1. Introduction
The Ayurvedic herb
Kalanchoe pinnata (Synonym-
Bryophyllum pinnatum), also known as the life plant, magic leaf, and cathedral bells, is recognized for its wide range of therapeutic properties [
1]. Natural resources, including medicinal plants, have gained increasing attention in healthcare as potential solutions for various ailments [
2]. Phytotherapy, utilizing plant-based treatments, has shown promising outcomes in numerous diseases, contributing to its growing popularity [
3].
Kalanchoe pinnata exhibits a wide range of pharmacological activities documented in scientific literature. It demonstrates potent anti-inflammatory properties by inhibiting inflammatory mediators and modulating key signaling pathways involved in the inflammatory response. It has also been established for its antioxidant, antileishmanial, hepatoprotective, nephroprotective, antimutagenic, antiulcer and antibacterial activities. Additionally, it shows promising analgesic and antinociceptive effects, potentially providing relief from pain and discomfort [
4,
5]
Within the context of the growing global predicament of obesity caused by factors such as excessive caloric intake and sedentary lifestyles, conventional interventions like anti-obesity pharmaceuticals face limitations and safety concerns [
6]. Consequently, exploring natural compounds with potential anti-obesity effects becomes imperative.
Kalanchoe pinnata, with its captivating attributes and untapped potential in combating obesity, presents a compelling avenue for investigation as it has not been studied for its anti-obesity potential till date.
Oxidative stress, closely linked to the development of ailments such as cancer, cardiovascular disease, and neurological disorders, can be mitigated through the deployment of antioxidant defense mechanisms encompassing both enzymatic and non-enzymatic antioxidants. Furthermore, the association between utilizing antioxidative traits derived from natural sources and the prevention of non-communicable diseases, including diabetes and obesity, has spurred interest in herbal medicines as potential therapeutics [
7,
8,
9,
10]
Given the existing evidence on phytochemicals, it is imperative to explore the potential of
Kalanchoe pinnata in combating obesity, an underexplored area of research. To address the gaps in this field, a hypothesis was formulated prior to the study’s design, aiming to explore all possible avenues. An optimized method of extraction and isolation was developed, followed by the selection of a potent fraction through a series of in vitro investigations, which will be further examined through in vivo experiments. Additionally, the most potent fraction will be identified and characterized using HPTLC MS/MS analysis, and the identified compounds will undergo in silico testing via molecular docking with specific proteins [
11,
12].
2. Results and Discussion
2.1. Bioactive Guided Fractionation and Their TLC Analysis
A total of eight bioactive fractions were obtained through flash chromatography, meticulously designated as fraction F1 to F8, which underwent subsequent rigorous examination via TLC analysis for further in-vitro investigations. The optimal solvent composition of a 6:4 ratio of toluene and ethyl acetate was precisely determined to yield optimal results in the TLC experiments. Notably, among the eight fractions, namely F1, F2, F3, and F4, a pronounced and distinct separation of compounds was prominently observed, underscoring their potential. Conversely, fractions F5, F6, F7, and F8 exhibited minimal to negligible separation of compounds. Based on the discernible separation of compounds, fractions F1-F4 were meticulously selected for subsequent in-vitro studies, recognizing their promise for further exploration and evaluation.
2.2. Phytochemical Assessment
In order to determine quantitative analysis of the fractions, three parameters namely total steroidal content, total phenolic content and total flavonoid content were used. Quantitative content values were determined individually for each fraction. Specifically, fractions F1, F2, F3, and F4 evinced flavonoid content of 78.79, 59.74, 31.68, and 39.71 QE mg/gm, respectively. Notably, the total phenolic content of fractions F1, F2, F3, and F4 was observed to be 61.58, 49.08, 18.21, and 11.28 GAE mg/gm, correspondingly. Additionally, the total steroidal content of fractions F1, F2, F3, and F4 was discerned to be 78.21, 59.27, 27.37, and 28.89 BSE mg/gm, respectively.
