Comparison of Lipid Peroxyl Radical Scavenging Activity and total Phenolic content of four selected Citrus species leaves Extract
Ramli Salfarina*
Department of Pharmacology and Pharmaceutical Chemistry, Faculty of Pharmacy, Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA Selangor, 42300 Puncak Alam, Selangor, Malaysia
*Corresponding Author E-mail: salfarina2892@puncakalam.uitm.edu.my
ABSTRACT:
In this study, water and 80% methanol extracts of Citrus aurantifolia, C. hystrix, C. maxima, and Citrofortunellamicrocarpa leaves were prepared. The lipid peroxyl radical scavenging activity of the extracts was determined by β-carotene bleaching method, whilst their total phenolic content was assessed by Folin-Ciocalteu method. An interesting pattern was observed where the scavenging activity and total phenolic content of Citrus water extracts are higher than that of the respective methanolic extracts. Scavenging activity in water extract of C. maxima leaves is the highest (81±1.6%), followed by Citrofortunellamicrocarpa (79.7±2.3%). These activities are not significantly different from the positive control—butylated hydroxytoluene (BHT) (82.91±0.9%)—indicating strong antioxidant activity of both Citrus extracts. The highest total phenolic contents showed by water extract of Citrofortunellamicrocarpa leaves (585.7 ± 5.8 mg GAE/100 g of fresh weight citrus leaves), whereas total phenolic contents of other water and methanol extracts are below 400 mg GAE/100 g of fresh weight citrus leaves. Importantly, the scavenging activity in both water and methanol extracts are positively correlated with the phenolic content. However, the correlation of scavenging activity with phenolic content is stronger in the 80% methanol extracts (r= 0.605) than in the water extracts (r=0.46). In conclusion, among all the citrus extracts, Citrofortunellamicrocarpa water extract exhibited higher total phenolic content and lipid peroxyl radical activity. Correlation analysis infers that the free radical activity is not only contributed by phenolic content of the water extract.
KEYWORDS: Chain reaction, antioxidant.
INTRODUCTION:
As a consequence, many studies have been devoted to a search for potent antioxidant from plant materials. To date, numbers of extracts from spices, vegetables, medicinal plants, ornamental plants and by products of food industries have been screened for its antioxidant properties using in vitro assays. The antioxidant properties investigated include the ability to donate hydrogen, have reducing capability, chelate metal and scavenge free radicals2. Irrefutably, intake of food rich in antioxidants resulted in lower risk of oxidative stress related diseases, such as cancer and cardiovascular diseases. Dietary antioxidants inhibit the imbalance formation of detrimental free radicals that adversely affect normal cellular processes and physiological functions. This has been supported by findings that demonstrated free radical scavenging activity of compounds from fruits and vegetables3.
Plants from genus Citrus can be recognized most easily from its lime-like smell of fruit or crushed leaves. In spite of the ethnobotanical usages of Citrus plant parts, a number of Citrus species are important crops planted for its juicy fruits and essential oil. Along with that, Citrus is a rich source of pectin suitable for pharmaceutical product4 and phenolic compounds including polymethoxy flavonoids such as naringenin, tangeret in, nobiletin and are abundant in Citrus genus5. Four citrus that are known locally were selected for this study. C. maxima is known for its tasty giant fruit that is usually eaten fresh or used in fruit salads, the juice of Citrus aurantifolia, Citrofortunellamicrocarpa and leaves of Citrus hystrix are used in cooking for its flavor or as a condiment, while the rinds are used traditionally as body scrub to reduce body odor or dandruff. Citrus hystrix pulp contained more aromatic compounds than Citrus aurantifolia due to difference in terpenes content thus explains the different aroma of both citrus fruits6. Previous study reported rich contents of total phenolics, tannins, and total flavonoids from fruit juices of C. hystrix and C. maxima. The finding suggested both fruit juices as good source for antioxidant agents7. The content of phenolic compounds and the antioxidant capacity of peels and leaves from seven selected orange varieties were reported. It was found that presence of phenolic compounds in peels and leaves were different among the varieties. A study highlighted that leaves are rich in flavonoids, flavonols, polymerized phenols and hydrolyzable tannins; more than peels. The study concluded that citrus have high amounts of phenolics and their efficiency as potent antioxidants was confirmed8. The effect of different area of collection to the antioxidant activity of peel and leaves extract of Citrus aurantifolia was found inconclusive, however the peels and leaves were suggested as an accessible source of bioactive compounds useful for the preparation of food supplements or for nutraceutical products9.
