Nutritional Composition and Pharmacological Activity of Musa balbisiana Colla Seed: An Insight into Phytochemical and Cellular Bioenergetic Profiling

Musa balbisiana Colla belongs to the family Musaceae which is well-known for its nutritional and pharmacological properties. Here, we have analysed the phytochemical content and evaluated the nutritional, antioxidant, anti-glycation, α-amylase, and α-glucosidase inhibition potential. Moreover, for the first time, we have studied the bioenergetic profiles of the bioactive fractions of M. balbisiana seeds extract against oxidative stress-related mitochondrial and cellular dysfunction using XFe24 extracellular flux analyzer. M. balbisiana seeds have high nutritional values with significant levels of carbohydrates, starch, protein, and minerals (Ca, Na, Mg, Cu, Fe, and Zn). Bioactivity-guided fractionation of the methanolic extract of M. balbisiana seeds revealed that the ethyl acetate fraction (EAF) showed the highest antioxidant, anti-glycation, and phytochemical content as compared to other fractions. Moreover, the EAF showed a lower α-amylase inhibition and a higher α-glucosidase inhibitory activity. Most importantly, our GC-MS analyses of EAF revealed the presence of unique and previously unreported 14 phytochemical compounds. A strong correlation between the biological activities and total phenolic/tannin content was observed. In addition, the bioactive fraction of M. balbisiana seeds (EAF) improved the bioenergetic profiles of free fatty acid-induced oxidative stress with a concomitant increase in ATP production, and respiratory and glycolytic capacity. Altogether, our findings suggest that M. balbisiana seeds can be used as a natural supplement to boost antioxidant levels and combat oxidative stress and non-enzymatic glycation.


Introduction
Antioxidant-rich fruits and their by-products promote good health by lowering the risk of non-communicable diseases such as diabetes, cancer, atherosclerosis and neurodegenerative disorders [1,2]. Numerous reports suggest that fruitderived antioxidants can protect against oxidative stress by Rajlakshmi Devi rajlakshmi@iasst.gov.in; rajiasst@gmail.com Island, and North-eastern states (Assam), where it has been incorporated with Assamese social culture since time immemorial [8].
Musa spp. has been used to remedy cough and cold, ulcers, diarrhoea and wound healing [2]. The pseudostem and flower are consumed as vegetables and it is believed to treat diabetes, fever, seizures and leprosy. Other parts like roots, peel and seeds are used for alleviating digestive disorders [9]. M. balbisiana fruit pulp is rich in both macronutrients and micronutrients such as carbohydrates, starch, protein, sodium, potassium, calcium, magnesium, zinc and iron [10]. Different parts of M. balbisiana have been reported for medicinal benefits with significant amounts of bioactive compounds such as gallic acid, epicatechin, rutin, chlorogenic acid, quercetin and apigenin [2,11]. Polyphenols-rich ripe pulp of M. balbisiana has cardioprotective properties and ameliorates cardiac hypertrophy through antioxidant and anti-inflammatory activities [12]. Apiforol, a flavanol compound isolated from the seeds of M. balbisiana, exhibited significant α-amylase and α-glucosidase inhibitory activity which facilitate cellular glucose uptake [13]. Various fatty esters of phytol and n-alkanols, β-sitosterol, stigmasta-5,22E-dien-3β-ol, and epiafzelechin are reported to be present in M. balbisiana seeds [14].
Surprisingly, there is a lack of reports on the nutritional and biological properties of seeds and phytoconstituents present in M. balbisiana seeds. Therefore, in the present study, we have investigated details of the bioactive compounds present in M. balbisiana seeds by GC-MS and their nutritional, antioxidant, anti-glycation, α-amylase and α-glucosidase inhibition potential. Furthermore, we have also studied the correlation between phytochemical content and biological properties. In addition, the bioenergetic profiles were estimated by monitoring the oxygen consumption rate (OCR) and extracellular acidified rate (ECAR) using XFe24 extracellular flux analyzer to understand the protective effect of M. balbisiana seeds against oxidative stressrelated mitochondrial and cellular dysfunction.

Materials and Methods
The materials and methods section is presented as a supplementary online resource.

