Introduction

More than 70% of the Earth’s surface is covered by water, serving as a habitat for a diverse range of aquatic plants with pharmaceutical significance (Penesyan et al. 2010). Extensive research has been conducted on these aquatic organisms to isolate novel compounds, resulting in the identification of over 15,000 new chemical compounds (Sun et al. 2015; Malve 2016; Rubiño et al. 2022). In the field of bioproducts, recent trends in natural drug research point to algae as a promising source of novel biochemically active substances (Cardozo et al. 2007; Shalaby 2011; Garayemi and Raeisi 2020). To thrive in a competitive environment, both freshwater and marine algae have developed defense strategies, resulting in a significant level of structural chemical diversity stemming from various metabolic pathways (Kim and Karagozlu 2011; Torres et al. 2014; Anbuchezhian et al. 2015; Shah et al. 2020). The exploration of these organisms for pharmaceutical purposes has unveiled important chemical prototypes, fostering the use of advanced analytical techniques and the development of new compound syntheses with biomedical applications (Goshtasbi et al. 2023). Additionally, algae hold promise as a source of both novel biologically active substances and essential compounds for human nutrition (Ghosh et al. 2022; Imran et al. 2023; Mishra et al. 2023). Consequently, there is a growing demand for algal extracts, fractions, or pure compounds within the economic sector (Babich et al. 2022; Birgersson et al. 2023). The pursuit of novel drugs has traditionally focused on natural products, and this domain continues to be a crucial area of exploration within the pharmaceutical industry (Vimala and Poonghuzhali 2017; Matharasi et al. 2018; Barkia et al. 2019; Jacob-Lopes et al. 2019; Hassan et al. 2022). According to (Adhoni et al. 2016) an increasing number of people are interested in using natural goods made from algae as sources of food, cosmetics, innovative medications, and therapeutic agents due to the wide structural diversity and complexity of these items. The chemical makeup of substances produced by freshwater algae has been studied and scrutinized less than that of marine algae (Imran et al. 2023). Therefore, the primary objective of this study was to assess the phytochemical components of the freshwater alga Nitella hyalina and emphasize their biological and/or industrial significance. Nitella, a genus of Charophytes, exhibits considerable variety with approximately 200 species and infraspecific taxa and has a global distribution (Sakayama et al. 2002). In the literature, only a few species of the genus Nitella have been evaluated for chemical composition and their role in pharmacology, such as Nitella tenuissima, studied by Hilda et al. (2014) for its phenols and antioxidant potential. Moreover, Seal et al. (2015) demonstrated that N. flagelliformis extracts contain the highest amount of flavonoids, flavonols, and phenolic compounds, which exhibiting strong reducing power and radical scavenging activity. Another study by Prashantkumar et al. (2006) reported a significant antibacterial potential of Nitella tenuissima. Nitella hyalina is a macroscopic green alga or stonewort that is widely distributed, specifically found in open sunny edges of lakes, pools, sea shores, and sheltered openings in reed stands with calcareous mud (García 1994). It is unique and remarkable specie and hence for the very first time, this study is conducted to evaluate its chemical composition and its pharmacological potential using different biological assays.

Materials and methods

Collection and extraction

Nitella hyalina a macroalgae was collected from Karachi, Sindh Province of Pakistan in December 2021. The collected algae was thoroughly washed with tap water to remove any sand or dirt. The cleaned samples were left in the shade to dry at a room temperature between 25 and 35˚C. After being fully dried, the algae were ground into fine powder and stored in plastic bags for further experimental uses. 300 g of powder was blended with a mixture of ethyl acetate and n-butanol in a ratio of 1:3 with a total volume of 1000 ml, and left undisturbed for 21 days. After this period, the resulting extracts were obtained and kept for further analysis.

Proximate nutrient composition

Standard methods of AOAC (Horwitz) were employed to determine the levels of crude fibers, ash contents, crude protein, crude fat, and moisture content. The carbohydrate contents were computed by summing up the percentages of crude fibers, ash contents, crude protein, crude fat, and moisture contents, and then subtracting the total from 100. The energy content was calculated by applying the “Atwater factor,“ which involves multiplying the quantities of carbohydrates, fat, and crude protein by 3, 9, and 3, respectively. The total energy content was obtained by summing up the individual outcomes (Onwuka 2005).

Gas chromatography Mass Spectrometry (GC-MS) analysis

Gas chromatography-mass spectrometry (GC-MS) analysis was performed using a Shimadzu Corporation QP2010 Ultra model instrument Kyoto, Japan. The analysis was conducted on a capillary column with an inner diameter of 0.25 mm and a length of 30 m. The stationary phase employed had a film thickness of 0.25 mm (Rtx-5MS, Restek Corporation, United States). Helium gas with a purity of 99.999% was used as the carrier gas, flowing at a constant velocity of 36.3 cm/sec. The sample, with a volume of 1 L, was introduced into the system via the AOC-20i + s auto-injector. The injection port was maintained in split-less mode at a temperature of 290 °C. The GC oven was preheated according to the following temperature program: initially held at 50 °C for 5 min, followed by a 10-minute hold at 300 °C, with a heating rate of 2 °C per minute. The total ion chromatogram was generated using an m/z range of 30 to 700. The identification of GC peaks was achieved by comparing their mass spectra with the National Institute of Standards and Technology’s (NIST) database and relevant literature. The relative percentage quantity of each constituent was calculated by comparing the peak area of each constituent to the total peak area of the chromatogram (Ayaz et al. 2017).

