Extraction yield, total phenolic and flavonoid content
The yield of methanol, hexane, and ethyl acetate fractions of the Lespedeza cuneata was shown in Table 1. Herein, a different solvent system was selected for the partition of L.cuneata. This different solvent system has the prime role in influencing the extraction yield and the quantity of the phytoconstituents, and it is also reflecting the upcoming findings. In particular, the ethyl acetate fractions have the maximum extraction yield of 5.59±2.01 mg followed by methanol (2.67 ±0.98 mg) and hexane (1.81±0.53 mg). It shows the ethyl acetate fractions were considered a suitable choice for the optimum extract yields from the leaves part of the L.cuneata. In addition, total phenolic and flavonoid content was measured by the standard spectroscopic method (Table 1). It revealed that the total phenolic and flavonoid content were significantly (p<0.05) higher in Lc-EAF than Lc-HF and Lc-MF. Comparatively, the Lc-EAF has a higher amount of total phenolic (395.54±5.04 mg/g extracts) and flavonoid content ( 209.63±0.63 mg/g extracts). Previous studies also revealed the L.cuneata has a higher amount of flavonoid content. Especially the ethyl acetate fraction has a higher amount of phenolic and flavonoid content due to its polarity nature. Our results endorsed these findings (Yoo et al. 2015, Zhang et al. 2016). In line with previous studies suggested that a higher yield of polyhydroxy compounds, glycones, and other organic compounds were extracted from the ethyl acetate fractions due to their polarity nature (Kifayatullah et al. 2015). These substances are responsible for various pharmacological properties, including antioxidants, antidiabetic, antiviral, anticancer, anti-inflammatory activities (Cho et al. 2009, Kim &Kim 2010, Lee et al. 2013, Yoo et al. 2015).
Antioxidants and free radical scavenging activity
A variety of in vitro studies assessed the antioxidant activity of the plant material. DPPH, ABTS, hydrogen peroxide scavenging, hydroxyl radical scavenging, and ferric reducing power are the most regularly used methods. Based on this, the free radical and antioxidants scavenging abilities of the active fraction of the L.cuneata associated DPPH, ABTS, hydroxyl radical and reducing power assay was studied (Table 2). We found that the Lc-EAF fraction was effectively inhibited the ABTS with the IC50 value of 58.32±4.21 µg/mL, whereas Lc-HF and Lc-MF showed the IC50 value of 149.86±10.73 µg/mL and 237.23 ±19.35 µg/mL, respectively. Likewise, Lc-EAF fraction (IC50 value of 99.54±4.43 µg/mL) were affording the dose-dependent inhibiting DPPH radical scavenging activity. On the contrary, the DPPH radical scavenging activity was comparatively higher than the ABTS assay. As a result, the scavenging activity of L.cuneata fraction in decreasing order was Lc-EAF > Lc-HE > Lc-MF. It showed that ethyl acetate fractions are more active than other fractions (p<0.05). These observations suggest a close linkage between the phytochemical content and antioxidant activity, such as the radical-scavenging effect on DPPH, ABTS assay (Fernandes de Oliveira et al. 2012). Similarly, dose-dependent hydroxyl radical scavenging activity was observed in our study. IC50 value the hydroxy radical scavenging properties of Lc-EAF (103.16±7.34 µg/mL), Lc-HF (198.44±6.11 µg/mL) and Lc-MF (452.95± 19.84 µg/mL). On the contrary, the IC50 value of the reducing power activity of Lc-EAF (254.37±35.52 µg/mL), Lc-HF (505.81±24.91µg/mL), and Lc-MF(952.62±15.67 µg/mL) showed to substantial inhibition activity. At the highest concentration of 1000 µg/mL, Lc-EAF, Lc-HF, and Lc-MF showed considerable antioxidant activity. These results revealed that the antioxidants and free radical scavenging potential of L.cuneata were increased with the increasing concentrations.
