Rhizomes of fresh Zingiber officinale and crude ginger extracts were purchased from a local market and herbalist in Jordan.

Gallic acid, quercetin, caffeic acid, rutin, and naringin hydrate were purchased from Geochem World (Mumbai, India). Reagents Include sodium nitrate (NaNO₂), and aluminum chloride anhydrous (AlCl₃) from Alpha Chemika (Mumbai, India). Sodium hydroxide pellets (NaOH) were supplied by Loba Chemie, (Milan, Italy), and the Folin-Ciocalteu reagent was purchased from Merck (Darmstadt, Germany). The solvents ethanol, methanol, ethyl acetate, dichloromethane, and n-hexane were purchased from Carlo Erba Reagents (Milan, Italy), and chloroform from Alpha Chemika (Mumbai, India). All reagents are of analytical reagent grade.

Digital balances (ADAM equipment PGW 453i, P.R China) were used for weighing purposes before and after the extraction of ginger different extracts. Buchi Rotavapor R-300 (Flawil, Switzerland) was used for both methods; maceration, and Soxhlet extraction methods. UV-visible spectrophotometer UV-1800 (Shimadzu, Japan) was used for the determination of absorbance, HPLC/UV, Stirrer, Sonicator, as well as water bath (Thermo Scientific) for the determination of TPC, and SYKAM.

Fresh Zingiber officinale were washed thoroughly to remove any trace amount of soil, then cut into pieces before grinding it in a blender. After that, stored in the refrigerator at -4 °C until required for extraction. The crude powdered ginger extract was bought from a local herbalist for the further extraction process. In this study, we use two methods for the extraction which are maceration and Soxhlet extraction methods using seven different solvents (ethanol, methanol, ethyl acetate, water, chloroform, dichloromethane, and n-hexane). After obtaining the extracts, the resulting solution was concentrated using a rotary evaporator (rota-vap) under the required conditions for each solvent at a certain vacuum pressure and temperature. Finally, the obtained crude extract was dried, weighed, and stored at -4 °C until use.

It’s well known that extraction is the primary step for the separation of the required natural extracts from raw materials. The plant extraction method is to prepare the rhizome of Zingiber officinale different extracts and different crude extracts of ginger using the Soxhlet and maceration extraction methods. In brief, the maceration extraction method was conducted by weighing 100 g and then soaked in 1 L of each solvent separately (ethanol, methanol, ethyl acetate, water, chloroform, dichloromethane, and n-hexane) in a container covered with aluminum foil for 72 hours with occasional shaking at room temperature (RT) in a dark condition. After that, the whole mixture was filtered in a conical flask and vaporized in a rota-vap, then dried and weighed for the final sample preparation (Abubakar and Haque 2020).

Soxhlet extraction method which is considered a high-efficiency automatic continuous extraction technology that takes less time and uses less solvent than other methods (Zhang et al. 2018). In brief, 40 g of fresh rhizome of Zingiber officinale was placed into the thimble and 200 mL (1:5) of the solvent to be used for the extraction was placed in the round bottom flask before fixing the thimble into the Soxhlet apparatus, then the heat is applied depending on the solvent extract. The temperature was adjusted to 90 °C for 9 hours of extraction for the solvents with agitation utilizing the magnetic stirrer at 400 rounds per minute (rpm). Lastly, the extraction is collected in the round bottom flask and applied into the rota-vap to remove any remaining solvent from the extraction. The same protocol was performed for the crude ginger extract with 30 g of crude ginger extract and 150 mL of solvents (1:5).

Two techniques, Maceration (MAC) and Soxhlet (SOX) apparatus were utilized to extract the active ingredients from fresh and powdered Zingiber officinale utilizing seven solvents including (ethanol, methanol, water, ethyl acetate, chloroform, dichloromethane, and n-hexane). Therefore, to select the best extraction yield of Zingiber officinale using different solvents, the following equation was used:

% Extractive Value of Solvent = (Weight of the extract / Weigh of the sample) × 100

Total phenol content (TPC) was measured using the Folin-Ciocalteu method by Wolfe et al. First, a dilution for each plant extract was done (10 mg/mL) with its corresponding solvent. Then, 4 mL of 7.5% sodium carbonate was added, mixed for 15 seconds in a vortex, and allowed to develop its blue color in the water bath at 40 °C for 30 minutes. Finally, calorimetric analysis at 765 nm was used to determine the phenolic concentration. The TPC was expressed as a gallic acid equivalent (GAE) (mg/g) using equation based on the calibration curve.

