INTRODUCTION:

Ginger, scientifically named Zingiber officinale Rosc., is a plant that grows in hot, tropical areas and belongs to the Zingiberaceae family. Phytochemicals are bioactive substances derived from plants. Many plant-based chemicals have been researched for their potential health effects in humans^1,2,3,4. The main components found in ginger rhizomes are phenolic compounds, terpenes, polysaccharides, fats, acids and fibers⁵.

Ginger contains natural chemicals called terpenes, such as β-bisabolene, zingiberene, α-farnesene, β-sesquiphellandrene, and α-curcumene. It also has phenolic compounds like gingerol, paradols and shogaol ². Gingerols are the main substances found in both fresh and dried ginger rhizomes. The quality of gingerol is influenced by other compounds like gingerol, zingerone and eugenol present in the ginger rhizomes. Among gingerol compounds, 6-gingerol is the most abundant form². Gingerols have various beneficial effects on health, such as reducing inflammation, antioxidant, preventing cancer, relieving pain, protecting the stomach, strengthening the heart, reducing fever, inhibiting blood vessel growth, preventing blood clotting and lowering blood sugar level^6,7. There is a high demand for 6-gingerol because it has low toxicity and promising medicinal properties⁶. 6-Shogaol, which is derived from gingerols through dehydration, is found in small amounts in fresh ginger roots but is more abundant in dried or heated rhizomes⁷. 6-Shogaol has various biological effects like antibacterial, preventing fouling and acting as an antioxidant⁶. On the other hand, 8- and 10-gingerol are spicy components and important active substances in ginger rhizomes but they are present in very small amounts in fresh ginger rhizomes⁸.

In the global marketplace, the unit price of gingerol, a bioactive compound found in ginger, has reached $270 per kilogram. According to export data, India has been recorded a total export value of USD 7,117,500, corresponding to a quantity of 109,500 kilograms of gingerol during the year 2016-17. Despite eastern India being the second-largest region in terms of ginger production, the average productivity and quality remain unsatisfactory due to the unavailability of necessary high-gingerol (≥2%) genotype variations. The production of gingerol is significantly influenced by environmental conditions. Therefore, achieving an optimal yield and quality depends only on cultivating genetically better-quality of Z. officinale. The best way to analyse the chemical compounds in ginger is by using high-performance liquid chromatography. HPLC is a fast and easy method used to separate, identify, and measure the compounds found in ginger rhizomes^9,10,^11.In spite of its high sensitivity, specificity, and accuracy in determining analyses in biological fluids and analytical media, this method is only used for quality control analysis¹². A new way to use HPLC has been developed to study gingerol levels in ginger rhizomes grown in fields and in the laboratory^13,14,15.Balladin and Headley concluded that the HPLC method works well for measuring and identifying gingerols extracted from dried gingerrhizomes¹⁶. Additionally, this HPLC method is better because it has low retention times, is very sensitive and gives accurate results¹⁷. This method can help to evaluate, detect and estimate the quality of ginger roots from various regions in eastern India with different climates and agriculture conditions.

MATERIALS AND METHODS:

Chemicals and reagents:

We obtained authentic [6]-shogaol, [6], [8] and [10]-gingerol from Sigma-Aldrich in Missouri, USA. The solvents used, including water, Methanol and Acetonitrile, were of HPLC-grade quality and sourced from Smart Biolab, Bhubaneswar. The purity of all standards exceeded 98%, as determined by high-performance liquid chromatography analysis.

Plant Materials, Extraction procedure of sample preparation:

Twelve ginger rhizome samples were collected from various districts of Odisha (Table 1). The fresh rhizomes were collected between April and June, the harvesting period for Z. officinale. The ginger rhizomes will be left to air dry at room temperature, then ground using a grinder. A total of 100 grams of powdered ginger rhizome was used for extraction with a Soxhlet apparatus (Borosil Glass Works Limited, Worli, Mumbai, India) at 40ºC for 8 to 9 hours. The extract was then filtered using Whatman No. 1 filter paper and concentrated at room temperature. All extracts were stored at 4ºC for further analysis.

Preparation of standard solutions:

We combined enough high-quality methanol with 5 mg of [6]-gingerol, 10 mg of [8]-gingerol, 10 mg of [10]-gingerol, and 5 mg of [6]-shogaol (Figure 1), which are all standard chemicals from Z. officinale. These mixtures were stored at 4°C.