Proximate Analysis of M. balbisiana Seed
Our analysis revealed that the seed constitutes about 10.4% of the total weight of M. balbisiana fruit. The moisture and ash contents are 9.02 ± 0.05 and 7.22 ± 1.1%, respectively. Carbohydrates were the major macronutrient present followed by protein (Table S1). We observed that, the total carbohydrate content was 27.0 ± 1.09 g/100 g dry weight out of which 19.6 ± 0.28 g/100 g was reducing sugar and 5.27 ± 1.03 g/100 g was starch content. Interestingly, the total carbohydrate content was found to be lower than that of the unripe Musa acuminata flour [4] but it was found to be higher than the raw pulp of M. balbisiana [10]. These observations might be due to the differences in the moisture content of the samples, where the seed and unripe banana flour have 9.02 and 0.9% moisure content respectively, and the raw pulp has more than 70% [4,10]. The crude and dietary fiber content (1.25g and 0.29 g/100 g respectively) of M. balbisiana seed was lower than the unripe pulp of Musa acuminata (21.0 ± 0.5 and 10.3 ± 0.2 g/100 g) where the unavailable carbohydrates were reported to reduce postprandial glycemic index, and promote colonic fermentation followed by the production of short-chain fatty acids [15]. The total protein content of the seed (3.09 ± 0.97 g/100 g) was lower than the unripe Musa acuminata flour but the lipid content was higher (1.10 g/100 g) [4]. As reported by Solis-Badillo et al. [16], the protein content of Musa spp. decreased with the increase in the maturity of the fruit. Micronutrients have an important role in promoting health despite their requirement in small quantities and a diet rich in certain micronutrients helps combat malnutrition [4]. Our results revealed that among the minerals present in M. balbisiana seed, Ca exhibits the highest concentration (17.87 ± 0.7 mg/100 g) followed by Mg (14.53 ± 0.49 mg/100 g), Na (8.96 ± 0.22 mg/100 g), and Fe (4.7 ± 1.17 mg/100 g) (Table S1). Ca, Na and Fe content was higher in M. balbisiana seed as compared to Berangan and Rasthali cultivars of Malaysian Musa spp., however, Mg content was lower [17]. Cu (0.53 ± 0.04 mg/100 g), Zn (0.58 ± 0.08 mg/100 g), and Mn (0.25 ± 0.06 mg/100 g) content of M. balbisiana seeds were similar to those reported from ripe M. balbisiana pulp (0.06 ± 0.01, 0.53 ± 0.03 and 0.21 ± 0.02 mg/100 g respectively) [10].

Antioxidant Activity
Oxidative stress occurs when the production of reactive oxygen species (ROS) exceeds detoxification by the antioxidant defence system. The antioxidant activity of M. balbisiana seed methanolic extract (ME) was measured using DPPH, ABTS, FRAP and phosphomolybdenum assay. Using a single method to determine the antioxidant capacity is inefficient since antioxidants act in different mechanisms either by donating an electron or hydrogen [3]. In the DPPH assay, the ethyl acetate fraction (EAF) showed significantly high antioxidant activity with an IC 50 value of 44.7 ± 0.47 µg/ml followed by n-butanol fraction (nBF) (56.9 ± 0.14 µg/ml), ME (80.6 ± 0.15 µg/ml) and water fraction (WF) (183.4 ± 0.6 µg/ml). However, EAF scavenged 3.7-fold lower than the standard ascorbic acid (IC 50 11.8 ± 0.14 µg/ml) as shown in Table S2. In the ABTS assay, EAF exhibited enhanced radical scavenging capacity (IC 50 13.8 ± 1.2 µg/ml) when compared to nBF, WF and ME which was not significantly distinct from ascorbic acid (IC 50 10.9 ± 0.38 µg/ml) (p > 0.05). The radical scavenging activity of M. balbisiana seed was higher than previously reported from M. balbisiana pulp, where the most active fraction of pulp scavenged 50% free radicals at 47.4 ± 2.4 µg/ml and 69.9 ± 5.7 µg/ml for DPPH and ABTS assay, respectively [10]. A consistent result was observed in FRAP and phosphomolybdenum assay where EAF showed the highest ferric-reducing capacity (IC 50 73.6 ± 0.28 µg/ ml) and molybdenum reduction (IC 50 55.4 ± 1.10 µg/ml) followed by nBF, ME and WF. There is a limited study available on the antioxidant potential of M. balbisiana seeds evaluated by DPPH assay [18].