The amount of total flavonoids was determined using the Saima et al. method. In brief, 10 mL volumetric flask, 0.1 mg/mL plant material extracts from each plant were diluted with 4 mL of water. Each volumetric flask was first filled with a 5% NaNO₂ solution of 0.3 mL for five minutes, followed by 10% AlCl₃ (w/w) for six minutes. Next, the addition of 2 mL of NaOH (1.0 M) to the volumetric flask and 2.4 mL of distilled water (DW) was followed, then thoroughly mixed in the reaction flask. At 430 nm, the reaction mixture’s absorbance was measured. The results were calculated in mg quercetin/g dry weight (Ghasemzadeh et al. 2010).

In the experiment, we investigated the ideal 9 hours of extraction in SOX apparatus, the extraction of active substances from ginger was higher than MAC for 72 hours for both fresh and powdered ginger. Additionally, SOX extraction time is shorter than the MAC technique which gives us a positive response in reducing the time consumption in the preparation process. The benefit of this extraction technique is to shorten the extraction time and achieve high active ingredients (Figs 2, 3).

Figure 2. 

% Yield of extracts obtained from fresh ginger rhizome using different solvents and methods of extraction, G1: Maceration, G2: Soxhlet.

Figure 3. 

% Yield of extracts obtained from ginger powder using different solvents and methods of extraction, G1: Maceration, G2: Soxhlet.

According to the results, the Soxhlet method is the best technique for isolating active ingredients from fresh ginger rhizomes and ginger powder extract by using different solvents. In addition, the results of fresh ginger shown in Fig. 2 for the MAC method in the ethanolic extract is low compared to the SOX method, and other shows some conflicting results, this could be an indication of the presence of water molecules in fresh ginger so this factor could affect on the extraction yield of the active substance in ginger. For instance, dichloromethane, chloroform, and n-hexane reveal significant variations, as demonstrated in Fig. 2, with MAC yielding more than SOX.

The results in powdered ginger extract in Fig. 3 show that the maceration method exhibits minimal extraction of the active ingredient, except for dichloromethane solvent the results are in the range of 3.3 in MAC and 3.2 in SOX. In this study, we used both types of ginger fresh and powdered, the high active components are achieved in powdered ginger rather than fresh ginger.

According to the results in Table 1, SOX shows the best extraction technique rather than MAC when compared with the time consumption. Moreover, the privilege of the usage of SOX apparatus is that it offers the best extraction yield for both types of fresh and powder ginger according to the result obtained.

According to Arawande et al., solvents are a measure of the potential extractive rate that could be obtained from a given sample; high extractive solvents are estimated to be effective in extracting bioactive compounds, while low extractive solvent results are estimated to have less impact on the identification of various bioactive compounds.

As a result, we have used seven solvents for the two types of ginger rhizome; fresh and powdered. Additionally, we used two extraction techniques (MAC and SOX), each of which has a unique extractive value. As demonstrated in Tables 1, 2, we can conclude from this experiment that SOX delivers the greatest outcome for ginger extraction for both types.

Comparison research of extraction yield by different extraction methods by Nourbakhsh Amiria et al., including MAC, SOX, subcritical water extraction (SWE), and further methods were utilized. The best yield was in SOX than MAC, and the operation time was 8 hours instead of 14 hours for the MAC method. In this study, a greater extraction yield for fresh ginger was obtained in methanol than chloroform in MAC, while in SOX the highest yield was developed in ethanol followed by methanol (Table 1). For powdered ginger in the MAC technique, the best result was shown in water followed by chloroform, as well as in the SOX technique also water then methanol (Table 2), respectively.