2.3. Antioxidant Assays
Different bioactive fractions derived from Kalanchoe pinnata leaves extract were assessed for their antioxidant activity using DPPH, nitric oxide, hydrogen peroxide, and ABTS assays to calculate their IC50 values. The results demonstrated that fractions F1 and F2 exhibited the most potent scavenging activity in all the assays. Specifically, the ABTS assay revealed that fraction F1 had an IC50 value of 1.41±0.25 µg/ml, while fraction F2 had an IC50value of 1.11±0.26 µg/ml, which were significantly more potent than fractions F3 and F4. Similarly, in the Nitric oxide assay, fractions F1 and F2 showed the significant scavenging activity, with IC50 values of 1.47±0.21 and 1.38±0.03 µg/ml, respectively, compared to fractions F3 and F4 with IC50 values of 3.08±0.28 and 2.58±0.25 µg/ml. Additionally, fractions F1 and F2 exhibited the most potent scavenging activity in the Hydrogen peroxide assay with IC50 values of 1.61±0.25 and 1.37±0.23 µg/ml, respectively, compared to the other fractions. Similarly, in the DPPH scavenging assay, fractions F1 and F2 demonstrated the potent scavenging activity, with IC50 values of 1.17±0.08 and 1.84±0.05 µg/ml, respectively, compared to fractions F3 and F4 (shown in
Table 1). In summary, the results indicate that fractions F1 and F2 possess remarkable antioxidant properties and exhibit potent scavenging activity in all assays. These findings suggest the potential use of Kalanchoe pinnata leaves extract fractions as natural antioxidants to combat obesity.
2.4. Enzyme Inhibition Assay
The enzymatic properties of four fractions (F1, F2, F3, and F4) of Kalanchoe pinnata leaf extracts were studied to evaluate their enzyme inhibition activity using pancreatic lipase, alpha amylase, and glucosidase assays. The results of the experiments demonstrated that F1 and F2 exhibited the most potent enzyme inhibition activity compared to fraction F3 and F4. Notably, F1 and F2 demonstrated remarkable enzyme inhibition activity in the pancreatic lipase assay, with IC50 values of 2.20±0.23 and 1.23±0.08 µg/ml, respectively compared to F3 and F4 with IC50 values of 4.07±0.28 and 2.12±0.17 µg/ml, respectively. Furthermore, F1 and F2 demonstrated significant activity in the alpha amylase assay, with IC50 values of 1.24±0.03 and 1.87±0.02 µg/ml, respectively, while F3 and F4 exhibited lower activity. The glucosidase assay produced similar results, with F1 and F2 demonstrating potent enzyme inhibition activity with IC50values of 1.12±0.25 and 1.17±0.37 µg/ml, respectively. These observations suggest that the F1 and F2 fractions of Kalanchoe pinnata leaf extracts possess potent enzyme inhibition abilities and have the potential to be developed into therapeutic agents for metabolic disorders (as shown in
Table 2).
2.5. Effect of Potent Bioactive Fractions on Body Weight
In the course of the study, it was observed that the corpulent mice exhibited a noteworthy augmentation in their body mass in contrast to the normal mice. However, selected optimized fraction of
Kalanchoe pinnata was administered at a dosage of 80 mg/kg, denoted as F1 and F2, respectively. The treatment with F1 and F2 fractions resulted in a marked mitigation of the escalating body weight, as compared to the control group. Interestingly, the fraction F2 demonstrated a more pronounced effect, displaying a significant decrease in the weight gain when compared to fraction F1, as illustrated in
Table 3. This finding highlights the potential of selected fractions in tackling obesity-related concerns.
2.6. Effect of Potent Bioactive Fractions Waist-Hip Ratio
Throughout the course of the experiment, there was a remarkable enhancement in the Waist Hip Ratio (WHR) across all treatment groups. Interestingly, a notable escalation in WHR was observed in obese mice when compared to their normal counterparts. However, the administration of selected fractions (F1 & F2) at a dosage of 80 mg/kg proved to be immensely effective in reducing WHR in comparison to the control group. In fact, the outcome was found to be similar to orlistat, a widely used standard drug. These findings are of immense significance as they demonstrate the therapeutic potential of
Kalanchoe pinnata fractions in the management of obesity (
Table 4).
2.7. Effect of Potent Bioactive Fractions on Biochemical Parameters
The study evaluated the impact of different fractions on various biochemical parameters in obese mice. The findings indicate that all treated groups exhibited significant reductions in total cholesterol, triglycerides, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) levels when compared to the obese control group.