α -amylase and α -glucosidase inhibition assay
To explore the inhibitory effect of L.cuneata, an in vitro α-amylase and α-glucosidase inhibition assay was performed, and the results were presented in Table 2. The inhibitory action of the α-amylase against the active fraction of L.cuneata was found to be dose-dependent from 10 to 1000 µg/ml concentrations. Lc-EAF showed the lowest value, which indicates the increasing inhibitory activity of the enzyme. The IC50 value of the α-amylase inhibitory activity of Lc-EAF (205.32±23.47 µg/mL), Lc-HF (407.85±25.54 µg/mL), and Lc-MF (682.23±30.86 µg/mL) showed the considerable enzyme inhibitory activity. IC50 value of the α-glucosidase inhibitory activity of Lc-EAF (105.32±13.93 µg/mL), Lc-HF (286.80±18.86 µg/mL), and Lc-MF (403.52±20.17 µg/mL). From the results, it was clear that the phenolic enriched ethyl acetate fraction of L.cuneata was much more effective in inhibiting the activity of α-amylase and α-glucosidase. Consequently, it might be envisaged to be an effective strategy to control or treat DM (Honda &Hara 1993). Other results were broadly in line with the phytocompound can inhibit the alpha-amylase and alpha-glucosidase by neither interacting nor inhibiting certain positions of the enzyme (Rohn et al. 2002, Unuofin et al. 2017).
To verify the biocompatibility nature of the Lc-EAF, we examined the WST based cytotoxicity assay in a non-cancerous cell line of NIH3T3 cells. NIH3T3 cells were subjected to increasing concentration of Lc-EAF, the cell viability was monitored for 24 h incubation. The results revealed that Lc-EAF has fewer cytotoxic effects on NIH3T3 cells. i.e., 1.97±0.87, 3.19±0.46, 6.60±0.18, 8.22±0.47, 9.49±0.69, 10.17±0.56 and 14.85±1.23 µg/mL at a concentration of 4.68, 9.37, 18.75, 37.5, 75, 150 and 300 µg/mL. From these findings, it could be suggested that Lc-EAF does not have any toxic nature compounds (Fig. 1a). Many previous studies revealed that most plant-based phytocompounds have biocompatible, non-toxic effects and immensely enhance cell viability. For instance, our previous studies reported that the active fraction of Helianthus tuberous considerably enhances the cell viability of non-cancerous cells (Mariadoss et al. 2021). The fermented and non-fermented extracts of L.cuneata exposure surmised that the Hs68 (human dermal fibroblast cells) viability does not significantly differ from the untreated cells. A similar finding was also documented by Park et al. (2020), who revealed that the aqueous extracts of A.manihot increase cell proliferation (Park et al. 2020).
Glucose uptake in IR-HePG2 cells
The liver is a vital metabolic organ of the body accountable for the normal metabolic pathway. The imbalance in liver metabolism, including glucose and lipid homeostasis, leads to diabetes mellitus through insulin resistance (IR). Hence, developing a stable and reliable IR hepatocyte model for researching the molecular mechanism of IR in diabetic treatment (Röder et al. 2016). Based on this, the liver cancer cell line of HepG2 was ideally used to examine IR because the hepatic cells have similar morphological and biochemical features. The hepatic embryonal cancer cells of HepG2 were ideally used cell lines to examine the IR because the hepatic cells have similar morphological and biochemical features (Donato et al. 2015). Several studies also endorsed this model. We were developed IR- HepG2 using a culture media containing high glucose medium and 5x107 M of Insulin, and these established cells were used for this study. Our results explored that the glucose uptake in the IR cells was much lower than in the control cells (p<0.001). In comparison to the IR cell, the glucose absorption was significantly boosted after treatment with Lc-EAf in a dose dependent manner (p<0.05). Among the tested concentration, 75 µg/mL of Lc-EAf showing about 68.23% of glucose uptake. On the contrary, the uptake levels were considerably lower for the concentrations 150 and 300µg/mL (Fig. 1b). However, in line with the findings of Nomura et al. (2008), it can be suggested that the bioactive compounds including quercetin, kaempferal, luteolin, and apigenin can suppress the IR signaling pathway through the activation of the AKT pathway and inhibition insulin phosphorylation (Nomura et al. 2008).