Fourier-transform infrared spectroscopy (FTIR)

The algae-derived sample was subjected to FT-IR spectroscopy analysis using a PerkinElmer FT-IR 2000 instrument based in Waltham, MA, USA. A small portion of the extracted material was mixed with KBr to create pellets for FT-IR analysis. A thin film was formed by applying pressure. Infrared transmittance data were collected over a wave number ranged from 4000 cm− 1 to 400 cm− 1. The plain KBr pellets were employed as a blank control. Subsequently, the spectral data were compared with a reference to identify the functional groups present in the sample(Hussein et al. 2015).

Determination of total phenolic and flavonoid contents

Determination of total phenolic content (TPC)

The folin-Ciocalteu method as reported byZohra et al. (2019), and. Odabasoglu et al. (2004) was used to assess the total phenolic content of Nitella hyalina extract. Solutions with concentrations ranging from 0.1 to 5 mg/mL using gallic acid as a reference were created. 100 µL of an extract with 500 µL of water and 100 µL of Folin-Ciocalteu reagent in each solution were added before incubating it for six minutes. After that, 1 mL of 7% sodium carbonate was added and let the mixture to sit at room temperature for 90 min. The total phenolic content was then determined by measuring the absorbance at 765 nm using a spectrophotometer and represented as milligrams of gallic acid equivalents per gm of dry extract (GAE/mg).

Determination of total flavonoid contents (TFC)

Following the procedure outlined by Chantiratikul et al. (2009), an aluminum chloride complex-based assay was used to calculate the total flavonoids in the Nitella hyalina extract. A calibration curve for quercetin was made by diluting a standard solution in methanol to concentrations ranging from 0.1 to 5 mg/mL. Following that, 500 µL of distilled water and 100 µL of 5% sodium nitrate were combined with 100 µL of each quercetin dilution, and the mixture was allowed to sit for 6 min. Then 150 µL of 10% aluminum chloride was added to the solution, and again was let to stand for 5 min. After that, 200 µL of a 1 M sodium hydroxide solution was added, and the solution absorbance was measured at 510 nm. Total flavonoid contents were expressed as mgQE/g of quercetin equivalent (Zohra et al. 2019).

Antioxidant assay

DPPH anti-radicals assay

Using the DPPH anti-radical assay as described by Shah et al. (2015), the antioxidant potential of Nitella hyalina extract was evaluated by examining their capacity to scavenge radicals and donate hydrogen. 0.1 mL of each extracts was mixed with 2 mL of a 100 µM DPPH solution and allowed to incubate for 30 min at 25 °C in the dark. The reaction mixture was then compared to a reagent blank using ascorbic acid as a reference standard. The absorbance was measured at 517 nm. The amount of sample required to lower the DPPH was determined by plotting the degree of discoloration versus the concentration of the sample extracts. A linear regression curve was plotted to calculate the IC50.

%RSA= [(Ac - As) ÷ Ac] ×100.

%RSA, Radical scavenging activity; Ac, Absorbance in Control; As, Absorbance in Sample.

Antimicrobial activity

The antimicrobial potential of Nitella hyalina extracts in ethyl acetate and n-butanol solvents against one fungus, Candida albicans, and four bacterial strains, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, were investigated using agar well diffusion method. The procedure used in this study was based on the protocols described by Ayaz et al. (2015) and Sani et al. (2021).

Culture of the Bacteria

The microbiology lab PCSIR Peshawar Pakistan provided pure bacterial and fungal cultures, which were maintained on a nutrientagar medium and sub cultured regularly to ensure their purity. The cultures were stored at 4 °C before their use in experiment.

Diameter of inhibitory zone (DIZ)

According to the method described by Mathabe et al. (2006), the agar well procedure was carried out with a few modification. To create an initial inoculum of 1.5 × 108 bacteria/fungal ml-1, bacterial and fungal colonies from the plates were cultured in Mueller-Hinton Broth (MHB) until they attained their respective OD at 600 nm. Each of the quadrants of the Mueller-Hinton Agar plates was identified and labeled. To ensure uniform dispersion of microorganisms, 6 ml of molten soft MHA was mixed with 100 µl of the inoculum, approximately 107 cfu, and then quickly poured onto the base layer of MHA. After that, the plates were allowed to set up for 10 min. In the marked area, four holes were drilled with a sterilized 5 mm borer. algal extract at concentrations of 100 mg/ml, 50 mg/ml, 25 mg/ml, and 12.5 mg/ml were used in each test sample, whereas Azithromycin 10 µL was added in the control. The test samples and bacteria-filled plates were incubated for 16 to 18 h at 37 °C. As stated by Ayaz et al. (2015), the zone of inhibition was then quantified using a Vernier caliper. Each experiment was run at least three times, and the formula indicated below was used to determine the percentage of inhibition.