From the previous studies and ethno pharmacology claims, plants from this genus are remarkable chemical factories with untapped potentials. In this study, a simple method of extraction was applied with aims to determine the lipidperoxylradical activity and total phenolic content of selected citrus leaves prepared in water and 80% methanol. The correlation between the activities with total phenolic content was also being evaluated.
MATERIAL AND METHOD:
Plant material:
The leaves of Citrofortunellamicrocarpa, Citrus aurantifolia, C. hystrix, and C. maxima were collected from home garden in Salak Tinggi, Selangor. All samples were authenticated by Prof. Dr. UmiKalsom Yusuf from the Department of Biology, Universiti Putra Malaysia, Serdang.
Extracts preparation:
The fresh collected leaves were cleaned under running tap water from any soil or dirt. Four grams of chopped leaves were extracted with 100 ml of distilled water or 100 ml of 80% methanol. Both extracts were agitated using orbital shaker for 2 hours at 60ᵒC. The extraction mixture was well decanted and filtered on paper.
Lipid peroxyl radical scavenging activity:
The activity was determined by adopted the β-carotene bleaching assay described by10. The experiments were performed in a minimal light environment with every apparatus used was covered in aluminum foil to prevent spontaneous oxidation. Butylated hydroxytoluene (BHT, 40 mg/ml) was used as a positive control, whilst 80% methanol was used as the control. β-carotene (0.2 mg) was dissolved in 10 ml of chloroform. A 3 ml of the solution was added into a flask with 60 µl linoleic acid, 600 µl Tween-20 and followed by 600 µl of 80% methanol, or crude extract or BHT. Chloroform was removed using rotary evaporator at room temperature for 10-15 min. Distilled water (150 ml) was added and mixed well to form an emulsion. Then, the solution was poured into test tubes and was immediately placed in a dry block for incubation at 50 ºC. The oxidation of the β-carotene emulsion was monitored using spectrophotometer (model Hitachi U2000) at 20 min interval at 470 nm for 120 min. The free radical scavenging activity was expressed as antioxidant activity (AA) using the following calculation:
AA= (Degradation rate of control - degradation rate of sample) / Degradation rate of control x100
Where; Degradation rate= ln(a/b) x l/t
Where;
ln = natural log, a = initial absorbance (470nm)
b = absorbance (470nm) at 120 minutes interval and
t = time
Determination of total phenolic content:
Total phenolic content was measured using Folin Ciocalteu assay11 with minor modification. Each of the citrus extract (0.1 ml) was added to 2 ml of 2 % Na2CO3. After 3 minutes, 50% (v/v) Folin-Ciocalteau reagent (0.1 ml) was added to the mixture and was mixed thoroughly before it was left for 30 min at room temperature. The absorbance was measured at 750 nm using a spectrophotometer. Gallic acid (Sigma) was used as a standard and the measurement of total phenolic content was based on the calibration curve of gallic acid (0.005 mg/ml to 0.1 mg/ml). Total phenolic content was expressed as gallic acid equivalents (GAE) mg/100 g) of fresh weight citrus leaves.
Statistical analysis:
All experiments were carried out in triplicates. A significant difference in antioxidant capacity and total phenolic content of studied samples was analyzed using one-way ANOVA. Comparison of the means was evaluated by Tukey post hoc test. The statistical probability was considered to be significantly different at the level of p<0.05. Data were also evaluated using Pearson’s correlation coefficients to identify relationships between phenolic contents and antioxidant.
