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Optimization protocol for the extraction of 6-gingerol and 6-shogaol from Zingiber officinale var. rubrum Theilade and improving antioxidant and anticancer activity using response surface methodology
© Ghasemzadeh et al. 2015
Received: 8 April 2015
Accepted: 11 June 2015
Published: 30 July 2015
Analysis and extraction of plant matrices are important processes for the development, modernization, and quality control of herbal formulations. Response surface methodology is a collection of statistical and mathematical techniques that are used to optimize the range of variables in various experimental processes to reduce the number of experimental runs, cost , and time, compared to other methods.
Response surface methodology was applied for optimizing reflux extraction conditions for achieving high 6-gingerol and 6-shogaol contents, and high antioxidant activity in Zingiber officinale var. rubrum Theilade . The two-factor central composite design was employed to determine the effects of two independent variables, namely extraction temperature (X 1 :50–80 °C) and time (X 2 :2–4 h), on the properties of the extracts. The 6-gingerol and 6-shogaol contents were measured using ultra-performance liquid chromatography. The antioxidant activity of the rhizome extracts was determined by means of the 1,1-diphenyl-2-picrylhydrazyl assay. Anticancer activity of optimized extracts against HeLa cancer cell lines was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
Increasing the extraction temperature and time induced significant response of the variables. The optimum extraction condition for all responses was at 76.9 °C for 3.4 h. Under the optimum condition, the corresponding predicted response values for 6-gingerol, 6-shogaol, and the antioxidant activity were 2.89 mg/g DW, 1.85 mg/g DW, and 84.3 %, respectively. 6-gingerol and 6-shogaol were extracted under optimized condition to check the viability of the models. The values were 2.92 and 1.88 mg/g DW, and 84.0 % for 6-gingerol, 6-shogaol, and the antioxidant activity respectively. The experimental values agreed with those predicted, thus indicating suitability of the models employed and the success of RSM in optimizing the extraction condition. With optimizing of reflux extraction anticancer activity of extracts against HeLa cancer cells enhanced about 16.8 %. The half inhibition concentration (IC50) value of optimized and unoptimized extract was found at concentration of 20.9 and 38.4 μg/mL respectively. Optimized extract showed more distinct anticancer activities against HeLa cancer cells in a concentration of 40 μg/mL (P < 0.01) without toxicity to normal cells.
The results indicated that the pharmaceutical quality of ginger could be improved significantly by optimizing of extraction process using response surface methodology.
Keywords6-Gingerol 6-Shogaol DPPH activity Zingiber officinale var. rubrum Theilade Response surface methodology
Herbs and natural products are precious sources of medicinal compounds and their benefits and importance for healing have been well recognized since ancient times. The characteristics and health effects of natural bioactive compounds, especially from plant sources including spices, have been extensively investigated. Phytochemicals are important compounds found in medicinal plants that are not essential for the normal functioning of the human body, but are active and exert positive effects on health or in amelioration of diseases. Many phytochemicals have been identified though a great many are yet to be identified . According to a report by the World Health Organization, 80 % of the population in developing countries depends on traditional medicine for their primary health care, and 85 % of traditional medicine is derived from plant extracts . In Malaysia, herbs and spices are generally consumed raw and fresh as vegetables (salad), especially by the Malay community. Ginger (Zingiber officinale Roscoe) is one of the most widely used spices in the world, especially in Malaysia and locally was known as Halia. Owing to its universal appeal, ginger has spread to most tropical and subtropical countries from China and India, where ginger cultivation has been prevalent, possibly since prehistoric times . In ancient times, ginger was highly valued for its medicinal properties and it played an important role in primary health care in ancient India and China. Ginger contains a variety of pungent and biologically active compounds, primarily 6-gingerol, 6-shogaol, zingerone, phenolics, and flavonoids . Between identified components, 6-gingerol was reported as the most abundant bioactive compound in ginger with various pharmacological effects including antioxidant, analgesic, anti-inflammatory and antipyretic properties [5–8]. The result of recent studies showed that 6-shogaol with lowest concentration in ginger represent more biologically actives compared to 6-gingerol [9–11] Dugasani et al.  reported 6-shogaol as a potent anti-inflammatory and antioxidant