Furthermore, the results of the overall biochemical analysis revealed that fraction F2 demonstrated the most potent activity as compared to fraction F1. It is worth noting that the specific compositions of these fractions were not disclosed and the precise mode of action that led to the observed effects remains unclear. Nonetheless, the findings suggest that Fraction F2 may have therapeutic potential in the management of obesity-related metabolic disorders.
This study demonstrates that the assessed fractions exerted beneficial effects on several biochemical parameters in obese mice, including reduced total cholesterol, triglycerides, LDL and VLDL levels. Additionally, the findings highlight the most potent activity of fraction F2 in comparison to fraction F1, indicating a potential avenue for further research and development of new therapeutic agents (as shown in
Table 5).
2.8. Histopathological Analysis
A meticulous histopathological examination was conducted to scrutinize plausible modifications and perturbations in the adipose tissue, liver, and kidney of different groups of animals. Examination of liver histology in the normal control group revealed a typical architectural composition and thriving hepatocytes, whereas the obese group evinced a profound state of steatosis characterized by an increased quantity of lipid droplets. Analysis of liver sections from mice treated with the conventional therapeutic drug (orlistat) demonstrated a reduction in the incidence of normative liver steatosis, ascertained by a decline in the count of lipid droplets (
Figure 1c). Upon comparison with the obese control group (
Figure 1b), the bioactive fractions (F1 and F2) were shown to exert a substantial effect on liver steatosis leading to a diminished number of lipid droplets, as explicitly depicted in (
Figure 1d,e). Adipose tissue histology showed that the obese control group had an accumulation of enlarged adipocytes (
Figure 2b). However, enlarged adipocytes were reduced and almost restored to normal in the standard and F2 treatment groups, as shown in (
Figure 2e). The bioactive fractions treated group F1 also showed smaller adipocytes than the obese group (
Figure 2d). The kidney tissue’s histology of mice in the normal group showed normal and healthy glomeruli, proximal and distal tubules (
Figure 3a), whereas the MSG- HFD (Monosodium glutamate- High fat diet) treated group showed distorted proximal and distal tubules along with shrunk glomeruli, as shown in (
Figure 3b). The test (
Figure 3c) and fraction F2-treated groups (
Figure 3e) had more repaired and healthy glomeruli and healthy proximal and distal tubules compared to obese treated group.
The overall investigation demonstrated that standard and fraction F2 and F1 treatment had a better effect on repairing liver, kidney, and adipose tissue. Fraction F1 also demonstrated a better effect than the obese control group, but the most potent activity was observed in the fraction F2-treated group.
2.9. HPTLC and HPTLC-ESI MS/MSn (Retro Diels-Alder Fragmentation)
The cutting-edge CAMAG TLC Visualizers 2 have proven to be a valuable tool in separating and analyzing compounds based on their Rf values. In this study, four compounds in the most potent fraction F2 were separated and further examined using mass spectroscopy. Through careful analysis of the resulting data, four of these compounds were tentatively identified as Quercetin (found m/z- 301, Rf- 0.24 and abundance -38.59), Stigmasterol (found m/z- 395, Rf-0.58 and abundance- 19.21), Sitosterol (found m/z-397, Rf- 0.71 and abundance- 12.21), and Thiamine (found m/z-266, Rf- 0.39 and abundance- 27.48), which were confirmed through retro Diels-Alder fragmentation data as shown in
Figure 4 and
Table 6. However, these compounds have already been established earlier. These findings were further validated by cross-referencing the obtained spectral data with Chemspider, a trusted reference source from the royal society of chemistry. The retro Diels-Alder fragmentation data and their corresponding peaks have been conveniently compiled and provided as Supplementary Files (
Figures S1–S4).
2.10. Insilco Molecular Docking Analysis
All the ligands under study displayed good binding affinities with all the proteins under study. The detailed binding affinities and the interactions have been presented in
Table 7.
