many more plants have been used to treat Alzheimer’s disease.

A valuable traditional medicinal plant in the Lamiaceae family is Colebrookea oppositifolia Sm. Subtropical areas of certain Asian nations, including China and India, are home to this thick, wool-like shrub. Acteoside, a phytoconstituent found in Colebrookea oppostifolia, has gained attention for its neuroprotective and anti-inflammatory properties, suggesting a potential role in addressing AD through dietary interventions. Verbascoside, also referred to as acteoside, is a phenylethanoid glycoside that is present in olives and several other plants, including species of Verbascum. Acteoside may have neuroprotective effects through modulating different cellular pathways, according to research. Because of its antioxidant qualities, it helps to counteract free radicals, which are linked to the oxidative stress seen in AD pathology [17]. The development of AD is significantly aided by chronic neuroinflammation. Acteoside has been shown to have anti-inflammatory qualities by lowering microglia activation and preventing the release of pro-inflammatory cytokines [18]. These anti-inflammatory properties might be extremely important in reducing the inflammatory processes linked to AD. The buildup of beta-amyloid plaques in the brain is one of the characteristics of Alzheimer’s disease. Acteoside has been investigated for its ability to impede the formation of toxic protein aggregates by interfering with the beta-amyloid aggregation process [19]. Examining acteoside’s historical and cultural applications in conventional medicine is part of its ethnopharmacological study.

2. Materials and Methods

2.1. Plant Procurement and Extraction

The plant containing the acteoside phytoconstituents (Colebrookea oppositifolia Sm.) was obtained from near Mukteshwar in the Nanital region of the Himalayan range. Two Soxhlet assemblies—one for the roots and one for the aerial portion—were utilized throughout the extraction process. Following shade drying, the two samples were broken up into tiny bits, crushed, and put in a Soxhlet device with ethanol acting as a solvent. Using a separating funnel, we gathered the filtrate containing the active ingredients separately. A rotary evaporator was used to concentrate the filtrated extract at low pressure and low temperature.

2.2. Chemicals Used

Ethanol, chloroform, DPPH, methanol, ascorbic acid, distilled water, glycerin.

2.3. Antioxidant Assay

2,2-Diphenyl-2-Picryl-Hydrazyl Radical Scavenging (DPPH) Assay

The potential of the extracts to scavenge free radicals was assessed through the DPPH assay. The plant extract’s ability to donate hydrogen atoms was determined by monitoring the decolorization of a methanol solution of DPPH. DPPH imparts a purple color to the methanol solution, which transitions to yellow in the presence of an antioxidant. To create the plant extract stock solution, the extract was dissolved in methanol at a concentration of 1 mg/mL. From this stock solution, diluted concentrations ranging from 100 to 500 g/mL were prepared. Similarly, a standard solution of ascorbic acid was prepared within the same concentration range. The DPPH solution was then prepared in methanol at 0.135 mM. One milliliter amounts of extracts at different concentrations were then combined with one milliliter of DPPH solution. After vigorous churning, the mixture was placed in a dark place for half an hour. After that, a spectrophotometer was used to test the mixture’s absorbance at 517 nm. In order to prepare a blank, no sample was added. To assess the plant extract’s antioxidant activity, its absorbance was contrasted with the concentration of the standard solution (ascorbic acid) [20].

3. Results

3.1. Percentage Yield of Plant Extract

The yield percentage of the aerial plant extract (

Table 1. Percentage yield (aerial part) by using hot continuous process (Soxhlet).

First Sample	Solvent Used	Sample Weight	Final Extract Obtained	Percentage Yield (w/w)
Aerial part	Ethanol	500 g	69 g	13.80

) was 13.80 percent (w/w), and for the root extract (

Table 2. Percentage yield (roots) by using hot continuous process (Soxhlet).

Second Sample	Solvent Used	Sample Weight	Final Extract Obtained	Percentage Yield (w/w)
Roots	Ethanol	500 g	70.5 g	14.10

) it was 14.10 percent (w/w) when the semi-solid residue was lyophilized to create a fine powder.

3.2. Antioxidant Assay (DPPH Assay)

The plant extract demonstrated notable DPPH radical scavenging activity in a manner dependent on the dosage.

Table 3. DPPH scavenging activity of ascorbic acid, root extract and aerial plant extract.

Antioxidant DPPH Assay
Concentration (μg/mL)	Percentage (%) Inhibition
	Standard Ascorbic Acid IC50 Value: 315.76	Root Extract IC50 Value: 423.62	Aerial Plant IC50 Value: 486.39	%RSA
0	0	0	0	0
100	27.5	21.4	19.2	72.5
200	40.8	30.5	25.4	79.6
300	52.4	38.6	32.1	82.53
400	60.5	47.2	41.8	84.87
500	68	55	50	86.4

and

Table 4. Descriptive statistical analysis of Table 3.