\(\% inhibition=100-\frac{test}{control}\)× 100

Hypoglycemic activity

Male albino rats weighing between 120 and 150gm were used for the experiment. Animals were obtained from the PCSIR Peshawar animal house and were handled in strict accordance with the ethical guidelines set by the Animal Ethical Committee Department of Pharmacy, University of Malakand (Ref: DREC/Pharm-DM/DD2-2020). The animals were kept at a constant room temperature of 25 °C, with equal duration of 12/12 hours of light and dark. In hygienic conditions, water and a rodent pellet diet were given to the rats. The rats were fasted for 16 h before receiving a single intraperitoneal injection of alloxan monohydrate in sterile saline at a dose of 150 mg kg-1to induce diabetes. Rats with hyperglycemia (glucose level greater than 200 mg dL-1) were chosen after three days and put into groups of six for the anti-diabetic investigation. Following the administration of alloxan for 72 h, the administration of plant extracts began. During this time, all groups were provided with a standard diet and had access to water.

Animals groups

The study comprised nine groups. Group 1 (the control group) received distilled water intraperitoneally (i-p), while the second group was the diabetic control group, which received 150 mg kg-1 of alloxan i-p. The third group received Gabinclamide (5 mg kg-1 i-P.) as a standard drug for diabetes management. The remaining six groups (groups 4–9) consisted of diabetic rats that had received varying dosages of n-butanol and ethyl acetate extracts. Ethyl acetate extract was administered to groups 4–6 at doses of 100 mg/kg, 200 mg/kg and 300 mg/kg, respectively. The n-Butanol extract was administered to groups 7–9 at doses of 100 mg/kg, 200 mg/kg and 300 mg/kg, respectively.

Statistical analysis

All the data were recorded in triplicate form. One-way ANOVA (analysis of variance) was conducted using Statistical Software (SPSS Inc. software version 13.0, Chicago, IL, USA). The data were presented as mean and standard deviation and the significance differences were indicated as p < 0. 001, p < 0.01 and p < 0.05. IC50 values were calculated between doses and effect.

Results

Nutritional analysis (proximate composition)

Significant variations were noted in the nutrient composition, as illustrated in Fig. 1. The ash content was found to be the highest (40.05 ± 2.40%), followed by crude protein and crude fat contents i.e. 24.66 ± 0.95% and 17.66 ± 1.42%, respectively. whereas,relatively low amount of crude fiber were noted i.e. 2.17 ± 0.91%. The moisture content was recorded to be 15.46 ± 0.48%. Furthermore, the calculated energy value was determined to be high at 173.50 ± 2.90 Kcal/100 g.

Fig. 1
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Assessment of proximate content in algal powder (in terms of dry weight)

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GC-MS analysis

The use of Gas Chromatography-Mass Spectrometry (GC-MS) analysis allowed for the tentative detection of approximately 23 compounds within the ethyl acetate extract of Nitella hyalina, as demonstrated in Table 1; Fig. 2. Among these compounds, Palmitic acid, methyl ester (C17H34O2) was the most abundant, accounting for 48.73%, followed by (Z) -9-Hexadecenoic acid, methyl ester, (6.88%), Methyl tetradecane (6.24%), 9-hexadecanoic acid (4.33%), Pentadecanoic acid (3.97%), Linolelaidic acid, methyl ester (2.67%), and 10- Octadecenoic acid, methyl ester (2.31%). moreover, The compound found to be the least abundant by weight was 2, 6, 11-Trimethyldodecane i.e. 0.18% of total extract. Similarly, approximately 15 compounds were identified in n-butanol extract (Fig. 3). Tetra decanoic acid (C14H28O2) was found to be the most abundant by weight i.e. 37.81%, followed by 9-hexadecanoic acid (21.69%), Methyl pentopyranoside (7.58%), undecane (6.08%), Palmitic acid, methyl ester (4.90%), Ethyl iso-allocate (4.20%), Lager acetal (3.77%), and Dodecane (3.50%). The least abundant compound was identified as 1-(5’-Chloro-2’-methylaminobenzoyl)-cyclohexane-1-ene, accounting for only 0.01%.

Table 1 Gas Chromatography Mass Spectrometry (GC-MS) analysis of ethyl acetate extract of Nitella hyalina
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Table 2 Gas Chromatography Mass Spectrometry (GC-MS) analysis of n-butanol extract of Nitella hyalina
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Fig. 2
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GC-MS chromatogram of Nitella hyalina ethyl acetate extract

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Fig. 3
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GC-MS chromatogram of Nitella hyalina n-butanol extract

