Extraction yield

Natural plant extracts have been extracted from many years for various purposes by numerous methods. Nowadays these extracts have been evaluated as an alternative for remedies as well as preservatives. To evaluate the extraction yield of natural plant material, the experimental soxhlet extraction conditions were applied on the ginger by using ethanol, acetone and ethyl acetate as solvents along with supercritical fluid extraction by using 3000, 3300 and 3600 psi pressure at 40°C for 2 hours. The different in extraction yield is given below in Table 3. The yield was calculated for each treatment as the mass of extract divided by the mass of material used for extraction. The extraction yield increased with the time due to more availability of solvent as well as with pressure due to more incorporation of carbon dioxide into dry material. The highest extraction was observed for ethanol 2.39% followed by ethyl acetate 2.16% and acetone 1.84% at 120 minutes whilst, the lowest was for at 60 minutes for ethanol, ethyl acetate and acetone, 1.91%, 1.27% and 1.64%, respectively. For supercritical extraction treatments maximum extraction was observed at 3600 psi (4.52%) and lowest at 3000 psi (4.08%).

Table 3: Extraction yield (%).

The outcomes of this experiment are in line with the findings of Imm et al. [26] that determined the extraction yield of ethyl acetate ginger extract and concluded that extraction yield was 2.5% for 3.23 kg of ginger in one liter of ethyl acetate. Nonetheless, Naghia [27] evaluated the extraction yield of supercritical extract of ginger and suggested that the extraction yield increased by the addition of ethanol as co-solvent that was 4.5% without co-solvent and upsurge to 4.8%, 5.9% and 6% by the addition of 6%, 8%, 10% of ethanol at 45°C temperature and 250 bar pressure for 2.5 hours. He also observed that by increasing flow rate, extraction yield increased from 3.5%-4.5% by varying the flow rate from 5-15g/mL.

Furthermore, Mesomo with his colleagues [21], assessed the effect of pressure and temperature at the extraction yield of ginger supercritical fluid extract and recommended that by increasing the pressure the extraction yield increased up to a limit and after that it decreased owing to acceleration in vibrational motion of molecules and the bioactive entities started to decompose. By their research work they suggested that by changing the pressure from 10 to 25 MPa the extraction yield changed from 1.68 to 1.90 at 293.15 K however, at 313.15 K the yield was 0.24%, 1.88% and 2.62% by using 10, 7.5 and 25 MPa pressure. Afterwards, Marrelli et al. [28] worked on the phytochemical screeningof ginger pulp and peel extracts and concluded that the extraction yield was 2.3% in both ginger peel along with pulp. One of their peers, Gan et al. [29] performed their research work on separation and purification of 6-gingerol from ginger supercritical extract and proposed that extraction yield of supercritical fluid was 3.7% without co-solvent.

Phytochemical screening test

Phytodrugs are the secondary metabolites acting as antioxidants that prevent and decelerate oxidation reaction both as in vitro and in vivo by eliminating the chain reaction of oxidation. Many crude plant extracts along with some pure extracted compounds are stated to have antioxidant and free radical scavenging activity. The higher accumulation and/or formation of free radicals in the body have the ability to accelerate the rate of lipids oxidation that results in bad health. To overcome these complications herbs and spices are used in food to provide health benefits along with flavor. In this context, ginger proved its phytoentities having moderate antioxidant capability as the pharmacological drugs to enhance the treatment for disorders [30].

Superoxide anion free radical scavenging activity: Gingtion to other phenolic antioxidant tests have the ability to reduce oxidative stress markers. Regarding superoxide, the means for solvents (Table 4) i.e., ethanol, acetone and ethyl acetate were 54.20 ± 1.84%, 32.07 er based bioactive compounds, in addi± 1.03% and 46.22 ± 1.39%, correspondingly whilst means regarding to time effect were 42.17 ± 1.35%, 49.33 ± 1.58% and 40.98 ± 1.32% at 60, 90 and 120 min, respectively. Maximum superoxide anion radical scavenging 57.80± 1.97 was noted in ethanol extract at 90 min. In addition to this solvents, the supercritical ginger extract showed maximum activity at 3300 psi (78.13 ± 2.50%) followed by 3600 psi (72.96 ± 2.19%) and 3000 psi (69.24 ± 2.35%) (Table 5).

Table 4: Means for superoxide anion radical scavenging activity(%) of ginger extracts.Valueshaving different superscripts differ significantly.

Table 5: In vitro antioxidant indices (%) of supercritical ginger extracts.

Table 4: Means for superoxide anion radical scavenging activity(%) of ginger extracts.Valueshaving different superscripts differ significantly.

