Effect of Boiling on Total Phenolic Content and DPPH Antioxidant Activity of DiabecineTM Water Extracts
Fadzilah Adibah Abdul Majid and Shahpour Khangholi etc
Abstract
DiabecineTM is a traditional herbal medicine for the treatment of diabetes among the community in Malaysia. It is consumed in a form of powdered herbal mixture packed in hard gelatin capsule. It is made of 5 medicinal herbs of temoelawak (Curcuma xanthorrhiza), cat whiskers (Orthosiphon stamineus), king of bitter (Andrographis paniculata), indonesian bay leaf (Eugenia polyantha or Syzygium polyanthus) and cinnamon (Cinnamomum zeylanicun). The determination of total phenolic compound (TPC) and free radical scavenging activity of this product is vital to indicate its functional and medicinal values. In this study, it was found that the value of TPC and DPPH radical scavenging activity of Diabecine™ water extract at 600C were 115.85mg/g GAE and 80% inhibition at 500mg/L respectively. However, Diabecine™ water extract at 1000C showed lower TPC of 84mg/g GAE while its DPPH value was similar. Boiling mode of extraction could have denatured some phenolic compounds in Diabecine™ water extract without affecting its free radical scavenging capacity.
Introduction
DiabecineTM is a traditional herbal medicine for the treatment of diabetes among the community in Malaysia. It is a proprietary formula of Nature Medic Supply and is marketed by Proliv Life Sciences Sdn Bhd. This product is currently under aggressive scientific investigation to validate its benefits and potential for medical application. It is made of 5 main herbs of temoelawak (Curcuma xanthorrhiza), cat whiskers (Orthosiphon aristatus), king of bitter (Andrographis paniculata), indonesian bay leaf (Eugenia polyantha or Syzygium polyanthus) and cinnamon (Cinnamomum zeylanicun). Based on the literature reviewed, all these five herbs are known scientifically to possess anti diabetes properties in their single form. The summary of review is presented in Table 1. Diabecine™ is consumed in a form of powdered herbal mixture packed in hard gelatin capsule (Figure 1). Recent finding has also indicated the potential of Diabecine as anti diabetes with multiple modes of actions to help lowering blood glucose (Abdul Majid et. al., 2012).
All these herbs are well researched and known for centuries for their safe use and with high antioxidant values (Table 2). However, the total phenolic content and the capability in scavenging free radicals of the herbal mixture is unknown. The value of TPC and DPPH radical scavenging are useful to predict the functionality and the medicinal value of DiabecineTM.
Diabecine™, being certified as herbal medicine under National Pharmaceutical Control Bureau, Malaysia is with low commercial value despite its great potential for diabetes treatment due to poor quality control over the source and quality of raw materials. In view of creating value and to safe guard the quality of this product, the development of new generation of Diabecine using standardized extracts is initiated in this study.
Aquous extraction is regarded as the safest extraction mode to extract phytochemicals for human application. In order to establish the most suitable extraction process for Diabecine™, to include the processing time, extraction with heating was selected. Heat was reported to influence the quality of the extract (ref). Traditionally, herbal decoction was either made by boiling or soaking in hot water like tea. Based on common practice by scientific community, boiling and heating at 600C are chosen in this study to prepare Diabecine water extract. The value of TPC and DPPH of both extraction modes were determined.
Materials and Methods
Materials
Bulk mix powder of DiabecineTM was directly supplied by Nature Medic Supply under the consent of Proliv Life Sciences Sdn Bhd. The powder was measured to the required weight according to extraction protocol. Powder was kept in the dark, cold dry place prior to use.
Safety note: Wearing safety gears such as mask, goggle, glove and lab coat are highly recommended while handling the powder material.
Extraction
Water extraction carried out at different temperatures; 600C and 1000C (boiling). Five hundreds gram of fine powdered of Jamu Diabetes was mixed with 1.5 liter of distilled water. The mixture was heated for 1 hour (extract 1000C) or heated at 600C for 3 hours (extract 600C). Then the mixture was filtered through muslin cloth. Extraction of the residue was repeated 2 times until a clear colorless liquid was obtained indicating that no more extraction was possible from the plant material.The extract was concentrated under vacuum evaporator at 80◦C. Final drying was done in oven at 45◦C. The extracts were preserved at -20◦C until utilization (Hena and Adamu 2010).
Total phenolic compound determination
Folin-Ciocalteu Method
Total phenolic content (TPC) of the extracts was determined based on the method described (Singh, Murthy et al. 2002). In brief, 5 mg of the Diabecine extract was dissolved in 10 (mL) distilled water or hydroalcoholic solution of methanol and water. 200 μL of extract solution was poured in each cuvette and then 1mL of 10-fold diluted Folin–Ciocalteu’s phenol reagent was added to each cuvette. After 5 minute a volume of 800 μL of 7.5% Na2CO3 were added to the mixture. Before readaing the 200 μL of each sample at 630 nm using microplate reader EXL800 the cuvettes were kept in dark place at ambient room temperature for 45 minutes. Blank sample was the same amount of reagents but water instead of extracts. Gallic acid solution in 8 different concentrations (300mg/L diluted 8 times) was used to prepare standard curve. In this regard the same amount of reagents that were used for extracts were applied for the gallic acid solution. The results expressed as gallic acid equivalent amount (GAEmg/g).