Phenolics have been acknowledged as a necessary component of a daily diet. In addition, their antioxidant properties because of their useful capacity to improve health (Tanweer et al. 2020). The calibration curve of gallic acid was constructed by mixing 500 mg in 50 mL of methanol as a stock solution with a concentration of 10 mg/mL, The stock solution was serially diluted (25 to 250 mg/mL). Based on the calibration curve, TPC was expressed as a gallic acid equivalent (GAE) (mg/g) using the following equation y = 0.0026x + 0.0317, R² = 0.9974, where Y is the absorbance and X axis is the concentration.

In each working test tube, 0.15 mL of plant solution was mixed with 5 mL Folin-Ciocalteu 10% reagent diluted with DW 1:10 v/v, and 4 mL of 7.5% sodium carbonate for 15 seconds in a vortex mixer, in the water bath at 40 °C for 30 minutes until color turns to blue. Then, the phenolic contents were determined calorimetrically at 765 nm. TPC for each extract was determined as GAE according to the linear regression equation obtained from the calibration curve of standard gallic acid.

Based on the research that has been done, the highest TPC for both types of Zingiber officinale fresh and powdered extraction are shown in Table 2, where by MAC starting from the fresh ginger was ethyl acetate, followed by n-hexane. As a result, in powdered ginger the highest phenolic content was found in ethanol, followed by methanol. Moreover, the highest TPC by SOX beginning with the fresh ginger was ethyl acetate followed by methanol. In addition, in powdered ginger the highest TPC was found in ethyl acetate-tracked methanol.

The findings of the current investigation are shown in Table 2. In fresh ginger the content varied within the range of 20 to 252.08 mg of GAE values in the MAC and SOX method, the highest TPC was found in ethyl acetate for the MAC and SOX were 252.08 and 131.6 mg/g in mean GAE values. Followed by n-hexane in the MAC technique where the value was 103.6 mg/g.

On the other hand, Ezez and Tefera studied a comparison result of solvents used to extract the ginger rhizome. In addition, the outcomes show that the high polarity solvents yielded extracts with the largest concentration of phenolic compounds, while methanol extract demonstrated a stronger ability to extract phenolic compounds from the ginger rhizome.

In the research work, a new trend was discovered, which indicates that fresh ginger has the second-highest TPC for low-polarity n-hexane. In the SOX technique, the second-highest phenolic content was in methanol the mean GAE values were 128.3 mg/g, respectively. Table 3 lists more results.

In powdered ginger among the solvents used, the highest TPC was found in ethanol using the MAC technique, with a value of 142.91 mg/g. Additionally, for the SOX technique, the highest TPC was discovered in ethyl acetate at a value of 110.83 mg/g in mean GAE values. Therefore, different intrinsic and extrinsic factors, including cultivars, type of soil and growing circumstances, maturation state, and harvest conditions, may be responsible for the variation in the amount of TPC (Ezez and Tefera 2021).

In Figs 4, 5 a summarized chart for the two types of Zingiber officinale, MAC is a higher result than SOX in two sources of ginger fresh and powdered with some exceptional solvents. As well as a literature review reported that the highest polarity of the selected solvent manifested a greater power to extract phenolics from ginger (Ezez and Tefera 2021). In this experiment, we have obtained a high value of n-hexane in fresh ginger, as we know that n-hexane has a low polarity. In addition, other literature data Eberle et al., studied two different extraction techniques MAC and SOX using dry and fresh ginger for ethanol solvent and the result was high in SOX rather than MAC, unlike our recent study of ethanol for fresh and powdered ginger were higher in MAC with a value of 42.08 and 142.91 mg/g.

Figure 4. 

Total Phenol Content (TPC) of fresh ginger in maceration and Soxhlet method for different solvents using GAE as a mean.

Figure 5. 

Total Phenol Content (TPC) of powder ginger in maceration and Soxhlet method for different solvents using GAE as a mean.

