Introduction

India is the world’s third-largest producer of citrus fruit, trailing only China and Brazil, which has serious social repercussions [1]. Citrus fruits are well-known for their nutritional and therapeutic properties, which are due to their high phytochemical content. Certain citrus species, such as galgal or hill lemon, are, however, largely unknown outside of local populations, leading to underutilization [2]. These plants include a plethora of bioactive substances, including polyphenols, antioxidants, carotenoids, dietary fibre, vitamins, and minerals [3]. Unfortunately, a lack of scientific research on the nutritional composition and health advantages of these foods has hampered their marketing and integration into modern enterprises [2,3,4].

Citrus fruits have been linked to a reduced risk of various chronic diseases owing to their abundance of bioactive phytochemicals [5, 6]. L-ascorbic acid (vitamin C), is a crucial physiologically active component found in many citrus fruits and vegetables. Vitamin C has a wide range of health benefits, including antioxidant, anticancer, and cardiovascular protection [7]. Polyphenols are the most prevalent phytochemicals in fruits and vegetables, consisting of phenolic acids, flavonoids, proanthocyanidins, stilbenes, and lignan [5, 8, 9]. Extensive research has established polyphenols’ diverse health benefits, including antioxidant, anticancer, anti-diabetic, anti-aging, and neuroprotective properties [10]. Furthermore, many fruits contain volatile chemicals that have been studied for their cardiovascular-protective qualities [11]. As a result, a thorough understanding of the phytochemical makeup and bioactivities of fruits and vegetables serves as a scientific framework for investigating their potential health benefits [12].

Various citrus fruits have been explored potentially for their remarkable bioactivity and industrial applications, however galgal fruit (Citrus pseudolimon) is an underutilized citrus fruit resource due to its scarcity and a lack of scientific proof about its nutritional worth and health advantages [13]. As a result, this wild fruit is not generally marketed or used in modern enterprises. Traditionally, in various parts of the Indian subcontinent, Citrus pseudolimon is considered an important medicinal plant [3, 12, 13]. Therefore, the current study was carried out to evaluate the physicochemical features, phytochemical composition, seed oil profiles, mineral content, volatile component profile, and antioxidant activity of galgal fruit in order to address this. Despite few studies existing on Citrus pseudolimon characterization, however, analysis of the fruit by employing various analytical techniques such as gas chromatography mass spectrometry (GCMS), GCMS-Headspace and inductively coupled plasma optical emission spectroscopy (ICPOES) is not reported. Thus, this study inferred a complete analysis of the underutilized fruit Citrus pseudolimon which could result in effective utilization in various food industries. Overall, the findings of this study will make it easier to use galgal fruit in the development of functional products for the food, nutraceutical, and cosmetic industries in the future.

Materials and Methods

Raw Materials

In the months of November and December, the ripened fruits were obtained from three distinct Indian states: Punjab, Himachal, and Haryana. The galgal from Himachal was acquired from the YS Parmar University of Horticulture and Forestry in Solan, India; the galgal from Punjab was bought from a fruit research farm run by the Punjab Agricultural University (PAU), Ludhiana, India; and the galgal from Haryana was procured from the CCS Haryana Agriculture University, Hisar, India. The fruits were sorted according to maturity and kept in a refrigerator (4–5 °C) until they were used.

Chemicals and Reagents

For the experimental study, analytical-grade chemicals were used. Sodium hydroxide, Folin-Ciocalteu reagent, sulfuric acid, Fehling’s solution, alkaline copper tartrate, hydrochloric acid, citric acid, ethanol, sodium sulphate, copper sulphate, methyl red indicator, petroleum ether, sodium carbonate, gallic acid, sodium nitrate, aluminium chloride, oxalic acid, sodium bicarbonate, 2,6-dichlorophenol indophenol dye, ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium persulfate, and potassium hydroxide were procured from Molychem, Mumbai, India. Whereas HPLC grade solvents, such as methanol, dimethyl sulfoxide (DMSO), and water (Thermo Fisher Scientific, India) were used. For elemental analysis, the digestion of samples was done in Suprapure nitric acid (Fisher Scientific, UK), and the standards for minerals were obtained from Thermo Fisher Scientific, India.

Morphological Properties

A basic random sample technique was used to analyse morphological properties. Ten samples were picked at random from the lot and analysed for morphological features. Several criteria were adopted to assess the morphological qualities of the fruit samples. Fruit yield, waste yield, and juice yield were evaluated, as well as fruit size, colour, peel thickness, and the quantity of seeds per fruit. Fruit colour was evaluated using a Hunter colour lab (Hunter Lab Colour, Flex, Hunter Associates Inc., USA), while fruit size and peel thickness were measured using a digital vernier calliper (Aerospace, Kovea Co. Ltd., India). The approach given by Kasabi et al. [14] was used to calculate the L*, a*, and b* values in order to determine the fruit’s final colour. By peeling 1 kg of fruits, the total fruit yield and waste yield were determined, and the percentage juice yield was determined using the peeled fruits.

