DETERMINATION OF TOTAL PHENOLICAND FLAVONOID CONTENT AND EVALUATION OF ANTIOXIDANT ACTIVITIES OF CUSCUTA REFLEXA
Abul Kalam Azad1 , Farahidah Mohamed2
1Faculty of Pharmacy, University College of MAIWP International, 68100 Batu Caves, Kuala Lumpur, Malaysia.
2Advanced Drug Delivery Laboratory, Department of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University Malaysia (IIUM), 25200 Kuantan, Pahang, Malaysia.
Background: The aim of the study was to identify functional groups and antioxidant activity of the Cuscuta reflexa (C. reflexa) stems extract. Due to the reach of medicinal components, it has been using among Indian subcontinent people to treat various chronic diseases for ancient era. However, there are lacks of study on antioxidant activities of C. reflexa with its potential therapeutic compounds, therefore this study motivated to investigate more on its stems extract for further explore.
Methods: Soxhlet extraction method was employed to extract phenolic compounds from C. reflexa stems. The effect of extraction time (1-4 h) and concentration of ethanol (45%, 60%, 75% and 90%) on the percentage of yield, total phenolic (TPC) and flavonoid content (TFC) was investigated. The functional groups of phenolic compounds were characterized by using Fourier Transform Infrared Spectrometry (FTIR). DPPH and ABTS•+ radical scavengers were used to evaluate antioxidant activity.
Results: Data showed the highest percentage of yield (10.22±0.14% w/w), TPC (64.11±0.17%, mg GAE/g d.w.) and TFC (41.08±0.34%, mg QE/g d.w.) at 3 h with 75% ethanol. FTIR results revealed the presence of functional groups associated phenolic compounds which are the band at 1260 cm−1 illustrations the C–O groups of polyols existence and C – O –C of ester for presence of quercetin which represent the hydroxy flavonoids in the extract. DPPH and ABTS•+ radical scavengers were showed very potent antioxidant activity with IC50 295.12±1.33 and 245.43±0.78 μg/mL.
Conclusion: Phenolic and flavonoids enriched C. reflexa extract may play a potential role as a natural nutritional and therapeutic source in Bangladesh.
Keywords: Antioxidant, Cuscuta reflexa, flavonoid content, phenolic content.
INTRODUCTION
C. reflexa is one of the well-known parasitic plants which is frequently utilized as a nutrient ingredient in traditional herbal tonics, functional foods and in alcoholic beverages1-2. C. reflexa belonging to Convolvulaceae family and commonly known as amarbel or dodder or algushi or swarnalatha. It is an enormously found traditional medicinal herb grown in Bangladesh3. In addition, this plant has been found most in tropical countries like Malaysia, Thailand, Afghanistan, and Indian subcontinent such as Bangladesh, India, Pakistan, and Nepal4. Different sorts of phenolic constituents were found in C. reflexa extracts namely glycosides, sterols, terpenes, unsaturated and saturated fatty acids, saturated aliphatic hydrocarbon5-6. The stem extract of C. reflexa containing phenol (23.49%)7, kaempherol and quercitin8. Furthermore, many biological active compounds were isolated from this plant extract previously like myricetin, quercetin and kaempferol9, which possess mainly antioxidant and antidiabetic activities. Moreover, some therapeutically active compounds also been reported like coumarin, α-amyrin, astragalin, linoleic acid, palmitic acid, isorhamnetol, oleic acid, luteolin, stearic acid, ß-sitosterol, n-hentriacontane, and sesamin10. The 4-vinylphenol isolated from this plant which is using as a flavoring agent11. The stem extract of this plant showed potent antibacterial, antioxidant, anti-inflammatory activities3. In addition, diuretic, anti-viral and anti-cancer12, fever, diaphoretic, insanity, demulcent, melancholy, and fits effects also been claimed for traditional use13. Besides, several studies were investigated with the extract of C. reflexa for antioxidant14, antitumor15, anti-epileptic16, antihyper-tensive17, anti-arthritic and nephro-protective18, anti-obesity19, antispasmodic20, antibacterial and anti-fungal21-22, hypoglycemic, hemodynamic, and antiviral effects23-26.