A.6-gingerol B.8-gingerol

C.10-gingerol D.6-shogaol

Figure 1. Chemical structures of the ginger compounds.

Table 1. Collection of ginger rhizomes from different geographic locations in Odisha.

SL. No.	Agro climatic Zones	Climate	Agricultural Districts	Accession no.	Latitude	Longitude	Altitude	Year of Collections
1	East and South Eastern Coastal Plain	Hot and Humid	Jagatsinghpur	G1	20.3079°N	86.4517°E	48	2023
				G2	20.3174°N	86.5274°E	4	2023
				G3	20.3145°N	86.4554°E	5	2023
				G4	20.3100°N	86.3820°E	7	2023
2	North Eastern Ghat	Hot, Moist and sub-humid	Rayagada	G5	19.1046°N	83.3490°E	91	2023
			Gajapati	G6	13.5833°N	79.3167°E	2551	2023
			Gajapati	G7	18.7655°N	84.0973°E	145	2023
			Ganjam	G8	19.4840°N	84.3936°E	433	2023
3	Eastern Ghat High Land	Warm and humid	Koraput	G9	18.4848°N	82.4244°E	888	2023
3	Eastern Ghat High Land	Warm and humid	Koraput	G10	18.3378°N	82.5789°E	1002	2023
4	South Eastern Ghat	Warm and humid	Malkangiri	G11	18.4225°N	82.5111°E	897	2023
5	Mid Central Table Land	Hot, Moist and sub-humid	Angul	G12	20.6710°N	85.0999°E	120	2023

Chromatographic system and HPLC analysis:

The Shimadzu HPLC system used for the analysis had several parts: an SPDM20A diode array detector (DAD), an LC-20AT binary pump, a CBM-20A lite system controller, a SIL-20A auto sampler, a DGU-20A5 degasser, and a filter. It also included an Agilent TC (2) C18 column (250 × 4.6mm, 5µm), which was kept at the controlled temperature by a CTO-10ASvp column oven. The mobile phase used during the analysis was a mixture of acetonitrile and HPLC water in a 40:60 volume ratio, with a flow rate of 1.0mL/min. 272nm was the wavelength at which detection was performed, and injection volumes ranged from 5 to 20µl. The column oven on the system was set up to operate at 40°C for a total of 60 minutes. The samples were analysed along with the standard compounds [6], [8], [10]-gingerol and [6]-shogaol. Their amounts were determined by comparing the results to a calibration curve

Sample analysis:

The method developed by Wang et al. was used to analyse the four compounds in twelve samples^.8. The quantities of all compounds were determined using their calibration curves. The peaks in the samples were identified by checking their retention times and UV spectra against those of known reference standards.

RESULTS AND DISCUSSION:

Optimization of extraction procedure:

In this study, powdered of Z. officinale rhizomes were tested with various combinations of solvents, including methanol, ethanol and chloroform, at different extraction durations (figure 2) (2h, 4h, 6h and 8h) to optimize the extraction procedure. These were analysed through reflux extraction method (using soxhlet apparatus) with temperature 50°C. Out of 3 combinations of solvents, methanol is the suitable solvent for the extraction procedure with 6h of extraction time. According to literature, methanol is the most effective solvent in extracting higher yields of Zingiberis rhizome extract as compared to other common solvents^18,19. Although, ethanol is commonly used for extraction, the contents obtained were inconsistent, relatively low in comparison to methanol^20,21. The extraction rate that could be possible to achieve from a particular material is measured by solvents²². Bioactive compounds can be extracted more efficiently from samples with high extractive solvents. Bioactive substances with low extractive solvent results are expected to have less of an impact on bioactive compound identification. Using ultrasonic extraction for 20 minutes provided the best outcomes, to optimize the extraction method, whereas the Soxhlet method is the most effective technique for extracting active ingredients from fresh ginger roots and ginger powder extracts using various solvents^23,24. The selection of the reflux extraction method (using soxhlet apparatus) was based on its simplicity and comparable results across different solvents.

Figure 2. A Content of different extraction method B Total time differences of four gingerols in zingiberis rhizome.