Chemical Properties

Total soluble solids (TSS) were measured using a hand refractometer (Erma, Japan: range 0–32 Brix). pH, titratable acidity, moisture, protein, fat, total sugars, reducing sugars, crude fibre, ash, and pectin content were also determined, following the method described by Bouhenni et al. [15]. Total phenol and flavonoid content were measured using the method given by Madaan et al. [16] and ascorbic acid was quantified using the titrimetric method, as outlined by Dinesh et al. [17]. The whole fruit and peeled fruit were ground homogeneously in a mixer grinder. In brief, a 1 g homogenized sample was applied on a refractometer to calculate the TSS of whole fruit and peeled fruit. The homogeneous paste of both peeled and whole fruit was taken in a beaker and analysed for pH. For estimating titrable acidity, 20 g of homogeneous paste was taken, 100 mL of distilled water was added and the content was to boil for 1 h thereafter, the final volume to 100 mL with distilled water, and 10 mL from this was taken for the estimation of titrable acidity. For the estimation of moisture content 10 g fruit slice (10 mm) was taken in a petri plate and kept in an oven at 65 °C and the weight changed to a constant reading was observed. For estimating total sugars 2.5 g of sample was taken in 40 mL of isopropyl alcohol and refluxed for 3 h. Thereafter, filter the content in a 50 mL volumetric flask and volume made up to 50 mL with isopropyl alcohol. From this stock solution, 0.5 mL was taken for the sugar estimation. For estimation of reducing sugars, total phenols, and flavonoids methanolic extract was prepared by taking 2.5 g of fruit sample in 40 mL of 80 % methanol and refluxing the sample for 3 h. Thereafter, the content and volume make up to 50 mL with 80% methanol. For estimating reducing sugars and total phenols 0.5 mL aliquot was taken from methanolic extract. For total flavonoids, 1 mL aliquot was taken and analysis was carried out using spectroscopy.

Macro Mineral Profiling using ICPOES

ICPOES was used for quantifying various components in fruit (peeled and unpeeled, separately). The iCAP 7000 (Thermo Fisher Scientific, USA) ICP-OES apparatus utilized was equipped with an autosampler model ASX-280. For instrument gas supply, high-purity argon (> 99.99%) was obtained from Sigma Gases, New Delhi. For sample preparation, a microwave-assisted digestion system (Multiwave Pro, Anton-Paar, India) with PTFE-TFM vessels (60 mL) and a 24-position digester block was employed. In digesting vessels, 0.5 g of sample was weighed, and 9.5 mL of Suprapure HNO3 was added. Then, a three-step heating program was used: (1) a 20-minute ramp to 180 °C; (2) a 20-minute hold at 180 °C; and (3) automatic vessel cool-down. Following digestion, the materials were diluted to a volume of 20 mL. Every experiment was carried out in triplicate. Analytical blanks were created in the same way but without the inclusion of samples in the digestion containers. The wavelengths 589.592 nm (Na), 279.553 nm (Mg), 766.490 nm (K), and 393.366 nm (Ca) were chosen to determine element concentrations. The mineral content results were given in parts per million (ppm).

Fatty acid Profiling of Galgal Seed Oil Using GCMS

Initially, the fatty acid methyl esters (FAMEs) were prepared from the extracted oil of fruit seeds using the direct FAME synthesis method [18]. 1 µL of FAMEs were injected into the GC-MS system (Thermo Fisher Scientific, USA), which was equipped with a gas chromatograph (GC 1300), autosampler (TriPlus RSH), mass spectrometer (TSQ Duo), and TGWAX MS column (30 m, 0.25 mm, 0.25 μm). The conditions for analysis were maintained as follows: injector temperature-240 oC, MS transfer line temperature- 240 °C, ion source temperature- 230 °C, helium flow rate-1 mL/min, initial oven temperature- 50 °C (with a 6 min hold), final temperature- 240 °C (with an 8 min hold), and ramp rate 7 °C/min. The spitless mode was adopted to run the samples. Ionization and fragmentation of the components were attained by 70 eV electron impact. The mass filter was adapted to a scan range of m/z 45 and 450. The obtained results were administered using the inbuilt Xcalibur Software.

