INTRODUCTION:

Reactive oxygen species (ROS) are naturally occurring byproducts of numerous metabolic processes that involve the consumption and utilization of oxygen. They are crucial in the regulation of cell survival, death, differentiation, signalling, and the generation of factors related to inflammation^1,2.

Then, the high amount of ROS generates oxidative stress, which causes a variety of diseases such as age-related illnesses, malignant growth, cardiovascular, inflammatory and neurodegenerative illnesses including Parkinson's and Alzheimer's³. Other than that, overabundance of ROS can harm cell lipids, proteins, or deoxyribonucleic acid (DNA), accordingly repressing sign transduction pathways and typical cell capabilities ⁴.

Antioxidants function as free radical scavengers which are substances that can stop and overhaul damage caused by ROS. By doing so, they have the potential to enhance immune defence and reduce the likelihood of developing cancer and other worsening diseases⁵. Antioxidants can be received in two ways which are spontaneously within the body, or it can be obtained through the consumption of external nutrients¹. Other than naturally occurring antioxidants, there are many treatments to treat diseases caused by oxidative stress includes the consumption of synthetic drugs. For example, N-acetylcysteine is used to treat acetaminophen toxicity by replenishing living glutathione, which is depleted as a result of oxidative reactions that produce more hydrogen peroxide during oxidative stress. However, the drugs have common toxic side effects such as nausea, vomiting, rhinorrhoea, rash, urticaria, pruritus, bronchospasm and tachycardia⁶. Therefore, in order to treat diseases caused by oxidative stress which produce minimal side effects, the natural product such as medicinal plants and herbs have been proven as a potential candidate⁷. Based on previous study, medicinal plants and bioactive compounds offer wide pharmacological activities without any cytotoxic effects^8-20.

Both Anacardium occidentale and Barringtonia racemosa are medicinal plants that are believed to produce many health benefits which potentially consumed as alternative treatment for oxidative stress-related diseases. A. occidentale or cashew nut, known as 'ketereh' or 'gajus' in the local Malay language, is a member of the Anacardiaceae family, just like the mango and are believed to treat cancer, diarrhoea, hypertension and diabetes mellitus²¹. Meanwhile, the benefits of B. racemosa consumption in Malaysia are believed to treat hypertension, relieve itchiness and chicken pox²². The plants also used to treat sore throat, asthma, and cough²³. In worldwide, the plants are applied for poisoning and used to treat malaria as well as eye inflammation^24-25. However, there is limited study in the antioxidant and toxicity effects of both plant extracts. Expectantly, this present research will provide new knowledge of the in vitro antioxidant and toxicity effects of A. occidentale and B. racemosa. This research is also to ensure both plants can be potentially consumed as an alternative way of treatment to oxidative stress-related diseases and reduce the dependence of synthetic drugs to avoid harmful adverse effects.

MATERIALS AND METHODS:

Collection, identification and deposition of plant samples:

The 400g raw leaves of A. occidentale and 800g raw leaves of B. racemosa were collected from Kuala Nerus, Terengganu and further identified by botanist. The plant voucher specimens were further deposited at South China Sea Repository and Reference Centre, INOS, University Malaysia Terengganu (UMT). Photo 1 presented the picture and number of plant voucher specimens.

Photo 1 The voucher specimen of plants; 1a) Anacardium occidentale (UMTP 1713), 1b) Barringtonia racemosa (UMTP 1714)

Preparation of plant extracts:

The leaves were washed, cleaned, and chopped in smaller size which then further dried into oven temperature. After that, the dried samples were ground into coarse powder using a mechanical grinder. Each powder of plant sample was soaked in the methanol for 2-3 days at room temperature with occasional stirring. The method was run in triplicate. After each maceration, the supernatant solution was collected by filtration method and the methanol was evaporated using rotavapor to prepare the crude extract. The resulting residue was stored in a sealed bottle in the refrigerator for further use²⁶.