2.10.1. Interaction Analysis of the Ligands in the Active Site of Protein 1LPB
The identified compounds β-sitosterol, stigmasterol, quercetin and Thiamine were docked with pancreatic lipase (PDB ID- 1LPB). The result section of the study discusses the crucial role of various amino acid residues in the efficient catalysis of human pancreatic lipase. Asp79 and Phe77 were found to play a critical role in stabilizing the oxyanion hole and orienting the substrate within the active site, respectively, through hydrogen bonding interactions. Meanwhile, hydrophobic residues, including Ala260, Ala259, Phe258, Leu264, Trp252, and Ile78, were found to create a hydrophobic environment within the active site, stabilizing the substrate and oxyanion intermediate during catalysis. The hydrophobic residues were also found to anchor the hydrophobic tails of the triglyceride substrate and stabilize the oxyanion intermediate during catalysis. These findings shed light on the complex molecular interactions that occur within the active site of human pancreatic lipase and provide a deeper understanding of the mechanisms behind lipid digestion and metabolism. The three-dimensional image of the highest-ranked molecule for this protein has been presented in
Figure 5.
2.10.2. Interaction Analysis of the Ligands in the Active Site of Protein 4W93
The identified compounds β-sitosterol, stigmasterol, quercetin and Thiamine were docked with alpha amylase (PDB ID- 4W93). The active site pocket of the carbonic anhydrase enzyme contains several residues, including rp59, Asp197, His201, Ile235, Glu233, and Asp197, that form hydrogen bonds with ligands, playing important roles in substrate or inhibitor binding and catalysis. Asp197 is particularly important in forming hydrogen bonds with the ligand, as it is often conserved in the active site of carbonic anhydrase enzymes and helps to orient the ligand in the correct conformation for catalysis. His201 and Ile235 also participate in hydrogen bonding interactions and stabilize the ligand within the active site. Trp59 and other hydrophobic residues anchor the ligand within the active site, while Glu233 acts as a proton acceptor in catalysis. These hydrogen bonding interactions are critical in stabilizing the ligand-enzyme complex and ensuring proper orientation and positioning of the ligand for efficient catalysis or inhibition. The three-dimensional image of the highest-ranked molecule for this protein has been presented in
Figure 5.
2.10.3. Interaction Analysis of the Ligands in the Active Site of Protein 7K9N
The identified compounds β-sitosterol, stigmasterol, quercetin and Thiamine were docked with co-crystal structure of alpha glucosidase (PDB ID- 7K9N) which binds to the active site of the protein and forms several hydrogen bonds with specific amino acid residues, including Asp640, His700, and Asp427. These hydrogen bonding interactions are critical in stabilizing the ligand-protein complex and determining the specificity and affinity of the interaction. Asp640, His700, and Asp427 are conserved residues that play essential roles in coordinating the binding of the ligand, positioning it for catalysis, and facilitating the enzymatic activity of PP1. Hydrophobic residues, such as Pro469, Leu701, Trp525, Trp423, Phe360, Phe673, Phe674, Ala429, Phe468, Trp562, Trp637, Ile452, Met565, Phe307, Phe571, Tyr675, Tyr676, and Val687, also play crucial roles in substrate binding, catalysis, and protein stability in the protein with PDB 7K9N. Overall, the hydrogen bonding interactions and hydrophobic residues in the active site of PP1 are essential for the enzymatic function of PP1. The three-dimensional image of the highest-ranked molecule for this protein has been presented in
Figure 5.
2.10.4. Molecular Dynamics Simulations Analysis
Molecular dynamics simulations were conducted for three protein-ligand complexes: Quercetin bound to pancreatic lipase (complex a), Quercetin bound to Human pancreatic alpha-amylase (complex b), and Thiamine bound to alpha glucosidase (complex c). The Root Mean Square Deviation (RMSD) was used to assess ligand binding to the active site. The RMSD analysis indicated stable complexes, with acceptable fluctuations within the range of 1-3Å for all the protein-ligand systems. Complex a showed significant RMSD values of 1.8Å-3.5Å, while complexes b and c exhibited values ranging from 1.0Å to 1.6Å (
Figure 6). RMSF analysis revealed minor fluctuations throughout the simulation, with green bars indicating protein residues involved in ligand interactions (
Figure 7). Hydrogen bond interactions were most prominent in complex b, with a higher number of H-bonds established compared to complexes a and c. The simulations demonstrated hydrophobic contacts, ionic interactions, and water-mediated linkages in all complexes, suggesting their stability (
Figure 8). The protein-ligand contacts timeline representation illustrated increased interactions between the ligands and proteins (
Figure 9). All the PLCs revealed good ligand atomic interactions (
Figure 10).