Sterol Profiling of Galgal Seed Oil Using GCMS

The unsaponifiable components from fat samples were extracted using a procedure similar to the method established by Rani et al. [19] for measuring sterol levels with slight modification. Initially, 0.5 g of sample was placed into a 15 mL screw-capped tube and also 670µL of internal standard of 5-alpha-cholestan-3beta-ol (1000ppb) was added. Subsequently, 5 mL of a 5% methanolic solution of potassium hydroxide (KOH) was added to the tube. The tube was then placed in a water bath set at 90 °C, and it was intermittently agitated every 5 min for a total of 30 min. After the incubation period, the tube was allowed to cool to room temperature using tap water. Following cooling, 1 mL of water and 5 mL of hexane were introduced into the tube. The tube was subjected to vortex mixing for 1 to 2 min, after which it was centrifuged at 2,000 rpm for approximately 5 min. The upper hexane layer was carefully transferred into a small beaker with a capacity of around 10 mL. Subsequently, the hexane was evaporated from the beaker to yield dried unsaponifiable residue. The resulting dried unsaponifiable matter will be redissolved in 1 mL hexane for GCMS analysis. The sterol content was calculated by the formula [20] given as:

$${\rm{Concentration }}\,{\rm{of}}\,{\rm{sterol}}\,{\rm{ = }}\,{{\rm{A}}_{\rm{x}}}\,{\rm{*}}\,{{\rm{M}}_{\rm{s}}}\,{\rm{*}}\,{\rm{1000}}\,{\rm{/}}\,{{\rm{A}}_{\rm{s}}}\,{\rm{*}}\,{\rm{ m}}$$

Where;

Ax = peak area of sterol.

Ms = mass of internal standard.

As = peak area of internal standard.

m = mass of oil.

Volatile Compounds Profiling Using GCMS-Headspace

To analyse the volatile components of galgal juice, a GCMS-Headspace system (Thermo Fisher Scientific, USA) equipped with GC (Trace 1300), autosampler (TriPlus RSH), mass spectrometer (TSQ Duo), and column (TG 5MS, 40 m, 0.15 mm, 0.15 μm) was used. 1 mL juice of individual samples was taken in 10 mL vials and sealed, which was then incubated at a constant temperature of 70 °C for 30 min in a GCMS agitator to segregate the volatiles in the headspace. The analysis conditions for GCMS were set as follows: injector temperature- 240 °C, mass transfer line temperature- 230 °C, ion source temperature- 220 °C, helium gas flow rate 0.7 mL/min, and sample volume- 10 µL. The components were ionized and fragmented using 70 eV electron impact. The mass filter was modified to accommodate a scan range of 45 to 450 m/z. The collected results were administered using the built-in Xcalliber Software.

Analysis of Fruits Extract Using GCMS

To obtain extracts from the edible fruit portions, DMSO (Dimethyl sulfoxide) was utilized. An appropriate ratio of solvent and edible fruit part (5:1) was taken in a flask and kept overnight in an orbital shaker incubator maintained at 37 °C. The content was then filtered, and vacuum evaporated to obtain dry extract mass. The mass was diluted in GC grade DMSO solvent (1:99) and placed in GC vials for analysis. The same GCMS system was used for this study, and the conditions of the system were set as follows: injector temperature-250 °C, MS transfer temperature- 250 °C, ion source temperature- 230 °C, sample volume- 1 µl, helium flow rate-0.7 mL/min, initial oven temperature- 70 °C (6 min hold), final temperature- 250 °C (18 min) and ramp rate- 7 °C/min. The spitless mode was adopted to run the samples, and the 70-eV electron impact ionized and fragmented components were set at m/z 45–450. The outcomes were administered by the inbuilt Xcalliber Software.

Antioxidants Potential Using DPPH Radical Scavenging Activity

The antioxidant activity of fruits was determined by the DPPH method [21]. A solution of 2 mM DPPH dye was prepared in ethanol, and this solution (3.9 mL) was mixed with a fruit sample (0.1 mL). The mixture was then incubated in a dark chamber for 30 min. A control was prepared using a DPPH solution (3.9 mL) and pure methanol as a blank. After incubation, the optical density was recorded at 517 nm (Systronic 2202: UV–VIS spectrophotometer) to measure the degree of discoloration of the dye, which is an indicator of the antioxidant activity of the fruit. The % of DPPH scavenging activity was calculated as:

$$\eqalign{{\rm{DPPH}}\,\left( {\rm{\% }} \right)\,{\rm{Radical}}\, & \cr & {\rm{Scavenging}}\,{\rm{activity}}\, \cr & = {{{\rm{Control}}\,{\rm{OD }}\,{\rm{-}}\,{\rm{ Sample}}\,{\rm{ OD}}} \over {{\rm{Control }}\,{\rm{OD}}}}\,{\rm{ \times }}\,{\rm{100}} \cr}$$