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FT-IR Spectrum Analysis

The FT-IR spectra of the ethyl acetate and n-butanol extracts were analyzed and the data were recorded as shown in Table 3; Figs. 4 and 5. The spectra suggested that hydroxyl groups were present in the samples due to the presence of a non-bonded O-H stretch at 3712.50 cm− 1 in both extracts. Similarly, the H bond and O-H stretch of hydroxyl compounds and alcoholic phenol were recorded in the region of 3373.50 cm− 1, 3360.00 cm− 1, and 3244.27 cm− 1 in the n-butanol extract, whereas these were absent in the ethyl acetate extract. Samples showed signature peaks at 2922.1623 cm− 1 and 2920.23 cm− 1 in the ethyl acetate and n-butanol extracts, respectively, corresponding to irregular stretching of -CH(CH2) vibrations, representing the presence of saturated aliphatic compounds and lipids. In addition, peaks at 2812.50 cm− 1 were also found in both extract due to the O-H stretching of the carboxylic acid. Two average bands were also recorded in the regions of 1714.72 cm− 1 and 1612.50 cm− 1 for the ethyl acetate extract, whereas for n-butanol, average bands were noted in the regions of 1743.75 cm− 1 and 1633.71 cm− 1, which correspond to the C = O functional group of carbonyl compound. C = C stretching of the aromatic moiety was measured at 1443.75 cm-1, 1417.68 cm− 1, and 1456.26 cm− 1 in the n-butanol and ethyl acetate extracts respectively. The functional groups C-N stretch and C-H with (CH, k) of amine and alkyl halides were recorded in the region of 1174.65 cm− 1 in ethyl acetate extract and were not found in n-butanol. For the phosphate ion spectrum, intense bands at 1020.34 cm− 1 and 1039.63 cm− 1 were seen in n-butanol and ethyl acetate extracts, respectively. Bands ranging from 872.12 cm− 1 to 723.31 cm− 1 represent the elongation of the -C-H bending of alkene. Low-size stretching for the C-Cl functional group of halogen compounds (chloro-compounds) was noted in region 713.66 cm− 1 and 684.73 cm− 1 of both n-butanol and ethyl acetate extracts, respectively. Stretching at 665.44 cm− 1, 584.43 cm− 1, and 667.37 cm− 1 demonstrates the C-Br functional group regions of halogen compounds, namely (iodol compound and chloro-compounds), and alkyl halide in both n-butanol and ethyl acetate extracts, respectively.

Table 3 Analysis of FT-IR spectral Peaks and functional groups in ethyl acetate and n-butanol extracts of Nitella hyalina
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Fig. 4
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FT-IR spectrum of Nitella hyalina ethyl acetate extract

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Fig. 5
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FT-IR spectrum of Nitella hyalina n-butanol extract

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Estimation of total phenolic and flavonoid content

The study was assessed to estimate total phenolic contents (TPC) and total flavonoid contents (TFC) in the selected algal extracts. TPC and TFC values were represented as mg of gallic acid equivalents (GAE) and mg of quercetin equivalents (QE) per gram of sample in dry weight (mg/g)Table 4. No significant difference was observed in both TPC and TFC values. The highest TPC value i.e. 1.663 ± 0.768 mg GAE/g was recorded in n-butanol extract., whereas ethyl acetate extract had 1.043 ± 0.961 mg GAE/g total phenolic contents. Similarly, the highest TFC value was recorded for n-butanol extract 2.050 ± 0.143 QE/g followed by ethyl acetate i.e. 1.730 ± 0.311mgQE/g.

Table 4 Quantification of Total phenolic and flavonoid contents and antioxidant potential of Nitella hyalina extracts
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Each value represents the mean and standard deviation of 3 replicates, whereas IC50 values were determined for all concentrations.

Antioxidant activity (DPPH anti-radical assay)

Antioxidant activity of extracts of Nitella hyalina was evaluated by DPPH radical scavenging. The findings presented in Table 4; Fig. 6. The results suggested that n-butanol extract of Nitella hyalina showed higher % of radical scavenging activity than that of ethyl acetate extract, i.e. ranging from 34.55 to 46.35% and 35.39–41.79%, respectively. Both extract exhibited the highest % RSA at 100 µg mL-1 (highest concentration), followed (by 75, 50, & 25 µg mL-1). The n-butanol extract showed 34.55%, 40.09%, 43.44%, and 46.35% of radical scavenging activity, whereas ethyl acetate extract showed 35.39%, 36.04%, 38.50%, and 41.79% activity at 25, 50, 75, and 100 µg mL-. IC50 values were calculated for each concentration, which suggest that n-butanol had better activity than ethyl acetate, as ethyl acetate IC50 was higher than n-butanol.

Fig. 6
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Percent radical scavenging activity (RSA) of ethyl acetate and n-butanol extracts of Nitella hyalina

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Antimicrobial activity

The antimicrobial properties of Nitella hyalina n-butanol and ethyl acetate extracts against fungal and bacterial strains are presented (Figs. 7 and 8; Table 5). The overall results of antimicrobial activity suggested that n-butanol extract was found to be more potent than ethyl acetate extract. Among the tested bacterial strains, P. aeruginosa was recorded as the most susceptible, which was followed by S. aureus and E. coli, whereas C. albicans was recorded as the least affected specie. In terms of concentration, 100 mg mL-1 was recorded as more potent, followed by 50, 25, and 12.5 mg mL-1. In the case of n-butanol extract 100.00%, 92.11%, 81.58%, and 70.18% growth of P. aeruginosa was inhibited by 100, 50, 25, and 12.5 mg mL-1, respectively. Similarly, ethyl acetate showed 100.00%, 90.35%, 80.70%, and 79.82% inhibition in growth of P. aeruginosa at the same concentrations. Overall results showed that n-butanol extract exhibited stronger antimicrobial activity compared to ethyl acetate extract, as evidenced by the enhanced activity against S. aureus, E. coli, and B. subtilis. Specifically, the inhibition percentages for S. aureus were recorded as 89.8%, 88.78%, 79.59%, and 78.57% for n-butanol and 77.55%, 66.33%, 63.27%, and 56.12% for ethyl acetate 100, 50, 25 and 12.5 mg mL-1, respectively. However, C. albicans and B. subtilis were the least inhibited strains by ethyl acetate and n-butanol extract respectively. A positive correlation between the concentration and zone of inhibition (ZOI) of extract was found. The highest zone of inhibitions was observed at 100 mg mL-1, whereas the lowest inhibition was observed at 12.5 mg mL-1. Moreover, there was no significant variation in the ZOI of n-butanol extract against C. albicans. Overall, the study suggests that ethyl acetate and n-butanol extracts possess antimicrobial potential against different strains, with n-butanol being the more potent extract.

