INTRODUCTION:

Remedies obtained from medicinal plants have been increasingly adopted by the masses over the past decade, as people believe that natural medicines are much safer than synthetic drugs^1,2. However, the notion that herbal medicines are totally safe is not only misleading but also wrong³.

In fact, it is becoming clear that they can have toxic sideeffects in animals, humans included. Moreover, there have been reports of increased cases of poisoning following the consumption of herbal medicines³. Therefore, in-depth toxicological evaluation of any medicinal plant before it enters widespread usage is crucial to ensure that it is safe for consumption and mitigate the health risk to the public⁴.

Phaleria macrocarpa, or known as ‘mahkotadewa’ in Malaysia,is an evergreen or small plantof the family Thymelaeaceaethat grows in tropical areas of Papua Island, Indonesia^5,6. This plant has been widely used as a medicinal herb in Malaysia and Indonesia⁷. P.macrocarpais generally considered a treatment for lifestyle diseases. Various parts of this plant have been used for disease prevention such as hypertension, cancer, diuretics, atherosclerosis, and infectious disease ^8,9. The fruitof P. macrocarpais one of the most utilised parts of the plant. Extract of P. macrocarpa’s fruit has been reported for many pharmacological activities, including antioxidant^10,11, antimicrobial¹², anti-inflammatory¹³, anti-hypercholesterolemic¹⁴, and male infertility management⁶. Phytochemical analysis of the fruit extracts of P. macrocarpa revealed the presence of tannins, lignans, saponins, phenolics, flavonoids, terpenoids, and alkaloids responsible for its various pharmacological activities^15,16. Moreover, the presence of icariside C3, magniferin, and gallic acid in the fruits of P. macrocarpa has also been reported¹⁷.

Despite its evidently widespread use, this plant's toxicological aspects have remained largely unexplored. Therefore, the present study aims to investigate the antioxidant activity and toxicity of the methanolic fruit extract of P. macrocarpa (PMFM) using brine shrimp. In addition, the protective effects of P. macrocarpa against oxidative stress will be further determined. The findings may provide valuable insights into the potential health hazards of using P. macrocarpa as a traditional medicine for treating various diseases.

MATERIALS AND METHODS:

Preparation of P.macrocarpafruit methanolicextract:

Three hundred grammes (300g) of dried slices of Phaleria macrocarpa fruits were purchased from a traditional plantation in Selangor. The PMFM extract was prepared using the maceration technique.The sliced fruits were cleaned, dried for three days in an oven at 40°C and groundto obtain a coarse powder. Then, 25g of P. macrocarpa dried powder was macerated with methanol using a 1:10 ratio. The mixture was macerated for three days with periodic agitation to dissolve the powder completely. After three days, the mixture was strained using Whatman filter paper no. 1. The maceration process was repeated three times. The obtained filtrate was concentrated using a rotary evaporator at 40°C. The extract was transferred into the container and further dried in the oven at 40°C for three days. Then, the extract was kept at 4°C until further use.

Phytochemical screening:

To detect the presence of saponins, 100mg of the PMFM extract was mixed with distilled water in a test tube. The formation of stable froth for at least 15 minutes indicates the presence of saponins. For the test of tannins and polyphenolic compounds, 100mg of the PMFM extract was mixed with a 1% ferric chloride solution. The formation of a blue-black colour indicates the presence of hydrolysable tannins, while brownish green indicates the presence of condensed tannins.

A total of 100mg of the PMFM extract was mixed with chloroform. To detect the presence of alkaloids, ammoniacal chloroform was added to the chloroform extract and then treated with 10% sulfuric acid. The mixture was then tested with Mayer's reagent. To test the presence of flavonoids, the chloroform mixture was dissolved in ether and shaken in a 10% ammonia solution. The chloroform mixture was tested using Liebermann-Buchard reagent to detect the presence oftriterpenes and steroids.

Antioxidant assays:

The PMFM extract was subjected to two commonly used antioxidant assays, namely the 1,1-diphenyl-2-picrylhydrazyl (DPPH) andferric-reducing antioxidant power (FRAP) assays. The DPPH assay is based on the reduction of the stable radical DPPH by an antioxidant compound, while the FRAP assay measures the ability of an antioxidant to reduce Fe(III) to Fe(II) in the presence of a complexing agent. The DDPH and FRAP assays were carried out according to previously described methods^18,19.

Brine shrimp lethality test:

The cytotoxic activity of the PMFM extract was evaluated using the brine shrimp lethality test with some modifications^20,21. Brine shrimp (Artemia salina) nauplii were used as test organisms. For hatching, brine shrimp eggs were incubated in salt water at 28°C with proper lighting and aeration. After 24hours, the collected nauplii were then used in the experiment. In brief, five different concentrations of PMFM extract (0.2, 0.4, 2, 0.6, 0.8, and 1mg/ml) were prepared in triplicates via serial dilution with 1% dimethyl sulfoxide (DMSO) in salt water. A total of ten nauplii were put in each of the well of the 24 well-plate containing 1 ml of the PMFM extract. The negative control wells contained ten nauplii and 1ml of 1% DMSO in salt water while the positive control wells contained ten nauplii and 1ml of 0.2mg/ml potassium dichromate. The well-plate was incubated at 28°C for 24hours. After 24 hours, the percentage of mortality of nauplii was calculated. The experiment was conducted in triplicates.