Antioxidants Potential Using ABTS Radical Scavenging Activity

ABTS method was also adopted to check antioxidant properties [22]. To prepare the stock solution, 7 mM of 3-ethylbenzothiazoline6-sulphonic acid (ABTS) and 2.45 mM potassium persulfate were mixed in a 1:1 ratio and left undisturbed for 12–16 h. The stock solution was then diluted with ethanol until an absorbance value of 0.7 ± 0.02 was obtained. For the estimation of antioxidant activity, a sample aliquot (0.1 mL) was mixed with ethanol (3.9 mL), followed by the addition of ABTS dye (1 mL). The solution was incubated for 6 min, and the final absorbance values were recorded at 734 nm (Systronic 2202 UV–VIS).

$$\eqalign{{\rm{ABTS}}\,\left( {\rm{\% }} \right)\,{\rm{Inhibition}}\,{\rm{ = }}\,{\rm{ }} & \cr & {{{\rm{Control}}\,{\rm{OD }}\,{\rm{-}}\,{\rm{ Sample}}\,{\rm{ OD}}} \over {{\rm{Control }}\,{\rm{OD}}}}\,{\rm{ \times }}\,{\rm{100}} \cr}$$

Statistical Analysis

All experiments were conducted in triplicate using three different lots of fruits. The standard deviation is presented along with the data. SPSS (Statistical Package for Social Sciences) software was utilized to analyse variance (one way) in all the collected data.

Results and Discussion

Morphological Characteristics

The results of morphological characteristics of three different galgal cultivars (PBG, HRG, and HPG) are presented in (Table 1). Herein, data includes various parameters such as weight, size, colour, peel thickness, number of seeds per fruit, and percentage yields of fruit, waste, and juice. The weight and size of the galgal cultivars varied significantly. HPG had the highest mean weight (333.4 g), which was significantly different from both PBG (147.05 g) and HRG (145.5 g). Similarly, the size of the galgal fruit was larger in HPG (Length: 110.18 mm, Breadth: 78.80 mm) compared to PBG and HRG. HRG showed an intermediate size (Length: 99.33 mm, Breadth: 82.69 mm), while PBG had the smallest size (Length: 72.55 mm, Breadth: 67.71 mm). Colour analysis revealed significant differences among the cultivars. HPG exhibited the highest L* value (64.83), indicating a lighter colour, which was significantly different from both PBG (61.97) and HRG (55.52). Additionally, HPG had the highest b* value (61.33), signifying a more intense yellow colour, while PBG had the lowest b* value (49.90). The a* values were not significantly different among the cultivars. Peel thickness varied among the cultivars, with HPG having the thickest peel (3.92 mm), significantly different from HRG (2.83 mm). PBG (3.63 mm) showed a peel thickness similar to HPG. The number of seeds per fruit also exhibited significant differences. HPG had the highest number of seeds per fruit (18), significantly different from HRG (10) and PBG (15). The percentage yield of fruit, waste, and juice varied among the cultivars. HPG had the highest percentage yield of fruit (72.14), followed by HRG (64.10) and PBG (60.36). Conversely, PBG showed the highest percentage yield of waste (39.63), while HPG had the lowest (27.85). The percentage yield of juice from both whole fruit and peeled fruit was highest in HRG (33.90 and 52.89, respectively) and lowest in PBG (24.32 and 40.29, respectively). The observed morphological results were discovered to be consistent with Lambani et al. [23] investigation of Pseudo limon.

Table 1 Morphological properties of different galgal cultivars
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The morphological properties of the galgal cultivars (PBG, HRG, and HPG) showed significant differences, with each cultivar having its unique characteristics. These findings can be valuable for growers, breeders, and consumers in selecting the most suitable cultivar based on specific preferences and requirements. However, further investigations into taste, nutritional content, and other agronomic traits are necessary to make informed decisions for commercial cultivation and consumption.

Chemical Characteristics

In the study, the chemical properties of whole fruit and peeled fruit were analysed for three different cultivars (PBG, HRG, HPG). The data is presented in Table 2 (whole and peeled fruit). In the whole fruit, PBG had the highest TSS content (6.60 ºB), followed by HRG (6.30 ºB), and HPG had the lowest (4.10 ºB). Similarly, in peeled fruit, PBG had the highest TSS content (7.80 ºB), followed by HRG (7.10 ºB), and HPG had the lowest (5.80 ºB). Overall, PBG exhibited higher TSS values than the other two cultivars in both whole and peeled fruit. In both whole and peeled fruit, PBG had the lowest pH value. In whole fruit, the pH values were 02.15, 02.04, and 02.01 for PBG, HRG, and HPG, respectively. In peeled fruit, the pH values were 03.10, 02.89, and 02.43 for PBG, HRG, and HPG, respectively. Thus, PBG exhibited the highest acidity among the three cultivars.