compounds in ginger. Various methods for the analysis of 6-gingerol and 6-shogaol in ginger extract have been reported [13–15] but, among these, high-performance liquid chromatography (HPLC) is most widely utilized. Extraction prior to component analysis is the main step for the recovery and isolation of bioactive phytochemicals from plant materials. Analysis and extraction of plant matrices are important processes for the development, modernization, and quality control of herbal formulations . In general, the first step of complete extraction is the selection of plant parts and careful preparation of plant extracts, and a thorough review of the existing literature to learn about the most suitable protocols for a specific group of compounds or plant species. Traditionally, the extraction of 6-gingerol and 6-shogaol compounds is accomplished by reflux or Soxhlet extraction . However, prolonged extraction at high temperature may degrade the 6-gingerol and 6-shogaol compounds, and involves high energy cost. Bhattarai et al.  recently reported that in acidic media and under high extraction temperature 6-gingerol can be degraded to 6-shogaol. Generally, the widespread use of ginger as a spice, dietary supplement, tea, cream, household remedy, as well as an ingredient of various herbal formulations, requires standardization of ginger formulations. A model for optimizing the most relevant operational parameters is required in order to achieve higher extraction yield. Response surface methodology (RSM) is a collection of statistical and mathematical technique that used to optimize the range of variables in various experimental processes with reducing the number of experimental runs, cost and time compared to other methods. Zingiber officinale var. rubrum Theilade is distributed mainly in Peninsular Malaysia, where it is known locally as halia udang, halia merah and halia bara. To the best of our knowledge, no other studies have been undertaken to optimize extraction condition of 6-gingerol and 6-shogaol from Z.officinale.var.rubrum Theilade. The aim of this study was to optimize the conditions for the extraction of a Malaysian ginger variety Zingiber officinalevar. rubrum Theilade namely Halia bara to achieve high 6-gingerol and 6-shogaol contents and high antioxidant and anticancer capacity by using response surface methodology with a central composite design (CCD).
Z.officinale var. rubrum Theilade rhizomes were collected from Bentong, Pahang, Malaysia. The samples were identified by herbarium of department of biology, faculty science, University Putra Malaysia. Rhizomes were washed with pure water and were soaked in a Mancozeb solution (0.3 %) for 30 min and were cut into 3–5 cm pieces containing 2 to 3 buds. After cutting, all pieces were planted 6 cm deep into the small pots filled with about 1 kg peat moss. Rhizomes were grown in a glasshouse for two weeks. Afterward, seedlings with 2 or 3 leaves were transplanted into polyethylene bags filled with a soilless mixture composed of burnt rice husk and coco peat (1:1). Ginger is a semi-shade loving plant and needs shade for growth and rhizome production. Then, the plants were grown under glasshouse conditions at the glasshouse complex of Universiti Putra Malaysia (UPM) where daily irradiance was approximately 790 μmol/m2/s (light intensity in outside was 1150 μmol/m2/s). Relative humidity was 70 ± 5 % and average temperature was 28 ± 1 °C. The plants were harvested after nine month, with the leaves, stems, and rhizomes separated. The rhizomes were shade dried and were powdered using grinder. These powdered materials were used for further analysis.
Levels of independent variables for reflux extraction condition based on CCD
X1: extraction temperature
X2: extraction time
Ultra High Performance Liquid Chromatography (UHPLC) analysis
The UHPLC system (Agilent, Model 1200) with Agilent C18 (4.6 × 250 mm, 5 μm) column was used for 6-gingerol and 6-shogaol analysis. In this system two mobile phases including: (A) water and (B) acetonitril (CAN) were used. The column temperature, flow rate and injection volume were adjusted at 48 °C, 1 mL/min, 20 μL. The UV absorbance was measured at 280 nm. To prepare the standard solution 6-gingerol and 6-shogaol (0.0625, 0.125, 0.250, 0.500 and 1 mg/mL) were dissolved in HPLC grade methanol. The linear regression equation were calculated with Y = aX ± b, where X was concentration of 6-gingerol and 6-shogaol and Y was the peak area of 6-gingerol and 6-shogaol obtained from UHPLC. Identification of the compounds was achieved by comparison of retention times with standards, UV spectra and UV absorbance ratios after co-injection of samples and standards. System suitability requirements: Perform at least five replicate injections of 6-gingerol and 6-shogaol. The requirements of the system suitability parameters are : (1) Symmetry factor (As) is not more than 1.5, (2) Percentage of relative standard deviation (RSD) of the retention time (tr) for 6-gingerol and 6-shogaol standards is not more than 2.0 %.