Nitric oxide radical scavenging activity: Nitric oxide radicals are produced by specific nitric oxide synthases in biological tissues which have the ability to metabolize arginine to citrulline via the formation of nitric oxide radical by involving five electron based oxidative reaction. Due to this reaction, at physiological pH i.e., 7.2 a compound namely sodium nitroprusside decomposed itself into aqueous solution and enhance the production of nitric oxide radicals. Although, under aerobic environment these radicals react with oxygen and convert into nitrate and nitrite, the stable compounds [7]. It is mostly used to assess the anti-oxidative markers that valued the antioxidant indices through free radical scavenging perspective of ginger. Means for nitric oxide (Table 6) depicted that free radical scavenging activity of ethanol extract was maximum 34.17 ± 1.16% followed by ethyl acetate and acetone extracts 28.81 ± 0.86% and 20.24 ± 0.65%, respectively. Among various ginger extracts, antioxidant activity of ethanol ginger extract was highest 38.23 ± 1.30% at 90 min whilst, lowest at 120 min 33.46± 1.14%. In supercritical fluid extraction of ginger maximum nitric oxide inhibition was 54.83 ± 1.75 at 3300 psi and lowest was 47.14 ± 1.68% at 3300 psi pressure (Table 5).

Table 6: Means for nitric oxide radical scavenging activity (%) of ginger extracts.Values having different superscripts differ significantly.

Hydroxyl radical scavenging activity: Mean hydroxyl radical scavenging activity (Table 7) regarding three solvents i.e., ethanol, acetone and ethyl acetate has revealed maximum activity 73.83 ± 2.51% in ethanol followed by ethyl acetate (64.56 ± 1.94%) and acetone (56.94 ± 1.82%). In the same way, time factor has also influenced hydroxyl radical scavenging rate of extracts that was highest at 90 min 67.80 ± 2.18% and lowest at 120 min 61.10 ± 1.96%. For hydroxyl radical scavenging of ginger maximum reduction was observed at 3300 psi 86.71 ± 2.77% and lowest at 3600 psi 77.30 ± 2.32% (Table 5).

Table 7: Means for hydroxyl radical scavenging activity (%) of ginger extracts.Values having different superscripts differ significantly.

Hydrogen peroxide: The presence of reducing agents accomplished with number and position of hydroxyl group also matter in this reduction mechanism to enhance antioxidant perception. Results (Table 8) have illustrated that ethanol extract has maximum H₂O₂ reducing power 47.09 ± 1.60 that was low in acetone extract 33.74± 1.12%. In the same way, significant effect was noted in time factor for each extract that was higher 45.01 ± .48% at 90 min while lowest 37.72 ± 1.21% at 120 min. Besides this the supercritical extract of ginger showed 69.36 ± 2.36% inhibition in hydrogen peroxide at 3000, 83.19± 2.66 at 3300 and 75.42 ± 2.26% at 3600 psi as depicted in Table 5.

Table 8: Means for hydrogen peroxide (%) of ginger extracts.Values having different superscripts differ significantly.

The finding of current research work are in accordance with the results of Dugasani et al. [31] who conducted the comparative study of antioxidant and anti-inflammatory effects of 6, 8, 10 gingerol along with 6 shogaol and suggested that superoxide anion radical scavenging activity of ginger extract was 5-40% by the addition of 1-6 μM ginger as a sample. Moreover, the hydroxyl radical scavenging activity and nitric oxide scavenging activity of ginger was 30-80 and 45-80% by using the same concentration of ginger sample. Moreover, another group of scientists, Amir et al. [32] investigated the phytochemical analysis and in vitro antioxidant analysis of ginger and depicted that the superoxide radical, hydroxyl radical, nitric oxide radical and hydrogen peroxide scavenging activity of ginger was 30-60% by using 10-50 μg/mL of ginger extract, 41-50% by addition of 5-25 μg/mL of ginger extract, 35- 50% by supplementation of 10-50 μg/mL of ginger extract and 35-65% by adding μg/mL of ginger extract, correspondingly.

Afterwards, Padamanabhan and Jangle [23], worked on the in vitro antioxidant potential of herbal blend by the addition of Aloe vera, Bacopa monniera, Moringa oleifera and Zingiber officinale and suggested that the superoxide, nitric oxide and hydroxyl radical scavenging activity of this blend was 19-66, 30-60 and 80-88% by the addition of 200-1000 μg/mL of that extract. At the same time, Oboh and his colleagues [33], worked on the antioxidant and inhibitory effect of red and white ginger and proposed that hydrogen peroxide and hydroxyl radical scavenging activity of ginger extract ranges from 10-40 and 10-70%, accordingly. At that juncture, Morakinyo et al. [34] insinuated that the superoxide anion radical scavenging of ginger extract ranges from 20-80% by addition of 10-500 μg/mL of ginger aqueous and ethanol extracts. Then and there another group of scientists, Kazeem et al. [35] determined the antioxidant and toxicological potential of ginger by using ethyl acetate, water and hexane as solvent and recommended the superoxide anion radical scavenging value of ginger ranges from 20-55%.

Nonetheless, Oranusi et al. [36] evaluated the antioxidant perspectives of some herbs and spices from Nigeria, amongst they concluded that hydrogen peroxide value as well as the nitric oxide value of ginger extract was 72.5-75.6 and 18.78-32.14% respectively by using different concentrations from 3-25 mg of ginger powder per mL of ethanol. Similarly, Jeena et al. [24] worked on the antioxidant potential of ginger essential oil and depicted that superoxide and hydroxyl radical scavenging activity of ginger extract was 25-65 and 3-40% by the addendum of 50-200 μg/mL of ginger extract. One of their peer, Ghosh et al. [5] assessed the in vitro antioxidant potential of ginger and believed that the superoxide and hydroxyl radical scavenging activity of ginger aqueous extract was 10-60 and 15-56% by the supplementation of 1-6 μM of ginger extract.