Analysis of phytochemical constituents:

a) The qualitative phytochemical screening:

The qualitative phytochemical screening of plant extracts for phenolic, flavonoid, tannin, coumarin, alkaloid, glycoside, cardiac glycoside, saponin, steroid, terpenoid, quinone, anthraquinone, were conducted based on methods by Soni et al., with slight modification²⁶.

b) The quantitative phytochemical analysis:

Quantitative phytochemical analysis of total phenolic content (TPC) and total flavonoid content (TFC) of plant extracts was performed based on the method of Derakhshan et al., with minor modifications²⁷. The TPC of the extracts was determined using the Folin-Ciocalteu method. Gallic acid was used as the standard equivalent, and the results are expressed as mg gallic acid equivalents (GAE) per g dry weight. The TFC of the crude extract was determined using the aluminum chloride colorimetric method. Quercetin was used as the standard equivalent. Total flavonoids were expressed as mg quercetin equivalents (QE) per g dry weight.

In vitro antioxidative assay:

In vitro antioxidative assay, which was DPPH radical scavenging activity and conducted referred on the standard protocol with slight modification²⁸. The results will be reported and calculated as equation below:

DPPH scavenging effect (%) = (A0− A1)/A0 × 100

A0: control (absorbance value), A1: extracts/standard (absorbance value)

The IC₅₀ values represent the concentration of each sample essential to decrease half of the DPPH absorbance. The IC₅₀ was determined from the graph of percentage of inhibition plotted against concentration of plant extracts.

In vitro toxicity assay:

In vitro toxicity assay was assessed by using brine shrimp lethality assay (BSLA) and conducted based on the standard method with slight modification²⁹. Portable mini aquarium air pump-automatic oxygenator device was provided by Faculty of Ocean Engineering Technology, UMT Firstly, artificial seawater was made and the pH was more than 7 to provide suitable hatching environment. Potassium dichromate is an oxidizing agent that was used as positive control for BSLA due to its’ highly toxicity properties. Ten brine shrimps were selected and transferred into each well. In two different 24 well plates, 0.5ml of potassium dichromate and 0.5 ml of the dilutions prepared with extract were added in triplicates. Potassium dichromate was also prepared for the same concentration as that of the extract. Seawater was used as negative control. They were incubated at 20–22°C for 1 day with the presence of the light. For each well, number of dead and number of survivors of nauplii were counted and the percentage of death were calculated,

% Death = Number of dead nauplii/Number of survivors nauplii + Number of live nauplii x 100

The lethal concentration 50% (LC₅₀) values represent the lethal concentration of each sample required to kill half of the populations used in the study. The LC₅₀ was calculated from the graph of percentage of mortality rates plotted against concentration of plant extracts.

Data Analysis:

Data were expressed as mean±standard error of the mean (SEM). Each sample was prepared in triplicate and obtained from at least three determinations. The significant between quantitative variables was determined using Statistical Package for Social Sciences (SPSS) software version 23.0. The statistical tests involved were one-way analysis of variance (ANOVA) and post hoc Tukey tests. The value of p < 0.05 was considered as significant. The IC₅₀ and LC₅₀ values were determined using Microsoft Excel.

RESULTS:

Plant sample analysis:

The data for plants analysis of A. occidentale and B. racmosa are presented in Table 1. The results present that wet weight and dry weight of A. occidentale and B. racemosa were 400g, 150g and 800g, 150g respectively. The moisture percentage for A. occidentale (62.5%) was lower as compared to B. racemosa which was 81.3%. The crude extracts and yield percentage for A. occidentale and B. racemosa counted almost similar amount which were 22g and 14.7% and 21.7g and 14.5%, respectively.

Table 1 Data analysis for plant samples

Extracts	Wet weight (g)	Dry weight (g)	Moisture percentage (%)	Crude extracts (g)	Yield percentage of crude extract (%)
A. occidentale	400	150	62.5	22.0	14.7
B. racemosa	800	150	81.3	21.7	14.5

Phytochemical analysis of plant extracts:

The results on the analysis of phytochemical constituents in A. occidentale and B. racemosa extracts are presented Figure 1 and Figure 2, respectively. The results present that A. occidentale and B. racemosa contained phenolic, flavonoid, tannin, coumarin, glycoside, cardiac glycoside, saponin, steroid, quinone and anthraquinone.