Anti-Glycation Activity
Under oxidative stress and hyperglycaemic conditions, proteins are non-enzymatically glycated, forming AGEs [19]. Here, we have designed our experiments to mimic the three stages (initial, middle and late) of glycation by BSA-fructose, BSA-methylglyoxal and arginine-methylglyoxal models, respectively. Our results indicated that EAF exhibited anti-glycation activity against all stages of glycation with IC 50 values lower than ME and other fractions. Interestingly, in BSA-fructose assay, the ME, EAF, nBF and WF inhibited 50% glycation at concentration ranges of 191.2 ± 1.5, 110.2 ± 0.2, 140.1 ± 0.9 and 296.6 ± 0.43 µg/ml, respectively, as shown in Table S2. The highest inhibition was observed in EAF, which was significantly higher (p < 0.001) than that of the standard, aminoguanidine. M. balbisiana seed extracts were previously reported to inhibit the protein glycation by glucose more effectively than ascorbic acid with an IC 50 value of 86.48 ± 0.751 µg/ml [13]. Protection of fructose-mediated protein glycation is vital in preventing AGE-associated diseases, as fructose is 10 times more reactive than glucose [20]. Fructose can be generated via a polyol pathway under high blood glucose level or through diet, which in turn glycate the proteins non-enzymatically to form AGE [19]. In the BSA-methylglyoxal model, EAF showed the highest inhibition (IC 50 373.7 ± 3.1 µg/ml) among all the samples. Aminoguanidine (IC 50 360.4 ± 1.4 µg/ml) was more effective in controlling the intermediate stage of glycation than EAF (p < 0.05). Arginine and lysine residues in proteins are prone to glycation by methylglyoxal whose interaction forms an AGE called argpyrimidine. The accumulation of argpyrimidine in the lens causes inflammation leading to the development of cataracts [20]. In the arginine-methylglyoxal assay, EAF inhibited 50% generation of fluorescent AGEs at the concentration of 490.10 ± 3.18 µg/ ml (Table S2). The anti-glycation activity increased in the order WF < ME < nBF < EAF < AG. As evident from the result discussed above, EAF lowered the initial stages of glycation more effectively than the standard drug (Table  S2). However, with the advancement in glycation, the effectiveness decreases. Altogether, our results suggest that EAF can be used as an early glycation inhibitor.

α-Amylase and α-Glucosidase Inhibition Activity
Inhibition of carbohydrate hydrolyzing enzymes is considered to be effective in controlling post-prandial hyperglycaemia [19]. M. balbisiana seed ME and its fractions have potent α-amylase inhibition activity. The nBF has the highest inhibition activity with IC 50 6.7 ± 0.4 µg/ml, which is not significantly different from the therapeutic drug acarbose (IC 50 5.96 ± 0.02 µg/ml) and ME (IC 50 12.4 ± 0.03 µg/ml) as shown in Table S2. Although EAF exhibited the highest antioxidant and antiglycation activity, it showed the least α-amylase inhibition potential (IC 50 210.5 ± 1.1 µg/ml) as compared to other fractions. Furthermore, α-glucosidase inhibition potential was evaluated and nBF has the highest inhibition and lowest IC 50 (20.6 ± 0.6 µg/ml), which is lower than acarbose (IC 50 23.8 ± 1.3 µg/ml, p > 0.05) and ME (IC 50 28.2 ± 1.0 µg/ml, p < 0.01). The α-glucosidase inhibition activity of WF (IC 50 93.3 ± 0.8 µg/ml) is lower than EAF (IC 50 83.9 ± 0.8 µg/ml, p < 0.01). Commercial drugs like Acarbose have strong α-amylase inhibition properties and less α-glucosidase inhibition resulting in the fermentation of undigested carbohydrates, raising different side effects including flatulence, bloating, diarrhoea and abdominal discomfort [19]. Therefore, drugs with strong α-glucosidase inhibition and lower α-amylase inhibition are more desirable and EAF fits these criteria. α-amylase and α-glucosidase inhibition potential presented in this study was higher than those reported from ethanol, water, hexane and acetone extracts of the M. balbisiana seeds [13]. This might be due to the differences in the use of extracting solvents, where choosing solvents that can extract a high quantity of phytochemicals is one of the critical factors in obtaining samples with higher biological activity [21].

Phytochemical Content
The highest phytochemical content was observed for the EAF as shown in Table S3. Total phenolic content (TPC) of EAF and nBF were not significantly different (61.53 ± 0.11 and 61.25 ± 0.21 mg GAE/g, respectively) followed by ME request by producing more ATP [25]. The incapacity of the FFA-treated cells to respond to an energetic demand was restored with EAF pre-treatment. The lower concentration of EAF (25 µg/ml) was more effective in protecting against mitochondrial dysfunction by increasing ATP production, MR and SRC than the higher concentration, EAF 50 µg/ml and the therapeutic drug, metformin (60 µM). This is the first study to report real-time OCR data of M. balbisiana seeds using an XFe extracellular flux analyser.