Table 5 Antimicrobial activity of ethyl acetate and n-butanol extracts of Nitella hyalina
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Fig. 7
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Percent antimicrobial activity of ethyl acetate extract of Nitella hyalina

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Fig. 8
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Percent antimicrobial activity of n-butanol extract of Nitella hyalina

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Hypoglycemic activity

The results about anti-diabetic effects of Nitella hyalina extracts have been shown in (Table 6). Results indicated that the diabetic control group experienced a significant increase in blood glucose levels (305.33 ± 7.74) on the third day following the alloxan injection, in contrast to the normal animals (99.67 ± 7.61). However, treatment with either the standard drug Glibenclamide or the Nitella hyalina extract (ethyl acetate and n-butanol) resulted in a substantial decrease in glucose levels, as reported in Table 6.

The results demonstrated that the n-butanol extract of Nitella hyalina had a significant blood glucose-lowering effect, with the most significant reduction observed at a dose of 300 mg/kg-1 after 6 h i.e. 241.50 ± 2.88, followed by doses of 200 and 100 mg/kg-1 (245.17 ± 3.43 and 250.67 ± 7.45, respectively). All extract were observed to significantly decrease blood glucose levels at α > 0.05, 0.01, and 0.001 at 24 h, with the n-butanol extract being more effective than ethyl acetate. A dose of 100 mg/kg-1 of ethyl acetate did not produce a significant effect, while a significant decrease in blood glucose levels at α > 0.05 was observed with a dose of 200 mg/kg-1, except at 24 h after treatment where a significant change in blood glucose levels was observed. Both the higher concentration (300 mg/kg-1) of ethyl acetate and all doses of n-butanol showed a considerable drop in glucose levels. These results imply that n-butanol extract could be regarded as an efficient standard medication, similar to the commercially marketed Glibenclamide. The onset of the anti-diabetic action of Glibenclamide at a dose of 5 mg/kg-1 was observed at 6 h i.e. 291.00 ± 3.54 and 24 h (291.33 ± 3.44).

Table 6 Antidiabetic potential of ethyl acetate and n-butanol extracts of Nitella hyalina
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Discussion

Macroalgae have been part of the diet in many countries across the world particularly in Asian regions. However, their consumption has experienced a significant increase in Western nations over the last few decades, driven by the acknowledged nutritional richness and health advantages associated with macroalgae (Buschmann et al. 2017). The nutritional compositions of numerous algae have been found to be rich sources of fatty acids, protein, dietary fiber, and a variety of minerals (Fernández-Segovia et al. 2018). The present study was conducted to analyze the nutritional profile of the macro alga Nitella hyalina. The results showed significant variations in the nutrient composition. The ash content was found to be (40.05 ± 2.40%), followed by crude protein (24.66 ± 0.95%) and crude fat i.e. 17.66 ± 1.42%. However crude fibers were comprised of 2.17 ± 0.91%. The moisture content was recorded to be 15.46 ± 0.48%. Furthermore, the calculated energy value was determined to be 173.50 ± 2.90 Kcal/100 g. Previously, (Nutautaitė et al. 2021) also reported protein and other essential nutrients from freshwater macroalga Cladophora glomerata biomass. Similarly, (Hossain et al. 2021) investigated the nutritional composition of three macroalgal species. He reported the protein contents i.e. 19.99% followed by fiber (26.82%) in Gracilaria tenuistipitata, crude fat (3.85%) in Spatoglossum asperum, and ash (14.71%) in Hypnea boergesenii. (Imran et al. 2023) reported almost the same results of nutritional analysis of Dictyota dichotoma. The results were also in accordance with those of (Das and Ghosh 2023) who documented the nutritional composition of five commonly available freshwater algae i.e. Cladophora, Microspora, Pithophora, Lyngbya and Spirogyra. The results revealed that the crude protein content of the algae ranged between 10.49 and 30.8% (w/w), whereas crude fiber and ash contents varied between 11.37 and 17.56%, and 16.32 and 20.21% (w/w), respectively. Similarly algae are highly effective at producing biologically active compounds, which evolved as a defense mechanism against a multitude of organisms living and interacting within the same complex environment. In the past thirty years, there has been a considerable increase in the discovery of metabolites with diverse biological activities, including antiviral, antimicrobial, antitumoral, insecticidal, antifungal, phytotoxic, antiproliferative, and cytotoxic actions (Benhniya et al. 2022). Thus, in the present study, the crude extracts of Nitella hyalina were subjected to GC-MS and FT-IR for detection and identification of bioactive compounds. Percentage composition of compounds in the crude extracts, has been given in Table-1 and Table-2. 23 compounds were identified, with Palmitic acid, methyl ester (C17H34O2) being the major compound in ethyl acetate. Similar results were reported by (Imran et al. 2023) by evaluating the chemical profile of Seaweed Dictyota dichotoma. The present result were also in line with that of Davoodbasha et al. (2018) who also reported Palmitic acid, methyl ester (C17H34O2) from microalga Scenedesmus intermedius. (Z)-9-Hexadecenoic acid, methyl ester was found to be the second most major compound. This is in agreement with those of Alreshidi et al. (2023), who reported 9-hexadecenoic acid, methyl ester from Sargassum sp, and evaluated its antimicrobial potential. Additionally, Methyl tetra decanoate was also reported in the extract, which is consistent with the finding of Marrez et al. (2022) and Sharma et al. (2022), who reported this compound from Oscillatoria princeps and Euglena tuba extract. 9-hexadecanoic acid reported from the red algae Hypnea musciformis by Sumayya and Murugan (2018). Similarly, in the n-butanol extract, 15 compounds were tentatively identified, with Tetradecanoic acid (C14H28O2) being the most abundant. This finding is consistent with Karimzadeh and Zahmatkesh (2021) who’s reported the same compound from red algae (Laurencia snyderiae). Similarly 9-hexadecanoic acid, Methyl pentopyranoside,, and undecane, was also reported to be the major compounds. This result is in agreement with those of Moshfegh et al. (2019) who also reported undecane in extract of Laurencia caspica, and reported to had antioxidant, anticancer, and antibacterial potential. Additionally, Palmitic acid, methyl ester and Ethyl iso-allocate was also tentatively detected through GC-MS. Kepel et al. (2021) supported the identification of Ethyl iso-allocate from Halimeda macroloba and Halimeda opuntia. The chemical classes identified in this study were in agreement with those stated in the literature for macroalgae from different environments (Santos et al. 2015). Although several studies have investigated the antioxidant, antihyperglycemic, and antimicrobial, activities of marine algae, limited information is available on their activities in different solvents depending on the harvest sites. Worldwide, over a thousand pharmacologically active compounds have been characterized from marine algae, with potential efficacy against various diseases such as bacteria, fungi, viruses, cancer, hypertension, and high cholesterol (Pérez et al. 2016).