Quantitative analysis of phytochemicals in plant extracts:

The Folin-Ciocalteau and aluminium chloride colorimetric methods were used to determine the total phenolic content (TPC) and total flavonoid content (TFC), respectively. The data for TPC and TFC values of A. occidentale and B. racmosa are presented in Table 2. Based on the results, A. occidentale yielded higher flavonoid content but lower phenolic content when compared to B. racemosa in methanol extraction.

Table 2: Total phenolic and flavonoid contents of A. occidentale and B. racemosa

Samples

TPC (mg GAE/g)

± SEM

TFC (mg QE/g)

± SEM

A. occidentale

25.64 ± 45.20

5.47 ± 2.46

B. racemosa

35.03 ± 56.55

5.27 ± 1.13

The data represent mean ± SEM of the total phenolic and flavonoid contents for methanolic extract of A. occidentale and B. racemosa, n=3. TPC: Total phenolic content; mg GAE: mg gallic acid equivalent; SEM: Standard error mean.

Figure 1 Phytochemical analysis of Anacardium occidentale. Each + sign represent the presence of the phytochemical constituent while - sign represent the absent of the phytochemical constituent.

Figure 2 Phytochemical analysis of Barringtonia racemosa. Each + sign represent the presence of the phytochemical constituent while - sign represent the absent of the phytochemical constituent.

Antioxidant assay by 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) method:

The antioxidant activities in plant extracts were determined using radical scavenging method which was DPPH method. Quercetin was used as positive control. There were vary concentrations of quercetin and plants tested obtained by serial dilution (7.8125 to 1000 µg/ml). The Figure 3 presents the concentration of plant samples plotted against percentage inhibition. Based on the graph, the inhibitory concentration 50% (IC₅₀)were calculated. Based on the results, it can be observed that the methanol extracts of A. occidentale and B. racemosa can inhibit the production of DPPH radicals in compatible to quercetin. The antioxidant properties of the A. occidentale and B. racemosa methanolic extracts were estimated by comparing the inhibitory concentration 50% of formation of DPPH radicals by the extracts and those of quercetin as positive control. Results show that IC₅₀value of A. occidentale extract which was presented higher amount (9.29±0.66µg/ml) and significantly different as compared to the value of quercetin (4.75±0.51µg/ml). Meanwhile, the antioxidative effects of B. racemosa extract (6.87±1.09 µg/ml) presented as effective as quercetin.

Figure 3: The percentage inhibition for quercetin, A. occidentale and B. racemosa. Each point represents the percentage inhibition of quercetin, A. occidentale and B. racemosa at concentration 1000, 500, 250, 125, 62.5, 31.25, 15.625 and 7.8125µg/ml.

Brine Shrimp Lethality Assay:

The results in Figure 4 presents the effects of potassium dichromate, A. occidentale and B. racemosa on mortality percentage rate of nauplii for 24hours. The findings presented that the lethality extention was directly related to the dose of the extract. After 24hours, there were maximum mortalities rate observed at the highest concentration which was at 10000µg/ml for A. occidentale, B. racemosa and potassium dichromate. However, in comparison to potassium dichromate, most nauplii were survived at 1000, 100 and 10µg/ml for A. occidentale and B. racemosa while some nauplii were still survived at 10µg/ml for potassium dichromate. The mortality rates of A. occidentale and B. racemosa are significant differences at concentration 100 and 10 µg/ml when compared to potassium dichromate which means A. occidentale and B. racemosa have low mortality rates of nauplii at concentration 100µg/ml and 10µg/ml when compared to potassium dichromate. The result on lethality concentration (LC₅₀) values which were 57.91±2.62µg/ml and 69.29±12.25µg/ml after 24 hours for A. occidentale and B. racemosa, respectively. LC₅₀value of A. occidentale and B. racemosa are significant difference in comparison to potassium dichromate (8.78±0.27µg/ml) indicate there are low toxicity effects exerted by the plant extracts.