Traditionally, people in Bangladesh and Nepal are consuming this plant or extract by using hot water decoction for the therapeutical benefits such as jaundice, tumor, skin infections, pain, and edema27. The present study was designed to extract from stems of C. reflexa using Soxhlet technique with different concentrations of solvent. Further, the concentrated extract was characterized by determine of total phenolic and flavonoids contents. The important functional group was identified using FTIR fingerprinting technique and determine in vitro antioxidant activity which leads this study’s novelty and important significance to audience.
METHODS
Sample collection
Fresh C. reflexa stems (500 g) were obtained from Dhaka district, Bangladesh in 2019. The plant was identified by a taxonomist in Bangladesh National Herbarium, Dhaka, Bangladesh with a voucher specimen (DACB Accession No. 41879). The stems were manually separated and clean with tap water. The plant sample was kept in a laboratory dryer chamber at 25°C until fully dried for one week. It was crushed by using normal a blender, separating uniform size of samples using a fine mesh strainer No. 100 (Sigma-Aldrich, St. Louis, Missouri, 63103, USA) in 0.149 mm. The separated fine sample powder was packed in a sealed bag for further usage.
Chemicals and reagent
Ethanol (99.5% purity) was obtained from Thermo Fisher Scientific (81 Wyman Street Waltham MA, 02454, USA), gallic acid, quercetin, Folin–Ciocalteu reagent, 2, 20-diphenyl-1-picrylhydrazyl (DPPH), sodium carbonate anhydrous, and aluminium chloride salt were procured from Sigma-Aldrich (Sigma-Aldrich, St. Louis, Missouri, 63103, USA) and all other reagents and chemicals used in the present study were of analytical grade.
Extraction of plant samples using Soxhlet technique
The C. reflexa stem powder sample (20 g) was placed in the Soxhlet extractor. Different concentrations of ethanol (30, 45, 60, 75, 90%) was employed with feed-to-solvent ratio (1:10, 1:15, 1:20, and 1:25 g/mL). The sample mixture was reflux by using heating mantle with different time point as 1, 2, 3 and 4 h26. After reaching the pre-determined extraction time, the extraction solution was left at 25°C to cool. The extraction solution was then filtered using filter paper and left in a beaker for some time. The mixture was further concentred to dry using a Buchi R-215 rotary evaporator (BUCHI Malaysia Sdn. Bhd. MY – 47301 Petaling Jaya, Selangor, Malaysia).
Determination of total phenolic content
The Folin-Ciocalteu reagent procedure was employed to determine the TPC of C. reflexa stem extract with partial modification of previous method described by Alara et al.,26. C. reflexa stem extract at 5 g/L (1 mL) and 200 μL of Folin-Ciocalteu reagent were mixed at room temperature. After 5 minutes, 0.2mM Na2CO3 (0.6 mL) solution was mixed well with the previously prepared mixture (Extract + Folin-Ciocalteu reagent). The absorbance was taken at 560 nm by UV-vis Spectrophotometer (Shimadzu UV-1800, Kyoto 604-8511, Japan). Subsequently, the mixture was kept at 25°C for two hours. TPC concentration of the extract was then measured using the gallic acid standard calibration curve (ranging from 50 to 500 mg/L). TPC was calculated as equivalent to mg GAE/g d.w. using below equation-
Total flavonoid content in the extracts
The quantitative determination of TFC for the extract of C. Reflexa was done by using the methods described in the studies of Alara et al.,26]. In brief, an aliquot of 100 μL (1 g/L) plant extract and the same volume of 2% AlCl3 (100 μL) solutions were vigorously mix. Afterwards, mixture solution was permitted to place at 25°C for 60 min. Then supernatant was collected from previous mixture, absorbance measured at 560 nm with UV-vis Spectrophotometer (Shimadzu UV-1800, Kyoto 604-8511, Japan). TFC was calculated using below equation-
FTIR analysis
To do FTIR, 1.5–2.0 g of plant sample was gently mixed with 200 mg of solid KBr and ground to make a pellet. The standard device was used to make pellets under vacuum and pressure (75 kN cm-2) for 2–3 minutes (Figure 1). These pellets were then used for spectral analysis of functional groups by FTIR. The spectral resolution was 4 cm-1 with a 400–4000 cm-1 scanning range27.