EAF Improves FFA-induced Glycolysis Stress
Changes in the glycolytic pathway or abnormal glycolysis impact mitochondrial functions and generate endogenous dicarbonyl compounds, which might lead to neurodegenerative diseases and ageing [26]. Cells can switch between glycolysis and oxidative phosphorylation depending on their energy demand. The glycolysis stress test profile is shown in Fig. S3a. We have observed that FFA-treated cells were not able to undergo glycolysis effectively even after injecting saturated amount of glucose (Fig. S3b). Next, we found that FFA treatment induced oxidative stress as well as hampered cell's ability to shift the energy production from mitochondrial respiration to glycolysis after oligomycin treatment. This treatment affected both glycolytic capacity (GC) and glycolytic reserve (GR) (p < 0.001) (Fig. S3c-d). However, a significant increase in glycolysis and GC was observed in EAF-treated cells (p < 0.01). Furthermore, EAF (25 µg/ ml) pre-treatment improved the glycolytic function of cells by increasing the GR (p < 0.05). Non-glycolytic acidification was also measured (Fig. S3e). A significant increase in glycolysis-induced cell proliferation in human adipose stem cells was reported to act as an anti-ageing effect [27]. EAF improved the overall metabolic dysfunction by improving the mitochondrial and glycolytic function. This is the first study to report real-time ECAR data using an XFe extracellular flux analyzer to support the efficacy of the bioactive fraction of M. balbisiana seeds in regulating stress-mediated glycolysis.

Conclusion
Musa balbisiana seeds are a nutrient-rich part of the fruit containing plenty of carbohydrates, protein, fats and mineral content. M. balbisiana has significant antioxidant, anti-glycation and anti-hyperglycemic effects via inhibiting α-amylase and α-glucosidase enzymes. In the present study, the GC-MS analysis of the bioactive fraction of M. balbisiana seed ME revealed several bioactive compounds having pharmacological properties including methyl palmitate, methyl linoleate and γ-sitosterol. There is a strong (59.5 ± 0.28 mg GAE/g) and WF (56.4 ± 0.71 mg GAE/g). However, total flavonoid content (TFC) was significantly varied among the tested samples. EAF has the highest TFC (6.00 ± 0.04 mg QE/g) followed by nBF, ME and WF (p < 0.001). The phenolics and tannin content of M. balbisiana seed ME was higher than the root, stem and inflorescence parts reported by Kalita et al. [22], but lower than the pulp [10]. In consistence with TPC and TFC, the tannin content was significantly high in EAF (59.43 ± 0.55 mg CE/g, p < 0.001). Tannins are particularly rich in fruits, nuts, vegetables and beverages such as tea, wine and cider. Although tannins have been misunderstood as anti-nutrients that compromise proteins and mineral absorption, recent studies reported several biological properties including antioxidant, anti-microbial, and anti-inflammatory [23].

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
GC-MS analysis of EAF is provided in the supplementary online resource.

Correlation Between Phytochemical Content and Biological Activity
Pearson's Correlation analysis is presented in the supplementary online resource.

Cell Viability Assay
The cell viability assay is presented in the supplementary online resource.

EAF Improves FFA-induced Mitochondrial Dysfunction
It is known that free fatty acid (FFA) induces oxidative stress by increasing ROS generation and hampering mitochondrial and cellular function [24]. Therefore, here we evaluated the effect of EAF on mitochondrial function and cellular bioenergetics under FFA-induced oxidative stress by directly measuring OCR in real-time on XFe24 extracellular flux analyzer (Fig. S2a). Our results revealed that the basal respiration of FFA-treated cells was significantly reduced (p < 0.01) due to the oxidative stress induced by FFA (Fig. S2b). Treatment with FFA significantly decreased maximum respiration (MR) (p < 0.001), ATP production (p < 0.001) and spare respiratory capacity (SRC) (p < 0.01) (Fig. S2c-e). MR showed the maximum rate of respiration cells could achieve by oxidizing substrates under physiological energy demand. SRC is the cell fitness indicator and offers the mitochondrial capacity to respond to an energetic correlation between phenolic and tannin content with biological activities suggesting the major contribution of phenolic compounds to the antioxidant and anti-glycation activities. Furthermore, we have shown that the bioactive fraction of M. balbisiana seeds improves the mitochondrial bioenergetics and glycolytic function of oxidative stressinduced cells by improving the OCR and ECAR. Our findings strongly suggest that M. balbisiana seeds could be recommended as dietary supplements and nutraceuticals to improve health and provide protection against chronic diseases. However, further research is required to characterize phenolic compounds present to understand the mechanism of action.