Concentration (g/mL)	Values	Standard Ascorbic Acid	Values	Root Extract	Values	Aerial Plant	Values
Mean	250	Mean	41.53	Mean	32.11	Mean	28.08
Standard Error	76.37	Standard Error	10.16	Standard Error	8.04	Standard Error	7.20
Median	250	Median	46.6	Median	34.55	Median	28.75
Standard Deviation	187.08	Standard Deviation	24.902	Standard Deviation	19.70	Standard Deviation	17.66
Sample Variance	35,000	Sample Variance	620.15	Sample Variance	388.42	Sample Variance	311.88
Kurtosis	−1.2	Kurtosis	0.390593	Kurtosis	0.268108824	Kurtosis	0.188460065
Skewness	0	Skewness	−0.92783	Skewness	−0.73187945	Skewness	−0.52811084
Sum	1500	Sum	249.2	Sum	192.7	Sum	168.5
Confidence Level (95.0%)	196.33	Confidence Level (95.0%)	26.13	Confidence Level (95.0%)	20.68	Confidence Level (95.0%)	18.53

and Figure 1 illustrate that the standard ascorbic acid displayed the highest scavenging activity, followed by the root extract and the aerial plant extract. However, upon comparing the two plant extracts, it is evident that the root extract exhibits superior antioxidant activity compared to the aerial plant extract. A lower IC₅₀ value indicates stronger antioxidant activity. The DPPH assay was performed in triplicate (n = 3) for each sample concentration.

4. Discussion

Acteoside, a bioactive compound present in various plant species, has gained attention for its potential antioxidant properties. This study aimed to investigate the antioxidant activity of plant extracts containing acteoside, specifically focusing on comparing the efficacy of root extract versus aerial plant extract using the DPPH (2,2-diphenyl-1-picrylhydrazyl) method. The results unveiled significant antioxidant activity in both extracts, with a noteworthy observation that the root extract displayed superior antioxidant efficacy in comparison to the aerial plant extract. The DPPH assay, a widely employed method for assessing antioxidant capacity, hinges on the capability of antioxidants to furnish hydrogen atoms to DPPH radicals, leading to the decolorization of the DPPH solution. The dose-dependent pattern exhibited by the plant extracts in substantial DPPH radical scavenging activity underscores the potency of acteoside in conferring antioxidant properties to these extracts. In Table 3 and Figure 1, the standard ascorbic acid served as a benchmark with the highest scavenging activity, aligning with its well-established antioxidant reputation. Subsequently, both the root extract and the aerial plant extract demonstrated antioxidant activity, albeit at varying levels. Acteoside, categorized as a phenylethanoid glycoside, is renowned for its antioxidant, anti-inflammatory, and neuroprotective properties. Its presence in plant extracts hints at a potential natural source for antioxidants with therapeutic implications.

Furthermore, the in vitro nature of the DPPH assay does not fully replicate the dynamic and complex conditions within a living organism. Consequently, the findings should be interpreted in the context of the experimental setup and may not entirely represent the physiological intricacies of in vivo antioxidant activity. The implications of this study are noteworthy for the potential utilization of these plant extracts as natural antioxidants in diverse applications, including pharmaceuticals, food, and cosmetics. The superior performance of the root extract suggests that targeted extraction methods focusing on specific plant parts enriched in acteoside could enhance antioxidant yields for practical applications. In the future, it will be crucial to investigate analytical methods that accurately ascertain acteoside concentration. Determining the identity and amount of this bioactive substance will be crucial in developing focused therapies. Advanced analytical methods, such as high-performance liquid chromatography (HPLC)/high-performance thin layer chromatography (HPTLC) or mass spectrometry, can be employed to discern acteoside levels with precision and reliability. This analytical method will help us better understand how the compound is distributed throughout plant tissues and make it possible to create standardized formulations that can be used therapeutically. In parallel, to evaluate acteoside’s therapeutic potential in the context of Alzheimer’s disease, future research efforts should incorporate in vivo parameters in addition to in vitro assays. Animal models, especially those that replicate the pathology of Alzheimer’s disease, can provide information about the compound’s capacity to cross the blood–brain barrier and confer neuroprotective effects. Furthermore, it is imperative to investigate the effects of acteoside on particular molecular pathways linked to the pathogenesis of Alzheimer’s disease. Because of its phenylethanoid glycoside nature, the compound has anti-inflammatory and neuroprotective qualities, making it a promising candidate for treating the various aspects of Alzheimer’s disease.

5. Conclusions

In conclusion, root extract containing acteoside had showed its antioxidant properties and potential in preventing Alzheimer’s disease. However, further in vivo experimental studies (behavioral, biochemical and histological studies) are required to continue this study. Initial results, such as a 14.10% yield using Soxhlet extraction and the DPPH antioxidant parameter assay, demonstrate its potential for medicinal use. To properly investigate its medicinal uses and confirm its effectiveness in treating Alzheimer’s-type dementia, further research is required.