Table 2 Chemical properties of galgal (whole fruits and peeled fruits)
Full size table

In whole fruit, PBG had the highest titrable acidity (4.50%), followed by HRG (4.05%) and HPG (3.87%). Similarly, in peeled fruit, PBG had the highest titrable acidity (5.48%), followed by HRG (5.84%) and HPG (5.01%). Overall, PBG consistently showed higher titrable acidity compared to the other two cultivars. In both whole and peeled fruit, PBG had the highest moisture content. In whole fruit, the moisture content values were 83.25%, 80.56%, and 80.23% for PBG, HRG, and HPG, respectively. In peeled fruit, the moisture content values were 84.37%, 81.23%, and 82.73% for PBG, HRG, and HPG, respectively. In both whole and peeled fruit, PBG had the highest total sugar content. In whole fruit, the total sugar content values were 8.45%, 7.56%, and 7.08% for PBG, HRG, and HPG, respectively. In peeled fruit, the total sugar content values were 10.26%, 10.12%, and 9.25% for PBG, HRG, and HPG, respectively. In both whole and peeled fruit, PBG had the highest reducing sugar content. In whole fruit, the reducing sugar content values were 4.01%, 3.75%, and 3.07% for PBG, HRG, and HPG, respectively. In peeled fruit, the reducing sugar content values were 4.77%, 4.00%, and 3.60% for PBG, HRG, and HPG, respectively. Non-reducing sugars play a crucial role in determining the sweetness and flavour of fruits. In this study, the non-reducing sugar content varied significantly among the galgal fruit samples. HRG exhibited the lowest non-reducing sugar content both in whole (3.81%) and peeled (6.12%) samples. On the other hand, PBG had the highest non-reducing sugar content in the whole fruit (4.44%) while HPG had the highest content in peeled samples (5.65%). These differences suggest that HRG might have a less sweet taste, making it potentially suitable for applications where lower sweetness is desired.

Dietary fibre is important for digestive health, and its content can influence the texture and mouthfeel of fruits. HRG displayed the highest crude fibre content in both whole (5.14%) and peeled (4.52%) samples, indicating its potential as a good source of dietary fibre. PBG and HPG had comparable crude fibre content in both whole (5.01% and 5.09%) and peeled (4.81% and 4.01%) samples. These findings suggest that HRG might offer better digestive benefits compared to the other two varieties. Pectin is a type of soluble dietary fibre that has various functional properties in food applications [24]. Interestingly, HRG had the lowest pectin content in whole fruit samples (4.10%) but the highest pectin content in peeled samples (4.90%). PBG had the highest pectin content in whole fruit samples (4.55%), while HPG had the lowest (4.32%). The levels of pectin in fruits may vary based on their respective varieties [24]. These differences suggest that HRG might be better suited for pectin-related applications after peeling, such as in jams or jellies. Protein content was relatively consistent across all samples, with no significant differences observed. This indicates that the protein content of these galgal varieties is similar and might not be a differentiating factor between them.

While fruits generally have low-fat content, HRG exhibited the highest crude fat content in both whole (3.22%) and lowest in peeled (0.68%) samples. HPG had the highest crude fat content in peeled samples (0.92%), whereas PBG had the lowest in peeled (0.80%) and second-lowest in whole (2.60%) samples. These slight differences suggest that HRG might have a slightly higher fat content, potentially contributing to its overall nutritional profile. Ash content represents the mineral content of fruits, providing insight into their nutritional value. PBG consistently exhibited the highest ash content in both whole (1.11%) and peeled (0.73%) samples. HRG consistently displayed the lowest ash content in both whole (1.04%) and peeled (0.65%) samples. These differences suggest that PBG might offer higher mineral content compared to HRG and HPG.

Phenolic compounds are known for their antioxidant properties [25] to potential health benefits. PBG consistently showed the highest total phenol content in both whole (81.33 mg GAE/g) and peeled (77.09 mg GAE/g) samples. HRG had the lowest total phenol content in whole fruit (74.35 mg GAE/g) and peeled fruit (69.83 mg GAE/g) samples. These findings indicate that PBG might have the highest antioxidant potential among the three varieties. Flavonoids are another class of antioxidants that contribute to the overall health-promoting properties of fruits [26]. PBG consistently had the highest total flavonoid content in both whole (39.88 mg QE/g) and peeled (35.31 mg QE/g) samples. HRG consistently had the lowest total flavonoid content in both whole (39.55 mg QE/g) and peeled (34.39 mg QE/g) samples. These results suggest that PBG might offer superior flavonoid content, potentially enhancing its health benefits. Ascorbic acid (vitamin C) is an essential nutrient with antioxidant properties [27]. PBG consistently exhibited the highest ascorbic acid content in both whole (58.25 mg/100 mL) and peeled (70.45 mg/100 mL) samples. HRG consistently showed the lowest ascorbic acid content in both whole (57.35 mg/100 mL) and peeled (68.28 mg/100 mL) samples. These findings indicate that PBG might provide the highest vitamin C content among the three varieties.