Polyphenolic compounds are known to have antioxidant activity, and the extracts’ activity is likely related to these compounds. This activity is thought to be mostly owing to their redox characteristics, which are significant in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, and degrading peroxides (Ghasemzadeh et al. 2010). Antioxidants like polyphenols are indeed present in high concentrations in many medicinal plants. Quercetin, for example, has anticancer properties and has been demonstrated in prior research to be able to stop the growth of cancer cells (Ghasemzadeh et al. 2010). Gallic acid has been shown to act as a free radical scavenger, stimulate differentiation, and trigger apoptosis in leukemia, lung cancer, and colon adenocarcinoma, as well as in healthy lymphocyte cells (Sohi et al. 2003). Gallic acid, like quercetin, is thought to be crucial in preventing cancer development and malignant transformation. Therefore, the findings of this study demonstrated that flavonoids are significant parts of this plant, and some of its pharmacological actions could be linked to the presence of these valuable constituents. These results are supported by the high cytotoxicity activity (IC₅₀<50 µg/mL) against HT-29 colorectal adenocarcinoma obtained (Table 9). These findings are in line with the literature, where Shailah et al. demonstrated that HT 29 CRC cells were more susceptible to the anticancer activity of the dried ginger extract, with an IC₅₀ (455 μg/mL) significantly lower than HCT 116 cells (496 μg/mL).

One of the substances that fall within the category of phenolic or polyphenol compounds is flavonoid compounds. Of all polyphenols, flavonoids have undergone the greatest research. This group consists of basic structure of two aromatic rings that are connected by three carbon atoms to create an oxygenated heterocycle (Maulana et al. 2019).

About 800 mg of quercetin was dissolved in 100 mL of ethanol. After that, serial dilution of concentration from 15 to 700 mg/mL was applied. The UV-visible device was used to measure the absorbance at 430 nm and the calibration curve was plotted using standard quercetin. The TFC of fresh and powdered ginger was calculated as mg of quercetin equivalents/mL using the following calibration curve equation y = 0.0009x + 0.023, R² = 0.9949, where Y is the absorbance and X axis is the concentration.

The flavonoid contents of Zingiber officinale extracts were quantified as a starting point. 1 mL of solvent extracts was diluted with 4 mL of DW and added to a 10 mL volumetric flask. 0.3 mL of 5% NaNO₂ solution was added for 5 minutes, followed by 10% AlCl₃ (w/w) for six-minute and 1.0 M NaOH (2 mL). Then, 2.4 mL of DW was added, and the absorbance of the reaction mixture was evaluated at 430 nm. By comparing the outcomes to the Quercetin standard curve, the results were given in mg quercetin/mL (Ghasemzadeh et al. 2010). The data were plotted as a chart scale for the two types of ginger fresh and powder in Figs 6, 7.

Figure 6. 

Total Flavonoid Content (TFC) of fresh ginger in maceration and Soxhlet method for different solvents using quercetin as a mean.

Figure 7. 

Total Flavonoid Content (TFC) of powder ginger in maceration and Soxhlet methods for different solvents using quercetin as a mean.

According to the research, TFC has shown various data trends compared to TPC. In fresh ginger, the results were from 530 to 26.67 mg/g Quercetin in the MAC method, while in SOX were by 406.67 to 10 mg/g, respectively. Dichloromethane and ethyl acetate had the highest TFC of Zingiber officinale extraction in the MAC technique for fresh ginger the results are explained in Table 3. As well as, in SOX the ethyl acetate and dichloromethane had the highest content. In this work, we indicate that the content could be affected by the polarity of the solvent used as discussed in previous literature data, as shown in Table 3.

When we used the MAC approach, the results of ginger powder ranged from 700 to 30 mg/g Quercetin, while those obtained from the SOX method ranged from 546.67 to 25.67 mg/g. Ethanol in MAC and ethyl acetate had the greatest levels of total flavonoid, respectively. On the other hand, ethyl acetate and dichloromethane had the greatest levels of flavonoid content in SOX the same result as fresh ginger. The advantages of employing the two extraction methods include learning the best method to utilize for extraction. In Table 3 the data are shown for the best solvents among other solvents for the TFC in powdered ginger.