Figure 4: The effects of potassium dichromate, A. occidentale and B. racemosa on mortality percentage rate of nauplii for 24 hours. n=3. * indicate p < 0.05 when compared to potassium dichromate.

DISCUSSION:

The present study presents that the dry weight of the both A. occidentale and B. racemosa samples were similar. However, the difference between dry and wet weights of B. racemosa leaves were larger compared to A. occidentale might be due to the high moisture content of B. racemosa leaves. High moisture content in most fruits and vegetables provides a conducive environment for growth and multiplication of microorganisms³⁰. In addition, the sample is highly perishable, and should thus be stored properly for longer shelf life³¹. Excessive moisture can lead to a higher tendency for spoilage and rotten products in the agricultural and food industries³².

The result percentage yield of both extracts were higher when compared to other previous study of³³ who presented the yield extraction of methanolic extracts of leaves of A. occidentale was 11.02%. This research investigated the difference in yield of extraction between three difference processes for the extraction where they found microwave assisted extraction gave the best yield, followed by Soxhlet and mechanical agitation. Similarly, the findings of³⁴ recorded the yield extraction of methanol extracts of leaves of B. racemosa was 13.9% that was lower from this present study. Besides, they also presented the difference of yield extraction between difference solvents extracts of the leaves where they revealed that methanol extracts had the highest yield, followed by the ethanol, ethyl acetate and lastly hexane extracts.

The presence of cardiac glycoside, phenolic and saponins in methanolic leaves extracts of A. occidentale strongly support the previously findings of³⁵ who study the presence of phytochemicals in different solvent extracts (hexane, ethyl acetate, methanol, water, chloroform and acetone extracts) of leaves and bark of A. occidentale. Meanwhile the presence of saponins, tannins, anthraquinones and flavonoids is concurrent with the findings of³⁶ who study the phytochemical analysis and antifungal activity of A. occidentale against root of Sesamum indicum L. in Benue State, Central Nigeria which used petroleum ether, ethyl acetate, methanol and aqueous extracts of leaves of A. occidentale. The phytochemical findings of B. racemose also supported the previous finding by³⁷ which reported the presence of similar phytochemical in their study of phytochemical evaluation and antioxidant properties of three medicinal plants (Leptadenia hastata, Barringtonia asiatica and Barringtonia racemosa) that used methanol extract of leaves of the plants. One of the possible factors that causes the difference in the findings between the previous study and the present study may be due to the differences in geographical location where the plants were collected. Therefore, the variance of the phytochemical composition of a certain variety or species of plant can be explained by differences in the type of soil, the levels of precipitation, the intensity of the light, and the humidity in that region³⁸.

Besides that, the extraction method used to extract the plants is also one of the possible factors that causes the variation in the phytochemical constituents. As an example the study by³⁵ used Soxhlet extraction method meanwhile the present study used maceration method to extract the plants. Maceration is a very simple extraction method that could be used for the extraction of thermolabile components. In comparison to maceration, the Soxhlet extraction is an automatic continuous extraction method with high extraction efficiency that requires less time and solvent consumption. However, thermal degradation of the bioactive compounds is more likely given the Soxhlet extraction's high temperature and lengthy extraction period³⁹. In addition, water able to extracts terpenoids in higher amount in comparison to other organic solvents⁴⁰.

In purpose to determine the amount of specific phytoconstituents present in plant extracts, quantitative analysis is a useful method. Phenolic compounds are a class of antioxidant agents which act as free radical inhibitors that has the abilities to chelate metals, inhibit lipoxygenase, and scavenge free radicals⁴¹. The present study reported that TPC of both extracts have higher values than the previous studies^33,42. Meanwhile, the TFC for both plant extracts were presented in moderate amounts and the result is quite similar amount with the previous work by⁴²who showed that TFC of B. racemosa was 2.39±2.02mg CE/g. However, the amount of TFC in A. occidentale was higher when compared with the past study reported by³³(0.77±2.95mg QE/g) which both used the similar method to determine the TFC of plants by using aluminium chloride colorimetric method. Therefore, in comparison to the previous study, the current study presented higher TPC and TFC values of B. racemosa and A. occidentale, respectively. Firstly, the differences in the findings may due to the differences in geographical factor of the plants collection. Secondly, the concentration of the solvents used to extract the leaves of B. racemosa were different as previous study used 70% methanol as solvent meanwhile the present study used absolute methanol as solvent for B. racemosa leaves extraction. Phenolic and flavonoid are polar compounds which higher extraction can be obtained by methanol as polar solvents⁴³. In the case of B. racemosa leaves, it can be concluded that absolute methanol more efficiently extracted higher phenolic and flavonoid content compared to 70% methanol.