In summary, the chemical composition of galgal fruit varied among different samples (PBG, HRG, and HPG) and between whole and peeled fruit. PBG consistently displayed the highest content of various compounds, including sugars, phenols, flavonoids, and ascorbic acid. HRG often exhibited intermediate values, while HPG generally had the lowest content of these compounds. These differences could be attributed to factors such as variety, ripeness, processing methods, and manual errors in analysis adopted [26].

Macro Mineral Profile

Macro minerals, including sodium (Na), magnesium (Mg), potassium (K), and calcium (Ca), play crucial roles in various physiological processes within the human body [28]. In this study, the cationic mineral content of different galgal cultivars was analysed, both in their unpeeled and peeled forms. The results, presented in Table 3, provide valuable insights into the mineral composition of these cultivars. The analysis reveals that the sodium content is relatively low across all samples, with the highest concentration found in the unpeeled HRG cultivar (32.41 ppm). The peeled samples generally exhibit lower sodium levels, indicating that peeling reduces sodium content. Among the samples, the unpeeled HRG cultivar displays the highest magnesium content (14.51ppm), followed by the peeled HRG (7.14 ppm), and unpeeled PBG (4.29 ppm) and HPG (3.41 ppm) cultivars. Peeling appears to slightly reduce magnesium levels in most cases. Notably, the unpeeled PBG and HPG cultivars exhibit the highest potassium content, with concentrations of 104.41 ppm and 54.49 ppm, respectively. Peeling again seems to lead to a reduction in potassium levels across all samples. The unpeeled HRG sample has the highest calcium content (43.61 ppm), followed by unpeeled PBG (36.91 ppm). Interestingly, peeling results in a significant decrease in calcium content in all samples, indicating that the skin or outer layer of the galgal cultivars is a notable source of calcium. The differences in cationic mineral content among the galgal cultivars and their peeled/unpeeled forms suggest potential variations in their nutritional value and health benefits. Unpeeled samples generally exhibit higher mineral levels, which could be attributed to the presence of these minerals in the skin or outer layer. Peeling appears to reduce mineral content, but it may also make the cultivars more palatable for certain consumers [28].

Table 3 Macro minerals content of various galgal cultivars
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Fatty Acid Composition of Seed Oils

The findings of a GCMS analysis of the fatty acid profiles of seed oil isolated from various galgal cultivars are shown in Table 4; Fig. 1 (a to c). The results showed that significant levels of palmitic, oleic, and linoleic acids were present in all cultivars. Particularly, it was found that PBG, HRG, and HPG contained, respectively, 22.87%, 22.39%, and 22.36% palmitic acid and 28.69%, 29.25%, and 27.77% oleic acid. The concentrations of linoleic acid in PBG, HRG, and HPG were 31.43%, 32.14%, and 31.43%, respectively, making it the most prevalent fatty acid. The oils also showed notable concentrations of linolenic acid. According to Kumar et al. [29] the fatty acid composition of the oils showed a high concentration of essential fatty acids and a superior ratio of SFA, MUFA, and PUFA providing better health effects. While the total MUFA and PUFA contents were calculated to be 31.83%, 32.09%, and 30.73% and 37.75%, 38.80%, and 38.13%, respectively, the total MUFA content of PBG, HRG, and HPG was found to be 29.06%, 27.98%, and 29.76%. The oils also contained minor fatty acids such as myristic, myristoleic, palmitoleic, margaric, and eicosanoic acids.

Table 4 Fatty acid composition of seed oil extracted from different galgal cultivars
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Fig. 1
figure 1

Chromatograms of PBG (a), HRG (b), and HPG (c) seed oil

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In summary, the fatty acid composition of the galgal cultivars’ seed oil indicates a mixture of saturated, monounsaturated, and polyunsaturated fatty acids. While there are variations among the cultivars, the overall content of MUFA and PUFA is generally favourable for human health. However, the relatively high SFA content suggests that moderation in consumption is important [30]. Additionally, the presence of essential fatty acids like linoleic acid and alpha-linolenic acid is beneficial. Further analysis and consideration of the omega-3 to omega-6 ratio would provide a more comprehensive understanding of the oil’s nutritional value.