Of note, the Lc-EA fraction was shown to have the most potent radical scavenging ability and intriguing antidiabetic properties. It could be owing to the enrichment of bioactive compounds. As a result, for the UPLC-QTOF-MS/MS analysis, Lc-EA fractions were used. The findings were presented with tentatively identified phytocompound along with formula, RT (min), (M-H)-, m/z, Response, Mass Error (ppm), and Fragmentation (m/z) (Fig. 2 and Table 3). The identified phytocompounds were characterized into five groups: Flavonoid, Flavonoid glycosides, Phenolic glycosides, Lignan glycosides, and saponin compounds. From the Lc-EA fractions, 28 compounds were identified by UPLC-QTOF-MS/MS assessment, including nine Phenolic glycosides (vanillic acid 4-O-b-D-glucoside, glucosyringic acid, trans-o-coumaric acid, ferulic acid glucoside, Isolariciresinol 9'-O-beta-D-glucoside, cuneataside A, cuneataside D and triterpene glycoside), nine flavonoid glucosides (Luteolin di-C-hexose, Taxifolin O-glucopyranside, Isorhamnetin-3-O-β-rutinoside, Apigenin C-pentosyl-C-hexoside, apigenin di-C-pentose, Apigenin C-hexoside-O-pentoside, Apigenin di-C-hexose, Apigenin O-hexose) and seven flavonoids ([Iso]Orientin, Quercetin-O-rhamnose-O-glucoside, [Iso]vitexin, Kaempferol-3-glucuronide, Nicotiflorin, Quercetin-3-O-β-D-glucopyranoside, Luteolin O-rutinoside, Roseoside). The bioactive organic fraction of Lc-EAF also contains lignan glycosides of Secoisolariciresinol-4-O-β-D-glucopyranoside and the saponin nature of the triterpene glycoside. Some of the identified phytocompound of Glucosyringic acid, Vanillic acid 4-O-b-D-glucoside, Ferulic acid glucoside trans-o-Coumaric acid, 2-glucoside along with the other phenolic compound has a significant therapeutic activity including antidiabetic activity (Shahidi and Yeo, 2018). The next category of flavonoids including Luteolin di-C-hexose, Taxifolin, Isorhamnetin-3-O-β-rutinoside, [Iso]Orientin, Quercetin-O-rhamnose-O-glucoside, [Iso]vitexin, Nicotiflorin, Kaempferol-3-glucuronide, Quercetin-3-O-β-D-glucopyranoside, and Luteolin O-rutinoside, which are to have a remarkable antidiabetic, antimicrobial, antimutagenic, and anticancer activity (Kumar &Pandey 2013, Middleton et al. 2000). In addition, there are four apigenin derivates abundantly present in the Lc-EA fractions. It is well known that flavonoid based phytocompounds has significant antidiabetic activity in several types of cell line and experimental animals (Malik et al. 2017, Qin et al. 2016).