Similarly, Fourier Transform Infrared (FT-IR) spectroscopy is a valuable analytical tool used for the identification and elucidation of chemical compounds in various fields, including pharmaceuticals. In recent years, FT-IR has gained significant importance in pharmaceutical analysis due to its unique spectral features that act as fingerprints and can be applied to a wide range of samples. This spectroscopic technique provides molecular-level information, making it possible to examine functional groups, molecular conformations, and intra- and intermolecular bonding types. Furthermore, FT-IR can be used for quantitative analysis, making it a versatile tool for studying chemical compounds in different areas of research (Cozzolino 2015). FT-IR analysis was carried out on both Nitella hyalina extracts to identify the characteristic infrared functional group absorptions, which were then compared to those found in literature sources. The crude extract of Nitella hyalina was found to contain a variety of functional groups, including Hydroxyl compounds, alcoholic phenols, saturated aliphatic compounds, lipids, Carboxylic acids, Carbonyl compounds, Aromatic compounds, Amines, alkyl halides, Phosphate ions, Alkenes, and Halogen compounds. FT-IR spectroscopy has been recommended as a reliable and rapid technique for analyzing the composition and authenticity of medicinal plants. The presence of Hydroxyl compounds and alcoholic phenols has been associated with potential antioxidative capabilities, as evidenced by previous studies (Munir et al. 2019; Palani et al. 2022). Furthermore, previous research has shown that compounds containing saturated aliphatic lipids possess encouraging activities i.e. antimicrobial, antidiabetic, and antioxidant effects (Raja et al. 2016; Torres et al. 2019; Wang et al. 2022). The FT-IR analysis of Nitella hyalina extracts revealed the presence of various functional groups, these compounds having similar functional groups have been associated with several properties, including anticancer, antioxidant, anti-inflammatory, antidiabetic, and antimicrobial activities in previous studies (Fernando et al. 2016; Donga and Chanda 2022). Aromatic compounds also detected in Nitella hyalina, have demonstrated antitumor, antimicrobial, antidiabetic, anti-inflammatory, and antioxidant properties in previous research (Jesus et al. 2019; Remya et al. 2022). The occurrence of phenol, alcohol, amine, alkenes, and aromatic compounds as detected in Chaetomorpha crassa, has also been widely used in skin care products (Kalasariya et al. 2021). Furthermore, the detection of chloro and iodo-functional groups in Nitella hyalina extracts is consistent with the production of halogen-containing compounds in many algae, which have pharmaceutical and nutritional applications, as reported in previous studies (Al-Adilah et al. 2022). Hence, the FT-IR analysis of Nitella hyalina extracts provides valuable insight into their potential pharmacological applications.