Optimization of Chromatographic conditions:

Chromatographic analysis was conducted using different combinations of mobile phases: water-methanol, water-ethanol, and water-acetonitrile. The optimal separation conditions were subsequently determined by testing these phases using different gradient programmes. When compared to water-methanol and water-ethanol, the water-acetonitrile combination showed the best separation and peak shape among of all three. Similar results have been found by Wang et al⁸. Different chromatographic parameters, such as column temperature (kept at 40°C) and flow rate (between 0.5 and 1.0mL/min), were carefully optimized. The initial efforts focused on finding the best concentration of acetonitrile. The best separation results were achieved using the Agilent TC (2) C18 column (250mm × 4.6 mm, 5μm) at a temperature of 40°C and a flow rate of 0.5mL/min. Within the given 60minutes for analysis, distinctive peaks representing the examined compounds were successfully separated based on the optimized chromatographic settings that were supplied. The most common analytical technique for component separation and quantification is HPLC, which is most usually used along with a UV detector^25,26,27. This technique, usually integrates UV absorbance, fluorescence, or UV absorption detectors to a mass spectrometer, is the most widely used for determining heterocyclic amines^28,29. However, in contrast to our results, HPLC analysis of various natural products in other studies utilized an octadecylsilane (ODS) column with methanol as the common mobile phase, instead of acetonitrile (ACN) and water of HPLC grade, which were selected as alternative mobile phases for analysing [6], [8], [10]-gingerol, and [6]-shogaol³⁰. Previous studies on [6], [8], [10]-gingerol and [6]-shogaol in ginger using high-performance liquid chromatography (HPLC) have mainly used a C18 column (250 × 4.6mm, 5µm)³¹. The chromatograms in (Figure 3) show the peaks for the twelve samples and the four standard compounds. The peaks were identified by comparing the retention times of the gingerols to those of the reference compounds separated under the same conditions.

Calibration of standard curve:

A change in location can affect a variety of parameters; including temperature, humidity, rainfall and geographical features which can lead to changes in agro-climatic conditions. Previous studies showed that variations in temperature and topography may affect the agricultural production as well as its quality. To create the calibration curves, at least four different concentrations of [6], [8], [10]-gingerol, and [6]-shogaol were measured (0.1, 0.26, 0.36, and 1.0μg/mL)^{30, 31} (Figure 4). The calibration figure was calculated using these concentrations, showing linearity in the 1–1000 μg/mL range. The linear regression equation (y =mx + c) was applied, in which x denotes the sample amount and y is the peak area. The formulas LOD = 3.3σ/s and LOQ = 10σ/s were used to calculate the limits of detection (LOD) and quantification (LOQ). In these formulas, σ represents the standard deviation of the intercept, and s represents the slope of the calibration curve. The following were the obtained LOD and LOQ values for the standards: LOD (0.032, 0.086, 1.439, 0.025 μg/mL) and LOQ (0.097, 0.263, 0.180, 0.076 μg/mL), respectively.

Figure 4. The amount of compounds in the ginger rhizome from different regions.

Determining the amounts of four compounds in twelve samples from various locations.