In vitro antioxidant activities of the extract
DPPH assay
Verification has been done on the hydrogen atom or donating ability of electron of extract from C. reflexa through DPPH assay3. Briefly, 200 µL of 0.1 mM DPPH was added with 200 μL of extract or ascorbic acid, respectively. The mixture was then serially diluted to different concentrations ranging from 100 to 500μg/mL. After incubating the mixture in a dark place for 30 minutes, the absorbance of the sample was measured at 560 nm. The IC50 of the sample was calculated through the sample absorbance, where each measurement was done as triplicate.
ABTS assay
Scavenging ability (ABTS) of C. reflexa extract or ascorbic acid was determined by previously described assay26. In brief, C. reflexa extract (150 μL) was serially diluted to different concentrations ranging from 100 to 500 μg/mL. Then, ABTS solution (285 μL) (2.45 mM potassium persul fate solution and 7mM ABTS) was added into the extract solution. After incubating the mixture in a dark place for 120 minutes, the absorbance of the sample was measured at 734 nm. Afterwards, the IC50 of the sample was calculated through the sample absorbance, where each measurement was done as triplicate. The data was represented as mean±SD. The ABTS radical scavenging activity was determined according to below equation-
Statistical analysis
Each experimental test was performed in triplicate. A statistical method analysis of variance (ANOVA, IBM, SPSS 20.0, Chicago, Ill., USA) with p<0.05 considered as significance difference.
RESULTS
Soxhlet Extraction
The determination of the recoveries of Soxhlet extraction was correlated with the yields of TPC and TFC. It is depending on the concentration of extraction solvent like ethanol and extraction duration. The extraction time and the concentration of ethanol are determined by the recoveries of extracts, TPC, and TFC from C. reflexa using the Soxhlet extraction technique. Duration of extraction is authoritative in minimizing cost and energy of the whole extraction process. Among the key factors, extraction time plays an important role in altering the recovery capacity of phenolic contents from herbal crude extract. The reason behind this is the overexposure of the plant sample on heating degrades the phenolic compounds. That is why, it is very important to fix the exact extraction duration to obtain maximum recovery of the targeted compounds. In this study, the Soxhlet extraction process was fixed with different concentration of ethanol at 1:10 g/mL feed-to-solvent ratio. The extraction time was varied as 1, 2, 3, and 4 hours. After completion of extraction time, the maximum yields of extract [Figure 1. (A)], TPC [Fig. 1. (B)], and TFC [Figure 1. (C)] were presented.
In this technique, the maximum yield was observed at 3 h with 75% of ethanol, therefore the TPC and TFC were also showed maximum quantity at the same time. However, 60% and 90% of ethanol extract exhibited almost equal quantity of TPC at 3 h. Interestingly, 45%, 60% and 90% of ethanol extract showed around same quantity of TFC at 3 h. Over all, the yields were declined drastically and it showed almost 0% at 4 h. Therefore, this study found that the % yields of extracts is directly correlate to the % yields of TPC and TFC and process also followed in time dependent manner.
FTIR
FTIR was used to determine the functional groups present in the plant extracts. The functional groups of the compounds were selected from obtained peak number in FTIR chromatograph. The representative absorption peaks value is reported in Figure 2.
The extract represents the phenolic component by the presence of a broad peak at 3471 cm−1 with O-H bending. Moreover, few peaks were found that can be attributed to the existence of lipid-carbohydrate in the sample due to the presence of lipids usually assigned in the peak area at 3000-2000 cm−1 region and 1500-1200 cm−1 represent for carbohydrate. In the extract, the peaks at 2123, 2963, 2960 and 2953 cm−1 specify the existence of lipids and 1449, 1428, and 1300 and 1260 cm−1 for carbohydrate. The band at 1260 cm−1 illustrations the C–O groups of polyols existence which represents the hydroxy flavonoids in the extract. In addition, the sharp peaks were found at 1085, 1010 and 1043 cm−1 that indicate the existence of secondary alcohols or ester groups in tested extract. Aromatic ring vibration could be associated with the peak value at 877 cm−1. Glycosides, flavonoids, and carboxylic acid could be associated due to the existence of C=O stretching and N-H bending that possible peak area at 1663 and 1607 cm−1.