Sterol Profiling of Seed Oils

The results obtained from the GCMS analysis of the compounds are presented in the Table 5, with their respective retention times and sterol percentages. Firstly, Tetraponerine T8, with a retention time of 10.62 min, was found to be present in the all cultivars at a relatively low concentration of 952.84 mg/kg. Squalene, eluting at 19.06 min, was detected in the sample at a very low concentration of around 41.08 mg/kg in all cultivars. Squalene is a natural hydrocarbon found in various organisms, and its low presence might suggest that the sample was not derived from a significant squalene-rich source [20]. 22-Ketocholesterol and Campesterol, with retention times of 29.60 and 29.81 min respectively, were detected in the samples at low concentrations of 52.12 mg/kg and 311.00 mg/kg, respectively. These compounds are important sterols found in various biological systems [20]. Their presence, although at low levels, suggests that the source material may contain a mixture of sterols. Stigmasterol, with a retention time of 30.90 min, was present in the sample at a concentration of 82.49 mg/kg. Like other sterols, stigmasterol plays a role in various biological processes, and its presence could have implications for the potential health benefits of the source material [31].

Table 5 Sterol profiling of extracted seed oil from galgal cultivars
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β-Sitosterol is a well-known plant sterol with potential health benefits, particularly in reducing cholesterol absorption in the human body [32]. β-Sitosterol, with a retention time of 33.70 min, was found highest in HRG (2010.93 mg/kg) followed by HPG (1911.43 mg/kg) and PBG (1813.70 mg/kg), respectively. Its higher concentration suggests that the source material may have nutritional or health-related significance. 9,19-Cyclolanost-24-en-3-ol, eluting at 36.61 min, was found in the sample at a concentration in range of 142.56 to 162.35 mg/kg. This compound is less commonly encountered in biological samples and may have specific significance in the context of the source material. Finally, peak for cannabinol were observed at retention time of 38.43 min, with highest concentrations of 2.71.95 mg/kg in HRG followed by 2.60.19 mg/kg in HPG and 249.91 mg/kg in PBG respectively. The presence of cannabinol suggests that the source material may contain cannabinoids, which are compounds known for their pharmacological effects [33].

Characterization of Volatile Components in Juice

The chromatograph and all the volatile components of HPG was analysed and the results are represented in Table 6; Fig. 2 (a to c). The study identified several common compounds that were found in all cultivars, including α-Pinene, Camphene, D-Limonene, 3-Carene, Linalool, Cyclohexasiloxane dodecamethyl, Caryophyllene, E, E, Z-1, 3,12-Nonadecatriene-5, 14-diol, Propanoic acid, Picrotoxin, 2-Dodecen-1-yl (-) succinic anhydride, and 1-Heptatriacotanol. PBG showed the presence of additional compounds such as 1,3-Diisopropoxy-1,3-dimethyl-1,3-disilacyclobutane, Elemene isomer, Globulol, c-Elemene, Humulene, α-copaene, 2-Butenal, 2-methyl-4-(2,6,6-trimethyl-1-cyclohexen-1-yl), Ingol 12-acetate, i-Propyl 5,9,17-hexacosatrienoate, 2-Dodecen-1-yl (-) succinic anhydride, and 7-Methyl-Z-tetradecen-1-ol acetate. HRG also had additional constituents, such as Terpinen-4-ol, α-Terpineol, cis-α Bergamotene, 7,8,12-Tri-O-acetyl ingol, 7-Methyl-Z-tetradecen-1-ol acetate, Spiro [4.5] decan-7-one, 1,8-dimethyl-8,9-epoxy-4-isopropyl, 5,6,6-Trimethyl-5-(3-oxobut-1-enyl)-1-oxaspiro [2.5] octan-4-one, and 10-Methyl-8-tetradecen-1-ol acetate. Similarly, HPG showed some variation in its composition with the presence of trans-(2-Chlorovinyl) dimethylethoxysilane, 1-Penten-3-one, 1-(2,6,6-trimethyl-1-cyclohexen-1-yl), 10-Methyl-8-tetradecen-1-ol acetate, 2,5-Furandione, 3-dodecyl, and Tricyclo [20.8.0.0 (7,16)] triacontane, 1 (22), 7 (16)-diepoxy. In summary, the analysis showed that each galgal cultivar had unique secondary metabolic components that possess specific therapeutic significance, as outlined by different workers [34,35,36,37,38].