Figure 1.
Histopathology investigation of liver carried out at 40 x magnification with hematoxylin-eosin’s staining. The red arrows on the images represent the lipid droplets whereas the black arrows are representing the hepatocytes. Normal group mice liver section (a) showing normal hepatocytes with no presence of lipid droplets. Obese control group (b) showing enlarged and damaged hepatocytes along with large lipid droplets formation on liver. treated group at Standard treated group (c) and test treated group (e) pathological condition is almost back to normal and showed normal condition whereas fraction 80 mg/kg. (d) is also showing normal and healthy observations as compared to obese control group.
Figure 1.
Histopathology investigation of liver carried out at 40 x magnification with hematoxylin-eosin’s staining. The red arrows on the images represent the lipid droplets whereas the black arrows are representing the hepatocytes. Normal group mice liver section (a) showing normal hepatocytes with no presence of lipid droplets. Obese control group (b) showing enlarged and damaged hepatocytes along with large lipid droplets formation on liver. treated group at Standard treated group (c) and test treated group (e) pathological condition is almost back to normal and showed normal condition whereas fraction 80 mg/kg. (d) is also showing normal and healthy observations as compared to obese control group.
Figure 2.
Histopathology investigation of adipose tissue carried out at 40 x magnification with hematoxylin-eosin’s staining. The black arrows on the images are representing the size of lipid droplets (adipocytes). Normal group mice adipose section (a) showing normal and smaller adipocytes. Obese control group (b) showing a prominent and large size of lipid droplets. Standard (c) and test treated group (e) are showing normal and lesser no of lipid droplets as compared to obese control whereas test group (d) is also showing smaller number of droplets as compared to obese control group.
Figure 2.
Histopathology investigation of adipose tissue carried out at 40 x magnification with hematoxylin-eosin’s staining. The black arrows on the images are representing the size of lipid droplets (adipocytes). Normal group mice adipose section (a) showing normal and smaller adipocytes. Obese control group (b) showing a prominent and large size of lipid droplets. Standard (c) and test treated group (e) are showing normal and lesser no of lipid droplets as compared to obese control whereas test group (d) is also showing smaller number of droplets as compared to obese control group.
Figure 3.
Histopathology investigation of kidney carried out at 40 x magnification with hematoxylin-eosin’s staining. The red arrows on the images represent the proximal tubules (PT) whereas the black arrows are representing the glomerulus (G) and blue arrow heads are showing distal tubules (DT). Normal group mice kidney section (a) showing normal and healthy G, PT and DT. Obese control group (b) showing distorted PT and DT along with shrunken G. Standard treated group (c) and test treated group (d & e) pathological condition showed healthy and normal condition.
Figure 3.
Histopathology investigation of kidney carried out at 40 x magnification with hematoxylin-eosin’s staining. The red arrows on the images represent the proximal tubules (PT) whereas the black arrows are representing the glomerulus (G) and blue arrow heads are showing distal tubules (DT). Normal group mice kidney section (a) showing normal and healthy G, PT and DT. Obese control group (b) showing distorted PT and DT along with shrunken G. Standard treated group (c) and test treated group (d & e) pathological condition showed healthy and normal condition.
Figure 4.
(A) Chromatograms of compounds in fractions (F2) with their RF value on HPTLC F254 plates at 254 nm developed with selected solvent mixtures of ethyl acetate: toluene in the ratio of 6:4 in prep HPTLC. (B) Densitogram of compounds in fractions (F2).
Figure 4.
(A) Chromatograms of compounds in fractions (F2) with their RF value on HPTLC F254 plates at 254 nm developed with selected solvent mixtures of ethyl acetate: toluene in the ratio of 6:4 in prep HPTLC. (B) Densitogram of compounds in fractions (F2).
Figure 5.
2D and 3D representation of the highest-ranked molecule in docking analysis, where a and b represent interaction of thiamine with alpha-glucosidase, c and d represents interaction of quercetin with pancreatic lipase and e and f represents interaction of quercetin with alpha amylase.
Figure 5.
2D and 3D representation of the highest-ranked molecule in docking analysis, where a and b represent interaction of thiamine with alpha-glucosidase, c and d represents interaction of quercetin with pancreatic lipase and e and f represents interaction of quercetin with alpha amylase.