The quantities of four bioactive compounds found in each of the twelve samples were identified using the HPLC technique. It was found that the twelve samples, which were collected from three different agro climatic zones of Odisha, had metabolic diversity. In our investigation, we observed varying levels of four significant bioactive compounds of Z. Officinale rhizome in different places. Sample G-3 had the highest overall concentration with 1.518 mg/ml of 6-gingerol, 0.878mg/ml of 8-gingerol, 0.828mg/ml of 6-shogaol, and 0.897mg/ml of 10-gingerol. In contrast, G-12 had the lowest amounts; 0.024mg/ml of 6-gingerol, 0.024 mg/ml of 8-gingerol, 0.002mg/ml of 10-gingerol, and 0.034mg/ml of 6-shogaol (Table 2). These findings underscore the variability in bioactive compound levels across different ginger samples, emphasizing G-3 as having the highest overall content and G-12 as having the lowest (Figure 5). According to the literature, ginger has the highest concentration of 6-gingerol, which ranges from 1.030 to 3.046mg per gram of ginger rhizome. In addition to 6-gingerol, ginger contains 8-gingerol in lesser amounts ranging from 0.078 to 0.0425 mg/g³². Another group of scientists reported that the gingerol content in the aqueous extract of dried ginger ranged from 1.17 to 2.08mg per gram³³. The concentration of gingerol was measured in twelve different varieties of ginger. Gingerol was determined using acetonitrile and water as the mobile phase^34,35. This made it possible to validate the ginger methanolic extract and determined that 2.10mg/g of 6-gingerol was present in the extract; small quantities of 8- and 10-gingerol were also found. Dried ginger had 29.2% of bioactive compounds overall^36,37. Subsequently, focused on the production and analysis of ginger extracts based on methanol and hexane^37,38. They found that while the retention period was the same at 738 minutes, the methanol extract had a greater peak at 25%, while the hexane extract had a peak of 235nm. Similarly, a C18 column with a flow rate of 1 mL/min was used to analyse the methanolic extract of dried ginger powder by HPLC, using acetonitrile and water as the mobile phase^39,40. Their report indicated that the methanolic extract of ginger contained 30 mg of gingerol per gram. There are some further studies which focused on different parts of the ginger crop^41,42,43. They used ultra-sonication to extract the ginger extracts for their research, and HPLC was used to quantify the results. The results of their study showed that the level of 6-gingerol in ginger roots was 2.98±0.06mg/g, while the concentration of gingerol in ginger leaves was 18.83± 0.28mg/g when acetonitrile was used as the mobile phase. The separated ginger extract contained 1.93–3.57 mg/g of gingerol based on the ginger profile. This high gingerol content gives it a strong and original flavour, which is important for enhancing the quality of spices and food products^44,45,46. Their experiment showed that when acetonitrile was used as the mobile phase, the concentration of gingerol in ginger leaves was 18.83± 0.28mg/g, while the level of 6-gingerol in ginger roots was 2.98±0.06mg/g. The ginger profile in this context and observed that the strong, distinctive pungency of the distinct ginger extract can be used for spices and food products to improve both flavour and taste as it contains 1.93–3.57mg/g of gingerol.

Our study highlighted the significant variations in the bioactive compounds of Z. officinale rhizomes across different regions, revealing the complex impact of agro climatic conditions on the phytochemical profile of ginger. A change in location can affect a variety of parameters, including temperature, humidity, rainfall, and geographical features indicative of the particular agro climatic zone. This could result in changes to the agro climatic conditions. Previous studies have shown that variations in agro climatic conditions and topographical change can affect agriculture in terms of quality and quantity^47,48,49.

A [6]-gingerol

B [8]-gingerol

C [10]-gingerol

D [6]-shogaol

Figure 5. Standard calibration curve of [A] represents [6]-gingerol, [B] represents [8]-gingerol, [C] represents [10]-gingerol and [D] represents [6]- shogaol.

Table 2. Quantitative detection of [6], [8], [10]-gingerol and [6]-shogaol.

Compound	Retention time, t_R(in minutes)	Peak Area	Oven temperature	Flow rate
6-gingerol	14.987	8652671	40°C	1ml/min
8-gingerol	25.206	1155797	40°C	1ml/min
10-gingerol	32.252	215400	40°C	1ml/min
6-shogaol	27.122	38614141	40°C	1ml/min

CONCLUSION:

The results of the investigation reveal that the four compounds were effectively separated and analysed using the HPLC analysis method: [6]-shogaol with [6], [8], and [10]-gingerol. The Soxhlet method proved to be the most effective technique for extracting active ingredients from fresh ginger rhizomes and ginger powder extracts using various solvents. Compared to ethanol and chloroform, methanol was the most effective solvent that we studied at maintaining the active components. The current findings indicate that the levels of the four main bioactive compounds in ginger rhizome are ranked as follows: 6-gingerol > 10-gingerol > 8-gingerol > 6-shogaol. The greater variation can be attributed due to the diverse ecological conditions, soil compositions, and weather patterns in these agro climatic regions of Odisha. This study not only contributes to the scientific understanding of ginger phytochemistry but also has practical implications for farmers, industries and researchers involved in harnessing the therapeutic benefits of this Z. officinale plant. More advanced research should be explored by the underlying mechanisms governing the variability in gingerol content and to elucidate the specific environmental factors influencing the phytochemical profile of Z. officinale. These agro climatic regions further can be recommended for large-scale ginger production having better gingerol content.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors are grateful to Prof (Dr). Sudam Chandra Si, Dean and Prof (Dr). Manoj Ranjan Nayak, President, Centre of Biotechnology, Siksha O Anusandhan (Deemed to be University), for providing all facilities.

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Received on 13.04.2024 Revised on 10.07.2024

Accepted on 23.10.2024 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1702-1708.

DOI: 10.52711/0974-360X.2025.00244

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