RESULTS AND DISCUSSION:
This study observed the antioxidant activity of four Citrus leaves species prepared in water and 80% methanol from their ability to scavenge lipid peroxyl radical generated of linoleic acid from bleaching the β-carotene. In the assay, a spontaneous oxidation of linoleic acid promoted by thermal induction at 50 °C produced lipid free radicals that later act with oxygen to produce lipid peroxyl radical in the emulsion, thus oxidizing the β-carotene. The discoloration ofβ-carotene in the emulsion indicated oxidation process is taking place.β-carotene is known easily bleached in temperature, however the β-carotene bleaching rate was fast in the presence of linoleic acid and slow in its absence, shows linoleic acid oxidation generated lipid peroxyl radical12.The presence of antioxidants terminates the chain reaction propagated in the emulsified aqueous system. Figure 1 shows the lipid peroxyl radical scavenging activity of BHT.BHT is a synthetic antioxidant primarily used as food additive to prevent food from deterioration by oxygen (oxidation) and sunlight exposure.BHT scavenging activity was 96.21% at 20th minutes and decreased to 82.91% at 120th minutes (Fig. 1). It was noted that only about 13.3 % of BHT activity decreased within 120minutes, indicating the ability of BHT to maintain β-carotene from oxidation. Although the amount of lipid radical generated from the linoleic acid was not determined, but more lipid free radicals were assumed to be produced at 120th minutes compared to at 20th minutes resulting further discoloration of β-carotene. At 120 minutes, BHT has the highest antioxidant activity (82.91±0.9%) as befits a positive control. The free radical activity of citrus extract at 120 minute was compared with BHT. The scavenging activity of water extracts of all tested citrus is higher than that of the respective water extracts (Fig.2). Amongst the water extracts, C. maxima has the highest scavenging activity (3% lower than BHT activity), followed by C. microcarpa (about 4% less than that of BHT). The activities of methanolic extracts are between 20 and 25 % lower than that of BHT. Methanolic extract of C. aurantifolia has the lowest activity, which is 50% less potent than BHT. Interestingly, no significant difference (p>0.05) was found between the antioxidant activity of BHT and the antioxidant activity of C. maxima and C. microcarpa in water extract. These observations indicate that C. maxima and C. microcarpa in water extract are good sources of antioxidant with activity that as potent as BHT in scavenging lipid peroxyl radicals.
Fig.1: Mean of free radical scavenging activity of BHT at temperature 50ᵒC.
Although the factors that contribute to the scavenging activity of the extracts cannot be discerned from the free radical scavenging results, phenolic compounds have been frequently reported to be correlated with antioxidant activity of extracts from various parts of plants. Total phenolic content was significantly different (p<0.05) among Citrus extracts (Fig. 3). Water extract of C. microcarpa has the highest total phenolic content amongst the extracts which is about 0.5% of 100 g fresh leaves, followed by methanolic extract of C. hystrix. The lowest total phenolic content was showed by methanolic extract of C. aurantifolia. It was also observed that water extracts of citrus leaves have higher phenolic content than that of 80% methanol extracts. This result shows a higher extraction capacity of water for phenolic compounds from Citrus leaves.
Fig. 2: Lipid peroxyl radical scavenging activity of BHT and Citrus extract at 120thminute.
Figure 3: Total phenolic content of citrus leaves extracts. Values are means ± standard deviation
It was observed that both water extracts of C. microcarpa and C. maxima with high total phenolic content showed high scavenging activity. However, it was also noted that C. hystrixmethanolic extract with an exceptionally high total phenolic content of 387.3 mg GAE/100g fresh leaves showed low activity inmaintaining β-carotene from the oxidation. Therefore, correlation analysis was carried out to explore the trend of association between the free radical scavenging activity and total phenolic content from two different solvent of extraction. It was found that higher total phenolic content was correlated with higher antioxidant in 80% methanol extract, with the value of Pearson’s correlation coefficient, R=0.605indicating a strong relationship. A positive relationship was also found in water extract, with the value of Pearson’s correlation coefficient, R=0.46indicating a moderate relationship between antioxidant activity and total phenol content in citrus water extracts.
β-carotene bleaching assay is one of the oldest methods employed for estimating antioxidant activityin oil-in-water emulsion. It is among routine essays for antioxidant determination. Although the assay has been criticized as not presenting the actual event of lipid peroxidation in food or biological system, the assay is sufficient to evaluate lipid free radical scavenging activity of potential antioxidant substances, as the extend of bleaching indicates the oxidation of β-carotene by free radical. Consequently,a number of plant extract have been evaluated for its antioxidant activity using this assay13.