Aerobic metabolism results in the production of free radicals and reactive speciesknown as oxidative stress which can harm proteins, DNA, amino acids, and lipids and cause degenerative diseases like atherosclerosis, cancer, and aging. Antioxidants are the chemicals required to stop or reduce this oxidative damage. Natural sources of antioxidants include flavonoids, phenolic acids, tannins, proanthocyanidins, anthocyanins, and other antioxidant compounds. These substances can reduce free radicals and restrain the oxidative processes that underlie degenerative diseases. Since the number of antioxidants in the human body is limited, external sources of antioxidants are necessary when there is a high concentration of radicals in the body. While synthetic antioxidants like BHT (Butylated Hydroxy Toluene) have been used in the past, their use is now restricted due to their toxicity and carcinogenic properties. Seaweeds or algae are a natural source of antioxidants and have been shown to contain bioactive compounds such as alkanes, fatty acids, ketones, phenolic compounds, acrylic acid, phlorotannin, and terpenoids, which have antioxidant properties. The DPPH test was used in this study to assess the antioxidant capacity of both extract of Nitella hyalina. The radical scavenging activity (RSA) values for the n-butanol extract and the ethyl acetate extract, ranged from 34.55 to 46.35% and 35.39–41.79% respectively. Since hydroxyl groups are known to be effective radical scavengers, their presence in the phenolic compounds of Nitella hyalina may account for their antioxidant potential. The present results are supported by earlier research on Nitella flagelliformis (Seal et al. 2015) and Cameroonian spice extracts (Bouba et al. 2010; Chai and Wong 2012), which demonstrated a positive correlation between total phenolic and flavonoid contents and antioxidant potential. Flavonoids and phenolic compounds are the primary antioxidant agents in plants due to their redox properties (Jimoh et al. 2011; Zakaria et al. 2011). Both compounds act as antioxidants by either scavenging or chelating the free radicals (Pourmorad et al. 2006). Based on the results obtained, it is apparent that Nitella hyalina contains significant amounts of flavonoids and phenolic compounds, making it a good antioxidant source. The TPC levels of n-butanol and ethyl acetate in the present study were found to be 1.663 ± 0.768 mg QE/g and 1.043 ± 0.961 mg QE/g, respectively, while the total flavonoid contents of n-butanol and ethyl acetate extract were 2.050 ± 0.143 mg QE/g and 1.730 ± 0.311 mg QE/g, respectively. These findings are similar to the results reported by Seal et al. (2015) for Nitella flagelliformis. Since the discovery of antibiotics and their utilization as therapeutic agents, there has been a prevailing belief within the medical community that these developments would ultimately lead to the complete eradication of infectious diseases (Amenu 2014). However, diseases and their causative agents, which were once thought to be effectively controlled by antibiotics, are now reemerging in novel forms that resist antibiotic treatments (Allen et al. 2014). Incidences of epidemics caused by these drug-resistant microorganisms have become a widespread global issue, giving rise to substantial public health concerns (Sibanda and Okoh 2007) The worldwide proliferation of multi-drug-resistant bacterial strains is progressively diminishing the effectiveness of current drugs and significantly contributing to treatment failures in infections (Pérez-Rodríguez and Mercanoglu Taban 2019). When infections develop resistance to first-choice or frontline antimicrobials, the medical community is compelled to turn to second- or third-line drugs, which are typically associated with higher costs (Sharma 2010). In many economically disadvantaged countries, the high cost of such replacement drugs is prohibitive, resulting in the inability to treat certain diseases in areas where resistance to first-line drugs is widespread (Bhatia and Narain 2010). Faced with such a challenge, there is need to develop alternative approaches in addition to the search for new antimicrobial compounds. Macroalgae are abundant and offer a variety of active compounds, which have potential commercial and therapeutic applications. These chemical compounds have numerous activities, such as antiviral, antioxidant, and antibiotic properties, among others, and their potential impact is still not fully understood. Different species of algae contain varying amounts of these elements, including great amounts of anti-inflammatory, antifouling, antimitotic, and cytotoxic, activities (Duraisamy and Selvaraju). The purpose of the present study was to investigate the biological potential of macroalgae by evaluating its antimicrobial activity. Previous research by De Quiros et al. (2010) and Priyadharshini et al. (2011) has shown that seaweeds are rich sources of various constituents such as tannins, flavonoids, phenolic acids, polysaccharides, carotenoids, bromophenols, which exhibit diverse biological activities. The choice of solvent for extracting these chemical compounds is crucial as different solvents can affect the antimicrobial activity of the extract. Therefore, it is necessary to extract the chemical compounds from various seaweeds using different solvent systems to optimize their antimicrobial potential (Salama and Marraiki 2010). The finding of the present investigation showed that the most potent antimicrobial activity was observed in the n-butanol extract, followed by the ethyl acetate extract. This results were in agreement with those reported by Buedenbender et al. (2020), who tested various extracts of Fucus vesiculosus against microbes and obtained similar outcomes. Ravikumar et al. (2016) evaluated Ulva reticulata against bacterial strains and found that n-butanol was the most effective solvent for extraction. Additionally, our research demonstrated that C. albicans was least inhibited at all concentrations, whereas P. aeruginosa was the most susceptible organism, followed by S. aureus and E. coli. The study’s findings align with previous research conducted by (Salvador et al. 2007). who discovered the antimicrobial properties of Iberian macroalgae and recognized P. aeruginosa as the most susceptible strain. El-Sheekh et al. (2014) and Bhuyar et al. (2020) also obtained comparable outcomes by evaluating the antimicrobial potential of Spirulina platensis and Padina sp., correspondingly. The GCMS examination of the extracts discovered numerous antimicrobial compounds, such as palmitic acid methyl ester, Tetradecanoic acid, (Z)-9-hexadecenoic acid methyl ester, methyl tetradecane, and methyl pentopyranoside, undecane, and ethyl iso-allocate. These results are consistent with the findings of Shobier et al. (2016) and Hu et al. (2015), which demonstrated that methyl esters of fatty acids possess antibacterial and antifungal properties. Moreover, the study conducted by KiliÇ et al. (2007) also emphasized the antimicrobial potency of palmitic acid methyl ester. Through GC-MS analysis it was recorded that heptadecane and hexadecane were the common hydrocarbons present in seaweeds (Sukatar et al. 2006). Several hydrocarbons detected in a recent investigation were found to have inhibitory effects on P. aeruginosa, as reported by Silici and Kutluca (2005) and Wagh et al. (2007). Additionally, Plaza et al. (2010) reported on the antimicrobial and antioxidant capabilities of oleic acids and hexadecane. Fatty acids, acting as anionic surfactants, have also been shown to exhibit antifungal and antibacterial potential (Hayes and Berkovitz 1979). Bakar et al. (2017) previously demonstrated that hexadecenoic acid, the main fatty acid in Sargassum granuliferum, which inhibits the growth of bacteria. Thus the present study concluded that the extracts of Nitella hyalina can be tested as alternative antimicrobial drugs.