Lipinski's rule was adopted to explore the drug-likeness properties of the isolated compounds from Lc-EAF using a web tool of SwissADME. Also, ADMET-SAR online server predicted the toxicological properties of the selected compounds (Sup. Table 1 & 2). The analysis revealed the selected compounds (Vanillic acid 4-O-b-D-glucoside, Glucosyringic acid, trans-o-Coumaric acid 2-glucoside, Ferulic acid glucoside, Roseoside and Isovitexin) are non-carcinogenic and had low rat toxicity and acute oral toxicity values. However, the phytocompound of trans-o-Coumaric acid 2-glucoside, Ferulic acid glucoside showed an AMES toxicity. Besides, the selected compounds also underwent the PASS online tool to screen the diabetic-related activities, and the potential compounds displayed a higher Pa value than Pi (Sup. Table 3). In silico docking analysis showed that selected phytocompounds were acts as a potential inhibitor of α-Amylase and α-Glucosidase. The interaction poses of Vanillic acid 4-O-b-D-glucoside, Glucosyringic acid, trans-o-Coumaric acid 2-glucoside, Ferulic acid glucoside, Roseoside and Isovitexin with the target protein of α-Amylase (Fig. 3) and α-Glucosidase (Fig. 4 and Table 4). Our studies revealed that among the tested compounds, trans-o-Coumaric acid 2-glucoside binds with the α-amylase with higher affinity with -9.99503 kcal/mol of docking score. Trans-o-Coumaric acid 2-glucoside were directly bound to the amino acid residue of Gly304, Arg 346, Thr 314, Asp 317, Arg 267 in IOSE. The other residual of Phe 348, Gly 309, Asp 353, Arg 303, Gln 302, Trp 316, Leu 313, Ile 312, Trp 269, Ala 310 and Gly 351 also showed a hydrophobic and other interaction with trans-o-Coumaric acid 2-glucoside. Besides, Glucosyringic acid has a docking score of -8.59kcal/mol with 3A4A (α-glucosidase). It was directly bound with the amino acid residues of Leu 434, Trp 402, Lys 400, Tyr 407, Asn 401 in 3A4A through the hydrogen bonding. The other residual of Val 404, Pro403, His 444, Phe 399, AlA 438, AsN398, Thr 358, Leu 439, Ile 437, Glu 435, Trp 402 also showed a hydrophobic and other interaction with Glucosyringic acid. The docking results of other tested phytocompounds docking results were shown in Fig. 3 & Table 4.
In vitro antidiabetic study
Diabetes is characterized by high blood glucose levels, excessive urination, excessive thirst, and weight loss despite increased appetite. Table 5 shows the blood glucose level, body weight, kidney, and liver weight of the experimental animals in each group. In the end, the STZ alone treated mice showed an increased blood glucose level (387.21±9.34 mg/dL). It indicates the ICR mice in diabetic status, whereas these levels were significantly reduced in Lc-EAF treatment. Besides, the body weight and organ weight were significantly decreased in diabetic animals compared to non-treated control mice. The STZ alone treated mice lost the bodyweight of -3.12±0.93, and the relative liver weight was found to be 4.17±0.81. These levels were significantly lower in the rest of the other experimental groups. Lc-EAF treatment has significantly balanced the body weight and organ weight in diabetic mice. We also monitored the animals' caloric intake and water intake daily while participating in the study. A significant reduction in body weight was seen in STZ-induced diabetic mice instead of their control counterparts(Saadane et al. 2020). Lack of Insulin may account for this, as it causes glucose to be unable to enter the cell, thereby increasing the percentage of sugar in the blood. To eliminate excess sugar, the body attempts to rid itself of the sugar through excretion in the urine (Cantley &Ashcroft 2015). An increase in urine production will lead to dehydration and weight loss. While hyperglycaemic STZ-induced ICR mice were found to have significantly increased food and water intake, they also found that the augmented food and water intake of these mice are likely due to a reduction in glucose utilization and significant loss of glucose in the urine, resulting in a stimulus to eat and drink (Data are not shown). The improvement in polyphagia, polydipsia, and preventing weight loss seen in STZ-induced ICR mice was strongly correlated with improved metabolic status and intestinal absorption in Lc-EAF-supplemented mice.
The low dose of STZ (50 mg/kg b.wt) causes pancreatic β-cells to be destroyed in rats, which results in insufficient insulin secretion. This model mimics the clinical condition of type 2 diabetes. The level of plasma glucose increased while the level of Insulin decreased (Table 5). Lc-EAF's ability to stimulate insulin secretion from the remnant β-cells and increase glucose utilization by the tissues is responsible for reducing fasting plasma glucose levels in diabetic rats. These findings were supported by evidence that insulin secretion increased in rats with diabetes due to Lc-EAF treatment and our histopathological analysis revealed a rise in the number of insulin-secreting cells in pancreatic islets. We also discovered that Lc-EAF could protect the pancreatic islets from free radical-induced damage by STZ by acting as a scavenger.