1,1-Diphenyl-2-picrylhydrazyl (DPPH) assay
Determination of anticancer activity
Retrieve the frozen cells from liquid nitrogen cell storage tank and thaw the cells in cyrovials rapidly. Carefully transfer the contents of the cyrovial to a centrifuge tube and add 10 ml of pre-warmed media slowly to the cell suspension. Spin down at 1000 rpm for 10 min and gently re-suspend pellet in 10 ml fresh media into culture flask. Incubate in 37 °C humidified incubator supplemented with 5 % CO2. After 24 h, the old medium was discard one day after seeding and adds 2-3 ml PBS to cover all surface and discard. Add 1.5-2 ml trypsining solution to cover the flask surface and leaved at room temperature for 3 min until most of the cells detach. Add 10 ml of complete medium. For MTT assay add 100 μl of cell into all the wells various concentrations of optimized (76.9 °C and 3.4 h) and unoptimized (80 °C and 4 h) extracts (10, 20, 40, 80 and 160 μg/mL) and incubate in 37 °C, 5 % CO2 incubator for 72 h. Prepare a stock solution of 5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in Phosphate buffered saline (PBS) and add 20 μL of MTT reagent to cell monolayer. Add 100 μl of DMSO (Dimethyl sulfoxide) to each well and mix thoroughly by pipetting 10–20 times to dissolved the blue formazan crystals. The absorbance of samples was read at 570 nm using ELISA reader.
Where Y is the predicted dependent variable; b 0 is a constant that fixes the response at the central point of the experiment; b 1 , b 2 , b 1 2 , b 2 2 , and b 1 b 2 are the linear, quadratic, and interaction coefficients, respectively. Graphical and numerical optimizations were performed to obtain the optimum conditions and predicted values for the response variables based on the response optimizer.
Results and Discussion
Effects of extraction conditions on 6-gingerol and 6-shogaol content
Experimental data and predicted content obtained for the dependent variables
6-gingerol content (mg/g DW)
Predicted content of 6-gingerol
6-shogaol content (mg/g DW)
Predicted content of 6-shogaol
Antioxidant activity %
Predicted content of antioxidant activity %
Regression coefficients, R2, adjusted R2 and F-values for dependent variables
Regression (F value)
Lack of fit (F value)
The result of previous study demonstrated that ß-hydroxy keto presence in gingerols structure is a sensitive to high temperature and promotes dehydration of gingerols at high temperature . The present results are consistent with observations by Bak et al.  who reported that when ginger was extracted at room temperature, the 6-gingerol content tended to decrease as the drying temperature increased up to 60 °C, whereas drying at 80 °C resulted in an increase in the 6-gingerol yield. The origin of the enhanced 6-gingerol content at 80 °C is not clear at this moment.
Effects of extraction conditions on antioxidant activity
Correlation between 6-gingerol, 6-shogaol and DPPH activity
Optimization of response
In order to obtain ginger extract with a high content of 6-gingerol, 6-shogaol, and high antioxidant activity, the optimal reflux extraction conditions were determined based on the combination of both responses. Multiple graphical and numerical optimizations were carried out to determine the optimum level for the independent variables with desirable response goals. One optimal condition was obtained for all responses, which was reflux extraction at 76.9 °C for 3.4 h. Under the optimum conditions, the corresponding predicted response values for 6-gingerol, 6-shogaol, and the antioxidant activity were 2.89 mg/g DW, 1.85 mg/g DW, and 84.3 % respectively.
Verification of the model
Verification of model for predicted optimum treatment conditions (78.9 °C and 3.8 h)
6-gingerol (mg/g DW)
6-shogaol (mg/g DW)
Antioxidant activity (%)
Evaluation of anticancer activity of optimized and unoptimized ginger extract
Response surface methodology was successfully implemented for the optimization of the experimental conditions for achieving high 6-gingerol and 6-shogaol content and antioxidant activity in ginger extracts. The results indicate that the extraction temperature and time affected the extraction yields of 6-gingerol and 6-shogaol significantly, and consequently the antioxidant activity of the extracts. Reflux extraction at 76.9 °C for 3.4 h was determined to be the most efficient condition for the extraction of 6-gingerol and 6-shogaol from Z.officinale var. rubrum Theilade rhizome to provide high antioxidant activity. Optimized extract showed a more distinct scavenging activity against the DPPH and also showed significant anticancer activities toward HeLa cancer cell lines in a concentration of 40 μg/mL without toxicity to normal cells.
The authors would like to thank the Ministry of Higher Education Malaysia and the Research Management Centre, Universiti Putra Malaysia (UPM) for sponsoring this work.
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