Lastly, the extraction method used to extract the phenolic and flavonoid compound in the leaves of A. occidentale as³³ used microwave assisted extraction (MAE) whereas the present study used maceration as extraction method. The use of MAE may reduce the extraction time and increase the extraction yield. However, MAE generates heat when reacting with polar compounds in the plant, thus, MAE may not suitable for use when considering heat-sensitive compounds because they will be denatured^39,44. The phenolic content and antioxidant activity of A. occidentale significantly decreased in high temperature process compared to fresh samples⁴⁵. The current study presented that TPC is higher than TFC in both A. occidentale and B. racemosa extracts. These findings are concurrent with the findings of ⁴⁶that presented the TPC of A. occidentale was 19.78 ±0.62 GAE which higher than TFC of A. occidentale 1.97±0.01 RE. Similarly, B. racemosa also contained higher TPC in comparison to TFC which is supported by the findings of ⁴² Based on the reported finding, the most common phenolic acids present in A. occidentale that contribute to the mentioned values were previously identified to be chlorogenic acid, coumaric acid, gallic acid, caffeic acid and ferulic acid⁴⁷. The presence of the chlorogenic acid, coumaric acid, gallic acid in leaves of A. occidentale were confirmed in high-performance liquid chromatography (HPLC) analysis in addition to sinapinic acid⁴⁸. The previous study showed that the presence of phenolic acids which were gallic acid, protocatechuic acid, ellagic acid identified by using HPLC analysis in B. racemosa⁴⁹.

One of the most unstable free radicals is DPPH (2,2-diphenyl-1-picrylhydrazyl), which is commonly used to evaluate radical scavengers in plants and foods. Antioxidants convert the unstable free radical DPPH to stable 2,2-diphenyl-1-picryl hydrazine, which can be observed by a colour change⁵⁰. The antioxidant activity of the extracts was measured in both in IC₅₀ as well as in percentage inhibition (%inhibition). The lower the IC₅₀ value, the more effective the extract as an antioxidant. The DPPH radical is scavenged more as the plant extract concentration increases, hence the DPPH concentration decrease⁵¹. Quercetin was used as positive control for DPPH scavenging assay as a potent antioxidant.

The IC₅₀ value of B. racemosa is in the same range with IC₅₀ value of quercetin indicated the effectiveness of B. racemosa as potential natural antioxidant. Although the IC₅₀ value of A. occidentale was statistically significant different compared to quercetin, but the value still low. Therefore, both A. occidentale and B. racemosa exhibit potential antioxidant activities. The findings of this present study supported the study by⁵² who investigated the DPPH scavenging activity of A. occidentale leaves extracts and obtained slightly lower values (IC₅₀ = 5.660.16g/mL for total leaves extract). It is expected for both A. occidentale and B. racemsosa to have high DPPH scavenging activity may be related with their high TPC and TFC. In addition, the high antioxidant property of B. racemosa when compared to A. occidentale may be attributed to its higher phenolic constituents. Previous study reported the antioxidant properties were in accordance with the TPC and TFC of the plant extracts. It is because the amount of flavonoid and phenolic compounds plays a significant role in the plants’ free radical scavenging capacity⁵³.