Table 6 Characterization of various volatile components of juices extracted from galgal cultivars using GCMS-Headspace
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Fig. 2
figure 2

Headspace chromatogram of PBG (a), HRG (b), and HPG (c)

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Analysis of Extracts

The chromatograph and volatile components of the extracts were characterized and the results are represented in Table 7; Fig. 3 (a to c). This study identified the compounds which include D-limonene and α-ocimene, which are known to have a number of health advantages [34, 39], and were among the compounds that were frequently identified in all cultivars. The other significant factors identified varied by cultivar. α-Myrcene, Caryophyllene, c-Elemene, Humulene, Solavetivone, 1-Methylene-2b-hydroxymethyl-3, 3-dimethyl-4b-(3-methylbut-2-enyl)-cyclohexane, 1-Penten-3-ol, 3-methyl, and 2,4,5-Trimethoxydiphenyl ether, for instance, were all present in the PBG extract. According to Chhikara et al. [40], these substances have antibacterial, antifungal, and antioxidant effects.

Table 7 Characterization of various components found in extract of galgal cultivars using GCMS
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Fig. 3
figure 3

Chromatogram of DMSO extract of PBG (a), HRG (b), HPG (c)

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Similarly, the chromatograph of volatile components of HRG was analysed and the results are represented in Fig. 3b. The extract from HRG was discovered to have different components, which were largely trans-α-Ocimene, Linalool, Acetaldehyde, bis(2-chloroethyl) acetal, (R)-lavandulyl acetate, 2,6-Octadien-1-ol, 3,7-dimethyl-, acetate, (Z)-, and trans-α-Bergamotene, α-Bisabolene. According to reports, these substances provide a range of therapeutic benefits, including analgesic, antimicrobial, and anti-inflammatory activities [40]. Because of the existence of these substances, HRG extract may also have significant health advantages. D-limonene, which has a number of health advantages including anti-inflammatory, antioxidant, and anticancer activities, was also discovered in significant concentrations in the HPG extract [34]. Other trace amounts of 3-Carene, α-Myrcene, linalool, Camphene, trans-α-Bergamotene, α-Bisabolene, 2,4(1 H,3 H)-Pteridinedione, 6-methyl, and 5-Ethyldecane were found in HPG extract. Various therapeutic capabilities of these substances, such as anti-inflammatory, antibacterial, and antiviral activities, have also been noted [40]. It is worth noting that the composition of each sample differed, which may be attributed to variations in cultivation regions, varieties, and climate conditions. Overall, the results of this extract characterization provide valuable insights into the potential health benefits (Table 8) of each cultivar, which could be further explored in future studies.

Antioxidant Potential of Extracts

The results represented in Fig. 4 (a) displayed the outcomes of the DPPH method utilized to determine the percentage of radical scavenging activity in galgal cultivars (whole fruit). The PBG cultivar demonstrated the highest percentage of radical scavenging activity (90.15%), followed by HPG (85.26%) and HRG (84.03%). This result is consistent with a study conducted by Rekha et al. [41] on another citrus variety. Figure 4 (b) demonstrates the antioxidant activity of galgals without the peel. The antioxidant activity was observed to decrease after peeling the fruit, and the order of antioxidant activity was consistent with that of the whole galgal. The PBG cultivar showed the highest activity (86.7%), followed by HRG (82.89%) and HPG (82.04%). In this context, Rekha et al. [41] predicted the DPPH percentage of radical scavenging activity of Citrus limon to be within the range of 80–95%, which is consistent with the obtained results. Figure 4 (c & d) exhibited the antioxidant activity by the ABTS method for whole fruit and fruit without peel. The PBG cultivar was found to have the highest antioxidant activity (46.28%), and HRG had the lowest (41.57%). Chen et al. [22] also reported similar findings of fruit pulp using the ABTS method.

Fig. 4
figure 4

The antioxidant properties of fruits with peel (a & c) and without peel (b & d) using DPPH and ABTS methods, respectively

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Conclusion

Based on research findings, the galgal fruit boasts a superior nutritional profile compared to other citrus fruits. It is rich in fibre, ascorbic acid, flavonoids, and total phenols, all of which are essential for maintaining a good health. Galgal fruit also has a significant amount of antioxidants, which work to fend off free radicals and prevent cancer. Several bioactive substances, including limonene, caryophyllene, α-phellandrene, α-pinene, linalool, and α-terpineol, were discovered when galgal juice was examined using headspace analysis. These substances all have antimicrobial, anticancer, antibacterial, antiviral, analgesic, antidiabetic, antifungal, and anti-inflammatory properties. PBG differed from other galgals in that it had significant concentrations of caryophyllene, compared to the modest concentrations seen in HRG and HPG. The fatty acid profile of fruit seed oil showed a balanced composition of SFA, MUFA, and PUFA and also contains a significant amount of omega-3 fatty acid. Galgal fruit is quite acidic, however, eating it has several advantages. The fruit can be suggested for use in pickles, chutneys, marmalades, nectars, cordials, teas, wines, and other forms due to its high nutritional profile.