Figure 6.
Root Mean Square Deviation (RMSD) graph of (a) Quercetin-pancreatic lipase (complex a), (b) Quercetin-Human pancreatic alpha-amylase (complex b), and (c) Thiamine-alpha glucosidase (complex c).
Figure 6.
Root Mean Square Deviation (RMSD) graph of (a) Quercetin-pancreatic lipase (complex a), (b) Quercetin-Human pancreatic alpha-amylase (complex b), and (c) Thiamine-alpha glucosidase (complex c).
Figure 7.
Root Mean Square Fluctuation (RMSF) graph of graph of (a) Quercetin-pancreatic lipase (complex a), (b) Quercetin-Human pancreatic alpha-amylase (complex b), and (c) Thiamine-alpha glucosidase (complex c).
Figure 7.
Root Mean Square Fluctuation (RMSF) graph of graph of (a) Quercetin-pancreatic lipase (complex a), (b) Quercetin-Human pancreatic alpha-amylase (complex b), and (c) Thiamine-alpha glucosidase (complex c).
Figure 8.
Plot (stacked bar charts) of protein interactions with the ligand supervised throughout Molecular dynamics simulation of graph of (a) Quercetin-pancreatic lipase (complex a), (b) Quercetin-Human pancreatic alpha-amylase (complex b), and (c) Thiamine-alpha glucosidase (complex c).
Figure 8.
Plot (stacked bar charts) of protein interactions with the ligand supervised throughout Molecular dynamics simulation of graph of (a) Quercetin-pancreatic lipase (complex a), (b) Quercetin-Human pancreatic alpha-amylase (complex b), and (c) Thiamine-alpha glucosidase (complex c).
Figure 9.
Specific contacts made by the Protein with the graph of (a) Quercetin bound to the active site of pancreatic lipase (complex a), (b) Quercetin-Human pancreatic alpha-amylase (complex b), and (c) Thiamine-alpha glucosidase (complex c).
Figure 9.
Specific contacts made by the Protein with the graph of (a) Quercetin bound to the active site of pancreatic lipase (complex a), (b) Quercetin-Human pancreatic alpha-amylase (complex b), and (c) Thiamine-alpha glucosidase (complex c).
Figure 10.
A schematic representation of detailed ligand atom interactions graph of (a) Quercetin-pancreatic lipase (complex a), (b) Quercetin-Human pancreatic alpha-amylase (complex b), and (c) Thiamine-alpha glucosidase (complex c).
Figure 10.
A schematic representation of detailed ligand atom interactions graph of (a) Quercetin-pancreatic lipase (complex a), (b) Quercetin-Human pancreatic alpha-amylase (complex b), and (c) Thiamine-alpha glucosidase (complex c).
Table 1.
The IC50 value of antioxidant activity of the different bioactive fractions using Nitric oxide assay, ABTS assay, DPPH assay and Hydrogen peroxide assay.
Table 1.
The IC50 value of antioxidant activity of the different bioactive fractions using Nitric oxide assay, ABTS assay, DPPH assay and Hydrogen peroxide assay.
Samples |
ABTS Assay |
DPPH Assay |
Hydrogen peroxide Assay |
Nitric oxide Assay |
Fraction1 |
1.41±0.25 |
1.17±0.08 |
1.61±0.25 |
1.47±0.21 |
Fraction 2 |
1.11±0.26 |
1.84±0.05 |
1.37±0.23 |
1.38±0.03 |
Fraction 3 |
3.19±0.22 |
2.8±0.15 |
3.99±0.22 |
3.08±0.28 |
Fraction 4 |
2.21±0.31 |
3.57±0.13 |
4.92±0.21 |
2.58±0.25 |
Ascorbic acid |
1.05±0.12 |
0.97±0.21 |
1.59±0.25 |
0.76±0.29 |
Table 2.
Enzyme inhibition assay of the different bioactive fractions using pancreatic lipase, Alpha glucosidase and Alpha amylase activity.
Table 2.
Enzyme inhibition assay of the different bioactive fractions using pancreatic lipase, Alpha glucosidase and Alpha amylase activity.