The antioxidant finding of B. racemosa is in agreement with other past study which used B. racemosa leaves against biological samples and it reported the ability of the aqueous extracts of B. racemosa to provide protection against oxidation of biological components. It also inhibited the formation of thiobarbituric acid reactive substance (TBARS) and lipid hydroperoxides and also delaying the time required to oxidize haemoglobin to methaemoglobin. The protective effect against oxidative damage is significantly influenced by the presence of polyphenols in the leaves⁴⁹. This result was further validated by using cell culture techniques in the study of antioxidant properties of B. racemosa leaves against oxidative damage in human hepatoma HepG2 cells, where it was found to suppress the activities of antioxidant enzymes (superoxide dismutase and catalase) during oxidative stress and inhibiting the production of reactive oxygen species and lipid peroxidation, protecting HepG2 cells from hydrogen peroxide-induced cell death⁵⁴.

The antioxidant properties of phenolic compounds are thought to be mediated through the few types of mechanisms which are scavenging radical species, suppressing radical species formation by inhibiting some enzymes or chelating trace metals involved in free radical production and upregulating or protecting antioxidant defence. Phenolic compounds also act as free radical acceptors and chain breakers which prevent lipids and other compounds from oxidizing by rapidly donating a hydrogen atom to radicals. Besides, the phenolic compounds have optimum structural chemistry for free radical scavenging activities due to the phenolic hydroxyl groups that are prone to give a hydrogen atom or an electron to a free radical and an extended conjugated aromatic system to delocalize an unpaired electron⁵⁵.

Toxicity testing is a useful method for determining the toxic effects of plants in a preliminary study, and it can be done using both in vitro and in vivo models⁵⁶. The toxic effect of a wide range of plant products has been determined using the brine shrimp lethality test (BSLT). The brine shrimp lethality test is a cheap and general bioassay that appears capable of detecting a spectrum of bioactivity present in crude extract. Many researchers have used the test sample's lethality in a basic zoological creature like the brine shrimp (Artemia salina) and found it to be a beneficial tool in screening various chemical compounds present in diverse bioactivities. The number of nauplii that survived after 24 hours was counted, and the percentage of mortality was calculated. The present study determined that the extent of lethality was directly proportional to the concentration of the extract⁵⁷. It has been used to screen the toxicity of plant extracts as an alternate bioassay technique⁵⁸.

In the present study, potassium dichromate was used as positive control. It is known as highly toxic compound which normally proves fatal when ingested orally as its fatal dose is very small⁵⁹. The total mortality rate was obtained by counting the naupili that died at each concentration. The mortality rates of A. occidentale and B. racemosa were directly proportional with the concentrations. The comparison of LC₅₀ values between A. occidentale, B. racemosa and potassium dichromate are significant difference which indicated the low toxicity effects of the plant extracts when compared to the potassium dichromate. Therefore, it can be concluded that the leaves of A. occidentale and B. racemosa are considered to have low toxicity. Additionally, the B. racemosa in this study presented to have higher values of LC₅₀ (69.29±12.25μg/ml) when compared to the study of⁶⁰as it reported LC₅₀ values of 42.968µg/ml when toxicity effects of methanol extracts of B. racemosa leaves was studied against brine shrimp. However, the LC₅₀ values of A. occidentale of this present study was reported to have lower (57.91±2.62 μg/mL) in comparison to the previous study.⁶¹.

CONCLUSION:

As a conclusion, the overall outcomes of this study suggest that both plants extract have antioxidant property and have a potential to develop as safe antioxidant agents. However, these results must be supported by another related advance investigations and needed to be commenced as these plants may able to acts as alternative treatment for oxidative stress-associated disease.

CONFLICT OF INTEREST:

The authors report no financial or any other conflicts of interest in this work.

ACKNOWLEDGEMENT:

This project was supported by Ministry of Higher Education through Fundamental Research Grant Scheme (FRGS/1/2024/SKK06/UNISZA/02/1). The authors also gratefully acknowledge the use of services at South China Sea Repository and Reference Centre, INOS, University Malaysia Terengganu (UMT) for plant identification and plant sample deposition.

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Received on 04.07.2023 Revised on 24.03.2024

Accepted on 30.07.2024 Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):8-16.

DOI: 10.52711/0974-360X.2025.00002

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