Interestingly, related plants of a same family or genus often shares similar types of phytochemicals, thus similar biological activity. However, a chemotaxonomy study on citrus leaves volatile oil from trees that were grown in the same pedoclimatic and cultural conditions revealed significant differences of metabolites level for sesquiterpenes, monoterpenes esters, and terpenes between the Citrus species14.Even among plants of same species, there are both biotic and abiotic factors that influence the distribution of phytochemicals in plants and later their biological activities. For example, there was a study that reported the effect of location, sessional variations and extraction solvent on the phytochemical and antioxidant activity of Ginkgo biloba leaf extract15. Moreover, extraction technique has to be taken into consideration as it resulted in varying profile of bioactive and associated biological activities16.Therefore, this study was carried out with anticipations that the lipid peroxyl radical activity of each different Citrus species will be greatly influenced by its phytochemicals, whereby the polarity of different solvents will have a significant consequence on the total phenolic content and free radical scavenging activity as well.
From the results, citrus leaves extracted with water showed better scavenging activity than those extracted with 80% methanol. Hightotal phenolic content was obtained from water extract compared to methanolic extract except C. hystrix. This indicates that water is a better solvent to extract phenolic compounds from C. microcarpa compared to 80% methanol, and the extractable compounds from water could be responsible for the radical scavenging activity of C. microcarpa extracts. The result of the study was similar to previous study where water was most effective solvent in preparing an extract of high total phenolic content and were found corresponding to their good biological activity17. Phenolic compounds are the vast majority of plant-based aromatic natural products that are often being associated with strong antioxidant potential. It constitutes of a large class of compounds comprises of simple phenols, flavonoidsand complicated structure of tannins. Some which ispolar made water a suitable solvent for extraction. For instance, phytochemical analysis of C. maxima water extract by High performance liquid chromatography with diode array detection (HPLC-DAD) detected and quantified gallic acid, catechin, caffeic acid, epicatechin, rutin and isoquercetin, and the major compounds detected from C. maxima leaves water extract wascaffeic acid. Those compounds are water soluble with antioxidant activity18 therefore are inferred as contributing to the observed free radical scavenging activity of C.maxima in this study.
A low total phenolic content of C. hystrixcould be due to the rich content of terpenes in C. hystrix leaves. Terpenes such as hemiterpenes, monoterpenes and sesquiterpenes are component of essential oils that are responsible for characteristic odor of fragrant plants. Limonene, α-Terpineol, 2β-Pinene, Terpinen-4-ol, γ-Terpinene, α-Terpinene, and α-Terpinolene are common terpenes in leaves of Citrus hystrix19.Those compounds are not water soluble and are commonly extracted by hydrodistillation. Nevertheless, the free radical scavenging activity of both C. hystrix water and 80% methanolic extract was not significantly different from each other (Fig. 2), suggesting a discrepancy if the seen free radical activity was contributed by its total phenolic content. According to correlation coefficients, total phenolic content seems to have contributed to the free radical scavenging activity of 80% methanolic extract but was not strongly correlated for water extract.
CONCLUSION:
The present study has demonstrated that both plant material and extraction solvent influenced the amount of total phenolic compounds and greatly affected their scavenging activity. Interestingly, a moderate correlation was found between TPC and the free radical activity of water extract of Citrus leaves suggesting that phenolic compounds are not the only contributors to the antioxidant activities of the extract. Results from this study indicated the effectiveness of Citrofortunellamicrocarpa and Citrus maxima water extracts as antioxidant in oil/water emulsion system. Since their activity is not significantly different from BHT, further study will be carried out to study the efficacy of both extracts in inhibiting oxidation of lipid in the oil-in-water emulsion foods.
ACKNOWLEDGEMENT:
The author would like to thank Dr Aedah Abu Bakar who has helped with the manuscript and Universiti Teknologi MARA for the support. A sincere appreciation goes to Prof Dr. UmiKalsom Yusuf for her kind guidance.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 12.10.2018 Modified on 17.11.2018
Accepted on 18.12.2018 © RJPT All right reserved
Research J. Pharm. and Tech. 2019; 12(4):1590-1594.
DOI: 10.5958/0974-360X.2019.00264.6