In vitro antioxidant activity of the extracts
DPPH and ABTS•+ radical scavengers were used to examine the antioxidant properties of C. reflexa extract with maximum extraction operating conditions. The results were compared with standard (Ascorbic Acid) shown in the Table 1. It has been found in several studies that the lowest IC50 value indicates the strong antioxidant effect. In this sense, the extract showed potent antioxidant in ABTS•+ assay with IC50 at 245.43±0.78 μg/mL compared to DPPH assay which showed IC50 at 295.12±1.33 μg/mL. The obtained data indicated the potent antioxidant properties of the extract. Dissimilar scavenging activities were observed between DPPH and ABTS•+ assay with the same extract. It might be due to the different assay followed by the different pathways to exhibit the antioxidant activities.
DISCUSSION
There are numerous methods to recover of antioxidants enrichment of bioactive compounds from natural sources, i.e., cold maceration, Soxhlet extraction, supercritical fluid extraction, microwave assisted extraction, and ultrasound assisted extraction. However, percentage of yield extraction and antioxidant capacity not only vary on the extraction technique but also on the solvent used for extraction. The existence of a variety of antioxidant enrichment bioactive constituents with their polarities and diverse of chemical characteristics may or may not be soluble in a specific solvent27-28. Polar solvents are commonly employed for regaining polyphenols from plant materials. Although, methanol, acetone, ethanol, and ethyl acetate containing aqueous mixtures are considering the most suitable solvents for extraction of polyphenols. However, ethanol has been known as a good solvent for polyphenol extraction and is safe for human consumption29-30. Do et al., studied with 50%, 75% and 100% ethanol Soxhlet extracts, while 100% extracted sample showed the highest TPC at 40.5 mg gallic acid equivalent/g and TFC at 31.11 mg quercetin equivalent/g from Limnophila aromatica crude extract31. Baba et al., reported the TPC at 45.17±1.70 gallic acid equivalents/g and TFC at 35±2.20 rutin equivalents/g using100% methanol in Soxhlet extracted up-to 4 h32.The current study found that the 75% ethanol extract showed the highest amount of TPC (65.32±1.07 gallic acid equivalents/g) and TFC at 3 h. However, the content recovery was drastically reduced at 4 h due to the maximum recoveries was obtained at 2 h and 3 h. The similar finding was reported by Tanruean et al., with 65.45 mg GAE/g extract of TPC in acetone extract of C. reflexa33. The possibility of bioactive compounds reacts with extracting solvent expanded with increase amount of extraction solvent, leading to higher rates of contents34. However, the percentage of yields of antioxidant rich active bioactive compounds will not continue to increase once equilibrium is reached. The solid-to-solvent ratio could considerably affect the equilibrium constant and considered the correlation between yield and solvent use as a steep exponential increase followed by a steady state to give the maximum yield35. Moreover, 75% ethanol extract with the highest antioxidant activity was observed by Turkmen et al.36,37, which is in line with the current findings. Patle et al., reported the band obtained at 1520-1500 cm-1, 1449-1400 cm-1 and 1260-1200 cm-1 due to be NO2 bending vibration, C–O, and C–O –C of ester for presence of quercetin in the sample extract37. Moreover, CH2 asymmetric and symmetric stretching group was found at 2963 and 2853 cm-1 in the sample indicated ethanol37. FTIR data revealed the characteristic of the fingerprints of C. reflexa extract using the Soxhlet extraction process thatis reflected the existence of various functional groups related to flavonoids or polyphenols compounds.
Limitations of the study: This study completed FTIR finger printing to determine functional groups, TPC, TFC and their antioxidant activities. Due to some limitation, present research project was not able to cover some biological assays such as antibacterial, anti-fungal to increase the depth of this research. Therefore, this study recommends to do further study for more on biological assays.
CONCLUSION
The study found that the time of extraction and solvent concentration had major role in recoveries of extract, TPC and TFC in Soxhlet extraction process. C. reflexa extract exhibited potent scavenging activities compared to ascorbic acid, suggested its possible potential use as the natural antioxidant.
AUTHOR’S CONTRIBUTIONS
A.K.A, conception and design of the work; A.K.A.; acquisition, analysis, interpretation of data; the creation of new software used in the work; provided funding acquisition, project administration, and resources and A.K.A.; FM; wrote the paper A.K.A.; review, editing A.K.A.; FM; All authors have read and approved the manuscript.
ACKNOWLEDGEMENT
The authors extend their gratitude to the Faculty of Pharmacy, International Islamic University Malaysia for supporting the research work by facilitating the necessary requirements.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
REFERENCES