Oxidative Stress Protection Assay:

The nauplii were obtained using the same method as the brine shrimp lethality test. Five non-toxic concentrations of PMFM extract were chosen based on the brine shrimp lethality test of the PMFM extract. A total of ten naupliiwere exposed to 1 ml of the PMFM extract in each well. The control group was exposed to 1ml of salt water. The well plate was incubated at 28°C for 1hour. After 1 hour, the nauplii were exposed to hydrogen peroxide (H₂O₂) diluted in salt water. The concentration of H₂O₂was chosen based on the LC₅₀ value of H₂O₂obtained during the brine shrimp lethality test. The well plate was incubated at 28°C for 24 hours. After 24 hours, the survival of naupliiwas observed using a microscope. The experiment was conducted in triplicate.

Statistical analysis:

One-way ANOVA and t-tests were used to evaluate statistical differences between the means using GraphPad Prism 9. A p-value of < 0.05 was considered statistically significantly different. Probit regression analysis was performed using Microsoft Excel. The LC50 values, which are the concentration that kills 50% of the sample population of the extract, were obtained from the best-fit line by regression analysis²².

RESULTS:

Phytochemical screening:

As shown in Table 1, the phytochemical screening of the PMFM extract revealed the presence of compoundssuch as saponins and hydrolysable tannins.

Table 1: Phytochemical screening of Phaleria macrocarpa fruit methanolic (PMFM) extract

Test	Result
Alkaloids	Alkaloids were not detected
Saponins	Saponins were detected
Flavonoids	Flavonoids were not detected
Tannins and polyphenolic compounds	Hydrolysable tannins were detected
Triterpenes	Terpenes were not detected
Steroids	Steroids were not detected

Antioxidant activity of PMFM:

The DPPH assay evaluates the ability of PMFM extract to serve as free radical scavengers or hydrogen donors, indicating the antioxidant activity of the extract. The results for the DPPH assay of PMFM extract and ascorbic acid are shown in Figure-1. The IC₅₀ values obtained for the PMFM extract and ascorbic acid were 0.3612mg/ml and 8.73µg/ml, respectively. This value represents the concentration at which 50% of DPPH radicals were reduced.The IC50 value of PMFM extract was higher than that of ascorbic acid, which is a well-known potent antioxidant.

(a)

(b)

Figure-1: Graph of mean percentage of inhibition of DPPH (%) versus log10 concentration of (a) PMFM extract (mg/ml) and (b) ascorbic acid (µg/ml)

In FRAP assays, the antioxidant activity of PMFM extract was assessed by its ability to reduce ferric ions. As shown in Figure-2, the reducing power increased with an increase in extract concentration. 0.5 mg/ml PMFM extract exhibited the highest FRAP reducing power at 21.00±2.75µg ascorbic acid equivalents (AAE)/µg and 217.19±54.25µg gallic acid equivalents (GAE)/µg.

(a)

(b)

Figure-2: Graph of mean of FRAP value (a) (µg of AAE/µg of extract) vs concentration of PMFM extract (mg/ml), (b) (µg of GAE/µg of extract) vs concentration of PMFM extract (mg/ml)

Oxidative stress protection of PMFM extract in brine shrimps:

Figure-3(a) showed the mortality percentage of the brine shrimp nauplii after being exposed for 24hours to different solutions and concentrations of PMFM extract. The percentage of nauplii mortality is directly proportional to the concentration of PMFM extract. The percentage of nauplii mortality increased from 3.33% to 83.33% when the concentration of the PMFM extract increased from 0.2 to 1.0mg/ml. The LC50 value (lethal concentration that kills 50% of nauplii) of the extract was 0.67mg/ml which was calculated through probit analysis. Meanwhile, Figure-3(b) displays the survival rate of brine shrimp naupliifollowing treatment with PMFM extract for one hour, and subsequent exposure to 7.24mM H₂O₂ for 24hours.The survival of nauplii was 50% when they were not treated with PMFM extract prior to exposure to H₂O₂. However, the survival of nauplii increased when treated with PMFM extract. The percentage of nauplii survival is directly proportional to the concentration of the PMFMextract. The percentage of nauplii survival increased from 63.33% to 76.67% when the concentration of the PMFMextract increased from 0.05 to 0.2mg/ml. This indicated that the PMFM extract could provide a protective effect to brine shrimp nauplii against oxidative stress induced by H₂O₂.

(a)

(b)

Figure-3:(a) Percentage mortality of Artemia salina after 24 hours being exposed to 1% DMSO in salt water, 0 2 mg/ml potassium dichromate and different concentrations PMFM extract. b) Percentage survival of Artemia salina after 24 hours being treated with PMFM extract and exposed to H₂O₂(b).*Indicates statistically significantly difference (p<0.05) compared to 1% DMSO in salt water.