Ethanol results from MAC and SOX were (700 and 513.3 mg/g), ethyl acetate (504.4 and 546.67 mg/g), and dichloromethane was (164.4 and 530 mg/g) so the MAC technique had manifested a greater result in ethanol, while for ethyl acetate the results were approximately close to the range. In addition, dichloromethane shows a crucial effect on the content of flavonoids in the SOX technique that offers a triple-fold increase in flavonoid content than MAC (Figs 6, 7). Further experimental work may be needed, but in comparison with the method, both were good to find TFC in plants extracted using these two solvents which also suggests a good polarity.

The MTT assay is a method used to assess cellular metabolic activity which in terms to indicates cell viability, proliferation, and cytotoxicity (Fig. 9). The concentrations vs. Survival% values were plotted, and the IC₅₀ value for the 28 ginger extracts was measured. Results were summarized (Table 9).

Figure 9. 

MTT cell proliferation assay Antiproliferative.1FM: Ethanol in fresh ginger MAC,2FM: Methanol in fresh ginger MAC, 3FM: Water in fresh ginger MAC, 4FM: Ethyl acetate in fresh ginger MAC, 5FM: Chloroform in fresh ginger MAC, 6FM: Dichloromethane in fresh ginger MAC, 7FM: n-hexane in fresh ginger MAC. 1PM: Ethanol in powder ginger MAC, 2PM: Methanol in powder ginger MAC, 3PM: Water in powder ginger MAC, 4PM: Ethyl acetate in powder ginger MAC, 5PM: Chloroform in powder ginger MAC, 6PM: Dichloromethane in powder ginger MAC, 7PM: n- hexane in powder ginger MAC. 1FS: Ethanol in fresh ginger SOX, 2FS: Methanol in fresh ginger SOX, 3FS: Water in fresh ginger SOX, 4FS: Ethyl acetate in fresh ginger SOX, 5FS: Chloroform in fresh ginger SOX, 6FS: Dichloromethane in fresh ginger SOX, 7FS: n- hexane in fresh ginger SOX. 1PS: Ethanol in powder ginger SOX, 2PS: Methanol in powder ginger SOX, 3PS: Water in powder ginger SOX, 4PS: Ethyl acetate in powder ginger SOX, 5PS: Chloroform in powder ginger SOX, 6PS: Dichloromethane in powder ginger SOX, 7PS: n- hexane in powder ginger SOX.

All 7 powdered ginger extracts extracted by maceration showed high cytotoxicity activity (IC₅₀<50 µg/mL) against HT-29 colorectal adenocarcinoma. Similarly, all powdered Ginger extracted by Soxhlet showed high cytotoxicity activity, except those extracted using water, which showed mild cytotoxicity activity (IC₅₀ 100–200 µg/mL).

In contrast, fresh ginger extracted by maceration, using methanol or n-hexane showed a lack of cytotoxicity activity. Only fresh extract using dichloromethane showed high activity. While extracts of ethanol, water, ethyl acetate, and chloroform showed mild cytotoxic activity.

Furthermore, fresh ginger extracted by Soxhlet using ethanol, methanol, and n-hexane solvents showed no cytotoxic activity against HT-29 colorectal adenocarcinoma (IC₅₀>200 µg/mL). On the other hand, ethyl acetate extracts of fresh ginger showed strong cytotoxicity activity (IC₅₀<50 µg/mL). While fresh extracted by water, chloroform, and dichloromethane showed mild cytotoxic activity.

These results are consistent with the literature, where Shailah et al. showed that HT 29 CRC cells were more vulnerable to the anticancer activity of dried ginger extract with an IC₅₀ (455 μg/mL) much less than HCT 116 cells (496 μg/mL). The cytotoxic activity is probably due to the presence of gingerol which is known to inhibit the growth of HCT 116 CRC cells and liver HepG2 cancer cells. Similarly, Al-Tamimi et al. found that ginger exhibited the lowest IC₅₀ among other studied plants, with 40 μL/mL on mucus-secreting HT-29 and 60 μL/mL on non- mucus-secreting HT-29 cells. In this work, the anticancer efficacy of Zingiber officinale extract shows a clinically significant, according to the outcomes at a safe dose that is clinically promising to the patients with minimal side effects.