Diabetes mellitus is a metabolic disorder with manifestation of hyperglycemia (Al-Awar et al. 2016). Chronic hyperglycemia causes damages to organs like the eyes, kidneys, nerves, heart, and blood vessels (Rahimi-Madiseh et al. 2016). The condition arises from either a genetic or acquired deficiency in insulin production by the pancreas or the ineffectiveness of the insulin produced. It is the result of insufficient insulin secretion, inadequate responsiveness of target cells to insulin, or a combination of these factors (Murugi et al. 2012). Managing this condition necessitates medical diagnosis, treatment, and lifestyle modifications. Predictions indicate that it will become a major contributor to global disability and mortality in the next two decades (Karim and Habib 2022). The challenge of managing diabetes persists globally, with no successful treatment yet identified (Shrivastava et al. 2013). While numerous synthetic medications have been developed for patients, it is noteworthy that complete recovery from diabetes has never been reported (Choudhury and Rajeswari 2021). Furthermore, modern oral hypoglycemic agents often lead to undesirable side effects. Therefore, there is an urgent need for alternative therapies, highlighting the necessity to explore various indigenous plant-based and herbal formulations (Zeng et al. 2020). Therefore in this study, certain compounds were identified through GC-MS analysis, and the hypoglycemic potential of the extract was assessed. The result showed that the blood glucose levels of diabetic animals treated with standard drug (Glibenclamide) and extracts (ethyl acetate & n-butanol) of Nitella hyalina were significantly lower compared to the control group, which had significantly elevated blood glucose levels. All extracts showed a significant decrease in blood glucose levels at α > 0.05, 0.01, and 0.001 at 24 h, with n-butanol being more effective than ethyl acetate extracts. These findings suggest that Nitella hyalina extracts could be useful in treating diabetes-related complications. Non-significant effects were noted for 100 mg kg− 1 of ethyl acetate, whereas 200 mg kg− 1 extract exhibited a significant decrease at α > 0.05 except after 24 h of treatment which showed a significant change in blood glucose level. The study found that higher doses of ethyl acetate (300 mg kg− 1) and all doses of n-butanol caused a significant decrease in glucose levels. The results were supported by (Akbarzadeh et al. 2018; Unnikrishnan and Jayasri 2018) who also reported certain seaweed for their antidiabetic potential. The anti-hyperglycemic action of Nitella hyalina in diabetic rats may be due to the increase in insulin release by interacting with the compounds which had the potential to activate the function of pancreas. This finding were in line with findings documented by (Lakshmanasenthil et al. 2014) and (Unnikrishnan and Jayasri 2018) who reported several components sourced from seaweeds have demonstrated anti-diabetic properties, including polysaccharides, fucoxanthin, polyphenols, as well as certain minerals such as zinc, magnesium, potassium, and calcium. Palmitic acid, a prevalent component in several seaweeds, has been suggested as a potential α-glucosidase inhibitor based on research on its inhibition properties (Xie et al. 2021).

Conclusion

The current research demonstrates the nutritional and phyto-pharmacological potential of Nitella hyalina. Nutritional composition revealed the presence of ash, proteins, lipids and fibers. Considerable amount of phenols and flavonoids were measured. Similarly, several phyto-constituents were detected and verified by GC-MS and FT-IR which contributed to the antioxidant, antimicrobial, and antidiabetic properties of Nitella hyalina extracts. Both the extracts subjected showed a dose dependent hypoglycemic potential in animal models. The positive correlation observed between the phenolic and flavonoids content of the algal extracts and their antioxidant activity, as assessed by the DPPH assay. Therefore, the organic extracts of Nitella hyalina could be used as dietary supplements as well as a promising source of antioxidants, antimicrobials, and anti-diabetic agents. It is recommended to isolate and characterize the bioactive constituents as lead molecules in the development of new drugs.