The current scenario has shown that elevated levels of hepatic lipids are commonly found in people with diabetes, which can serve as a valuable risk factor for cardiovascular problems. Additionally, increased concentrations of fatty acids also promote the oxidation of fatty acids, resulting in more acetyl CoA and cholesterol, which causes hypercholesterolemia (Martín-Timón et al. 2014). As indicated by increased plasma cholesterol, TGs, LDL, and diminished HDL, dyslipidemia was detected in STZ-induced ICR mice in the current study. Because of this, we discovered that Lc-EAF could treat hyperlipidemia by reducing cholesterol, TGs, LDL, and increasing HDL levels in diabetic mice (Table 6). Because of an increase in insulin secretion, there was a reduction in cholesterol synthesis, which accounts for the anti-hyperlipidemic effect. In addition, Lc-EAF is thought to affect lowering cholesterol levels by inhibiting the uptake of cholesterol from the intestines by binding to bile acids, which subsequently increases bile acid excretion and decreases cholesterol absorption from the intestines. These findings concur with Kim et al. , who reported that hesperetin reduced the hepatic lipid profile in hypercholesterolemic hamsters, resulting in reduced blood lipid levels (Kim et al. 2010).
We assessed microscopic histological observations of the endocrine pancreas to learn whether biochemical modifications led to structural changes. After a treatment period with menthol, histological assessment of the pancreas revealed significant improvement in both the changes in the islets and the numbers of pancreatic β-cells. The number of insulin-producing β-cells in STZ-injected ICR mice were diminished, which lowered the amount of insulin in the blood. Interestingly, the application of Lc-EAF to STZ-induced ICR mice resulted in improvements in islet cell rejuvenation and increased insulin secretion, suggesting that Lc-EAF can defend and repair pancreatic β-cells from free radical exploitation (Fig. 5). Because of this, Lc-EAF could be of great help in helping to repair pancreatic β-cell damage and assist in the production of Insulin. Increased glucose utilization in diabetic rats mediated by the promotion of b-cell regeneration and insulin secretion in the pancreas explains the antihyperglycemic effect of Lc-EAF.
Although in the current assessment, diabetic rats have demonstrated hepatic damage as well, current research also points to liver dysfunction and changes in circulating enzymes as additional contributors to hepatic injury. Decreased blood insulin, primarily due to leakage of these enzymes from the liver cytosol into the bloodstream, led to elevated ALT, AST, and ALP levels in the serum (Ollerton et al. 1999). Experimental ICR mice have significantly higher levels of ALT, AST, and ALP compared to normal mice. Our results showed that administration of Lc-EAF prevented the rise in hepatic injury enzymes beyond normal levels, which may be due to the hepatoprotective effects of Lc-EAF (Table 7). Histological studies revealed that Lc-EAF improves cellular liver damage, and so it successfully handles diabetic complications (Fig. 6).
Next, we found out that BUN and creatinine are excreted in the urine along with urea nitrogen. The presence of this waste product may indicate enhanced protein breakdown in both the liver and plasma in experimental diabetes (Ozcan et al. 2012). The current study discovered that elevated BUN levels are indicators of renal dysfunction in hyperglycemic mice. In diabetes, increased serum creatinine levels indicate a decreased GFR (Table 7). This study's finding suggests that extracts possess the potential to attenuate renal injury caused by a hyperglycemic state, which is linked directly to the antioxidant capacity of this extract. Our study's findings on Lc-EAF, as well as cutting-edge research on this extract and its significant benefits for treating diabetic nephropathy, have been proven to be accurate. The administration of Lc-EAF showed significantly improved STZ-induced histopathological changes in the kidneys of the ICR mice and minimal tubular damage and less necrotic damage (Fig. 7). The biochemical findings corroborate histopathological findings, and the biochemical results indicate the potential nephroprotective properties of Lc-EAF.