At the moment, Lin with his colleagues [22], conducted a chemical study of [6] gingerol for its protective effect against hydrogen radical and superoxide anion radical scavenging activity and depicted that IC₅₀ for hydrogen peroxide was 70.39 whilst, for superoxide it was 228.40. Recently, Nile et al. [37], evaluated the chromatographic, antioxidant and anti-inflammatory activities of ginger extracts and reported that by using 1 μg/mL of ethanol and ethyl acetate extracts the IC₅₀ for hydrogen peroxide was 20 for ethyl acetate and 34 for ethanol extract. Nevertheless, Marrelli et al. [28] performed a research work on ginger pulp and peel for its phytochemical analyses and advocated that by the application of 1 mg/mL of ginger extract the nitric oxide radicalscavenging activity was 36.74% for pulp however the ginger peel did not show any activity against nitric oxide scavenging.

Quantification by high pressure liquid chromatography

HPLC (High Performance Liquid Chromatography) quantification of the powdered ginger revealed that among all the pungent ingredients 6-gingerol was four times more intense. The resultant peaks obtained from HPLC regarding to 6-gingerol were inferred with the help of standard spectral exploration, peak area and retention time. HPLC assessment for ginger (Table 9) proved that the highest gingerol concentration was present in supercritical extracts as 7.81 mg/g in 3300 psi pressure, 6.19 mg/g at 3600 and 5.63 mg/g in 3000 psi followed by ethanol, ethyl acetate and acetone 5.74 mg/g, 4.56 mg/g and 3.20 mg/g respectively.

Table 9: HPLC quantification for gingerol (mg/g) in ginger extracts.

The current findings are in harmony with the outcomes of Aly et al. [38] who characterized ginger by high performance liquid chromatography (HPLC) and concluded about the quantity of 6-gingerol. They suggested that the quantity of gingerol varies in rhizome, roots and shoots as the highest quantity of 6-gingerol was found in rhizome (42.64 mg/g) followed by shoots (7.46 mg/g) and roots (6.40 mg/g). Furthermore, Naghia [27] worked on the supercritical extraction of ginger oleoresin by using specific flow rate and co-solvent and depicted that the by quantifying the supercritical extract of ginger by HPLC ranged from 179 mg/100 g-221 mg/100 g. After that, another group of scientists, Anisa et al. [39], evaluated the quantity of 6-gingerol and 6-shogaol in ethanolic extract obtained by soxhlet apparatus by HPLC and resolved that the quantity of 6-gingerol was 8406.996 μg/g as well as 716.760 μg/g for 6-shogaol.

Nonetheless, Sonale et al. [40] did research work on the characterization of gingerol and its analogues in supercritical fluid extracts and recommended that the HPLC percentage portion of 6-gingerol was 30-35% in crude ginger extract and 75-79% in purified supercritical extract. One of their peers, Cho with his colleagues, [41] performed analysis of 6-gingerol in Zingiber fresh, powder and in different commercial products by using HPLC and concluded that the 6-gingerol content in root of Zingiber officinale ranged from 13.24± 0.55-17.09 ± 0.10 mg/g. Moreover, Guo et al. [42] conduct a research to evaluate the decomposition of 6-gingerol during processing and concluded that during processing gingerol decreased and shogaol content increased. Amongst all gingerol series, 6-gingerol decreased to 2.11 ± 0.2 mg/g from 9.72 ± 0.03 mg/g that was the concentration of 6-gingerol in unprocessed fresh ginger.

Even so, Murthy et al. [43] determined the composition of ginger oleoresin along with its bioactivity and usage as bio-preservative. In this research work they concluded after soxhlet apparatus extracting of ginger oleoresin by using acetone, 6-gingerol is the chief bioactive ingredient that accounts up to 12.80% in ginger extract. Recently, Ito et al. [44] determined the composition of ginger extract and quantified it through RP-HPLC by using water and acetonitrile as mobile phase in gradient system under UV detector (280 nm) against standards of 6-gingerol and 6-shogaol. Conclusively, they suggested that in fresh ginger extract the main ingredient was 6-ginger (110 mg/g) followed by a lesser amount of shogaol (5 mg/g).

Conclusively, supercritical extract of ginger showed maximum health boosting properties by highest inhibition of free radical. In previous literature many papers are present related to redox potential of conventional extract however it is the first paper describing redox potential of supercritical extract. The quantity of bioactive moiety decreased at higher pressure due to increase in vibrational motion within molecule. This paper will ensure the usage of supercritical based nutraceutical in routine diet.

Conflict of Interest

No external funding was applied to this project.

This work was carried out under Pak-US Science and Technology project for establishment of Functional and Nutraceutical Research Section at University of Agriculture, Faisalabad-Pakistan. The financial and technical assistance under the framework of this project is highly acknowledged.