DISCUSSION:

Despite its widespread application in traditional medicine, there is a general dearth of exploratory studies regarding the antioxidant activity and toxicity of Phaleria macrocarpa. Therefore, in this study, DPPH radical scavenging activity, ferric reducing antioxidant power assay, and brine shrimp lethality assay were employed to evaluate the antioxidant and toxicity of the P. macrocarpa methanolic fruit (PMFM) extract.

In the present study, the PMFM extract exhibited good antioxidant activity, especially at the highest concentration used (0.5mg/ml). In the DPPH assay, the antioxidant capacity of PMFM extract was quantified by measuring the change of purple chromogen radicalsinto the corresponding light-yellow hydrazine (DPPH)^23,24. The discoloration of DPPH is proportional to the number ofelectrons gained, resulting in the presence of a free radical scavenger. The IC₅₀ value for the DPPH scavenging activity of PMFM extract was estimated to be 0.3612mg/ml, while the IC₅₀ for ascorbic acid was 0.00873mg/ml. According to classification by Blois et al.²⁵, samples with an IC₅₀ or EC₅₀ less than 50g/ml are considered very strong antioxidants, and a low IC₅₀ value indicates a significant radical scavenging activity. The analysis revealed that the extracts are capable of proton donation, resulting in their ability to function as radical scavengers. These results are in accordance with several authors who have demonstrated that P. macrocarpais an efficient free radical scavenger^24,26,27.

The FRAP assay was conducted to determinethe total antioxidant capacity of the PMFM extract. In this assay, the reducing power of a substance is determined through direct electron donation in the reduction of a ferric ion (Fe³⁺) to a ferrous ion (Fe²⁺).The results revealed that the ferric reducing antioxidant activity had a linear relationship with the PMFM extract concentrations. The present study demonstrated that the ferric reducing antioxidant activity was highest for 0.5mg/ml PMFM with 21.00±2.75µg AAE/µg of PMFM extract and 217.19±54.25 µg GAE/µg of PMFM extract.The extract showed a higher reduction capacity compared to ascorbic and gallic acids. The relationship between the concentration of PMFM extract and FRAP values observed in this study suggests that the extract possesses a high potential to reduce ferric ions, making it an effective antioxidant agent.The presence of bioactive compounds could possibly contribute to the good antioxidant activity of PMFM extract. The plant with high levels of phenolic compounds has been reported to possess higher antioxidant activity, which makes it a good candidate for the prevention of oxidative stress-induced cell injury^28,29. In the present study, the PMFM extract tested positive for the presence of saponins, tannins, and polyphenolic compounds,which might attribute to the reduction of DPPH free radicals and FRAP activity. However, further detailed analysis of the compounds present in the PMFM extract is required to determine the exact mechanism of protection.

Many studies have demonstrated that brine shrimp may suitably be used as an alternative model system, for screening the possible cytotoxic properties of drugs andchemicals²¹. Moreover, it has been established that the cytotoxic compounds generally exhibit significant activity in the brine shrimp lethality assay, and this assay also hasa good correlation with the human solid tumour cell lines³⁰. Results obtained from the present study demonstrated that incubation of brine shrimp nauplii, in a medium containing PMFM extract caused cytotoxicity in a dose dependent manner. The 24 h-LC₅₀ value of the PMFM was found to be 0.67mg/ml, suggesting that the plant extract is potentially toxic to brine shrimp nauplii. On the contrary, PMFM extract (0.0.5 - 0.2mg/ml) able to provide protection against oxidative stress induced by H₂O₂ in brine shrimp. The results demonstrated that the number of nauplii (pre-exposed to H₂0₂) that survived increased as the concentration of PMFM extract increased. Our findings suggested the potential protective effects of PMFM by reducing oxidative stress. However, due to the potential for PMFM extract to cause toxicity, the supplementation of a high dose of P. macrocarpa extract should be cautious as it might produce unwanted effects, particularly when this extract is taken for a long time.

CONCLUSION:

In conclusion, the present study demonstrated that PMFM extract possesses good antioxidant activity. Supplementation with low doses of PMFM extract prevented the H₂O₂-induced oxidative damage. However, further study is required to determine the exact mechanism responsible for the toxicity of this extract. Moreover, identification of theexact active compounds responsible for causing the toxicity of PMFM is also required. The findings from the present study may provehelpful in providing valuable insights into the potentialhealth hazards of using P. macrocarpa as a traditional medicinefor treating various illnesses.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGMENTS:

The research was financially supported by UniversitiTeknologi MARA through the Supervisory Research Grant (600-RMC/GIP 5/3 (128/2023)).

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Received on 03.07.2023 Modified on 19.10.2023

Research J. Pharm. and Tech 2024; 17(2):585-590.

DOI: 10.52711/0974-360X.2024.00091