DPPH radical scavenging assay
Ability to scavenging free radical DPPH (2,2-diphenyl-1-picrylhydrazine1,1-diphenyl-2-picryl-hydrazyl is a fast and reliable way to assess the overall antioxidant capability of a compound (Aviram, Dornfeld et al. 2007). Evaluation of free radical scavenging capacity was performed according to Marinova and Batchvarov 2011((Marinova and Batchvarov 2011). Using dilution procedure 100 μl of decreasing concentrations of Diabecine extracts in range of 500 (mg/L to 4mg/L were prepared in 96-well microplate. 100μl of 0.2 mmol DPPH in methanol was added to each well. After that the mixture incubated at ambient temperature in dark place for 30 minutes. Then absorbance was measured at 490 nm using ELX800 micoplate reader. The control will be same volume of methanol instead of the extract into the DPPH solution. The percentage of antioxidant activity calculated using the following equation
%SC=Ac-As Ac ×100
Where SC is scacenging capacity and Ac and As are Absorbance of control and sample respectively.
Result and discussion
Two extraction methods were used in this study to yield Diabecine extract. These extracts were 1) water extraction at boiling temperature 1000C, and 2) water extraction at 600C. The yield of each extract was determined according Folin-ciocalteu method.
Total Phenolic Content of DiabecineTM
Folin-Ciocalteu method was used to estimate the amount of total phenols in the extracts. Gallic acid in known concentrations was employed to prepare standard calibration curve (figure 1). The amount of phenols in the test samples was estimated using equation Y=0.0086x-0.0186 with R2=0.9989 (Table1).
Figure 1: Gallic acid standard curve for estimating total phenol contents
The results of the experiments revealed that water extraction at 600C yields highest amount of the phenolic compound with a value of 115.85 (mg/g). The amount of phenolic compounds in water extract at 1000C was 84.29 mg/g GAE which could be related to the effect of temperature on extraction (Figure 2).
Figure 2: Total Phenolic Content of DiabecineTM in gallic acid equivalent value using different solvents
DPPH radical scavenging capacity
The results of the DPPH radical scavenging activity of Diabecine show that extract obtained from different methods possess high antioxidant value. The percentage of DPPH radical scavenging of water extract 600C was 83.86% inhibition while water extract 1000C was 81.84% inhibition at the concentration of 500 mg/L. Both extracts display a reductive potential. DPPH solution, which is dark violet in color, contains free radicals which simply accept an electron or hydrogen ion in reaction with proper reducing agents after quenching the color changes to yellow. This change is measurable via spectrophotometer absorbance at 517 nm antioxidant (Karagözler, Erdag et al. 2008).
According to the theory of the DPPH assay it is estimated that Diabecine water extracts contain substances with reducing properties which are able to quench the free radicals and terminate the radical chain reaction. This may be due to donating electron or H ion.
A large body of experiments have proved the relationship between polyphenolic content and the antioxidant assays. Figure 3 reveals the antioxidant activity of the Diabecine is adose-dependentmanner. Results demonstrate a positive correlation coefficient between the total phenolic content and DPPH assay of plants extracts in each solvent. The Pearson’s correlation coefficient for water extract 60, and water extract 100 are r=0.79 and r=0.81 respectively.
Figure 3: The correlation between total phenolic content and DPPH free radical-scavenging activity of Diabecine in different Solvents.
Despite the fact that water 60 had the most amount of the total phenolic compound (Figure 2) but didn’t show significant differences (Figure 4) with the DPPH scavenging capacity. However the least half maximal inhibitory concentration (IC50) was recorded for water 60 (40 mg/L) and water 100 (45 mg/L) and the last one was hydroalcoholic extract with 65 mg/L (Figure 5).
Figure 4: Comparison of DPPH radical scavenging between extracts of the different condition of extraction of Diabecene
Figure 5: Thehalf maximal inhibitory concentration of the Diabecine using different condition
In comparison with other solvents and with regarding to high correlation coefficient in methanolic solvent it seemed that, in hydralcoholic extract may be other compounds rather than polyphenolic compounds have contributed in antioxidant activity or may be Methanolic extract contains different profile of polyphenolic compound. It has been shown that the kind of polyphenol has important role in antioxidant activity which is related to their structure (Michalak 2006).
Conclusions
Based in the results it can be concluded that Diabecine possesses polyphenolic compounds with high antioxidant properties. Therefore Diabecine is potentially a source of natural antioxidant which can be considered to improve diabetes complication. Water extract at 60 C showed most amount of phenolic compounds but the antioxidant activity were not distinguish between solvents. Further studies are needed to elucidate other aspects of the Diabecine antioxidant activity.
Acknowledgement
References
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