Samples |
Pancreatic Lipase assay |
Alpha amylase assay |
Alpha glucosidase assay |
Fraction 1 |
2.20±0.23 |
1.24±0.03 |
1.12±0.25 |
Fraction 2 |
1.23±0.08 |
1.87±0.02 |
1.17±0.37 |
Fraction 3 |
4.07±0.28 |
4.25±0.12 |
4.21±0.13 |
Fraction 4 |
3.99±0.26 |
3.89±0.21 |
4.01±0.28 |
Orlistat |
2.12±0.17 |
---------- |
-------- |
Acarbose |
------- |
0.68±0.17 |
0.92±0.17 |
Table 3.
Effect of fraction F2 and F3 on changes in Body weight of different groups of mice.
Table 3.
Effect of fraction F2 and F3 on changes in Body weight of different groups of mice.
Days |
Normal group (Weight in grams) |
Obese group (Weight in grams) |
Standard (orlistat) (Weight in grams) |
(Fraction 1) 80 mg/kg/bw (Weight in grams) |
(Fraction 2) 80 mg/kg/bw (Weight in grams) |
1st
|
25.21±0.26 |
55.65±0.18*** |
54.15±0.2 |
55.12±0.03 |
54.12±0.21 |
7th
|
26.23±0.26 |
57.12±0.16*** |
48.32±0.3 a
|
49.14±0.02 |
48.21±0.12 |
14th
|
25.38±0.57 |
56.23±0.18*** |
46.54±0.06 b
|
45.21±0.03 |
42.07±0.19 |
21st
|
26.93±0.26 |
55.74±0.16*** |
39.00±0.23 c
|
420.03±0.08 |
35.09±0.12 |
28th
|
28.21±0.18 |
55.00±0.23*** |
31.21±0.32c
|
38.02±0.06 |
31.17±0.22 |
Table 4.
Effect of fraction F2 and F3 on changes in Waist hip ratio in different groups of mice.
Table 4.
Effect of fraction F2 and F3 on changes in Waist hip ratio in different groups of mice.
Days |
Normal group |
Obese group |
Standard |
(Fraction 1) 80 mg/kg/bw |
(Fraction 2) 80 mg/kg/bw |
1st
|
0.4 ± 0.02 |
1.4 ±0.05** |
1.4 ±0.011 |
1.4 ±0.023 |
1.4 ±0.012 |
7th
|
0.5.±0.05 |
1.3 ±0.020** |
1.3 ±0.010 |
1.3 ±0.003 |
1.2 ±0.009 |
14th
|
0.4.±0.04 |
1.4 ±0.03** |
1.2 ±0.003 a
|
1.1 ±0.016 |
1.2 ±0.012 a
|
21st
|
0.5.±0.08 |
1.3 ±0.01** |
0.9 ±0.080 a
|
1.0 ±0.020 a
|
0.9 ±0.003 b
|
28th
|
0.4 ±0.07 |
1.4 ±0.05** |
0.7 ±0.030 b
|
0.8 ±0.240 b
|
0.8 ±0.011 c
|
Table 5.
Effect of fraction F1 and F2 on biochemical parameters of different groups of mice.
Table 5.
Effect of fraction F1 and F2 on biochemical parameters of different groups of mice.
Parameters measured |
Normal group |
Obese group |
Standard |
(Fraction 1) 80 mg/kg/bw |
(Fraction 2) 80 mg/kg/bw |
Serum cholesterol (mg/dl) |
87±0.06 |
208±0.16 *** |
115±0.10c
|
131±0.06 b
|
113±0.01 c
|
Serum triglyceride (m mol/L) |
47±0.03 |
121±0.21*** |
95±0.14 b
|
107±0.03 |
95±0.02 b
|
HDL Cholesterol (mg/dl) |
68±0.02 |
48±0.14*** |
51±0.11 c
|
61±0.01 c
|
59±0.03 c
|
VLDL cholesterol (mg/dl) |
8.1±0.06 |
28±0.13** |
16.21±0.08a
|
21.01±0.09 |
20.2±0.07 a
|
LDL Cholesterol (mg/dl) |
14±0.03 |
65±0.21*** |
35.08±0.06 b
|
51.27±0.06 |
39.27±0.01 b
|
Cholesterol/HDL ratio (mg/dl) |
1.27±0.01 |
4.33±0.02* |
2.25±0.1 a
|
2.14±0.03 a
|
1.91 ±0.03 |
LDL/HDL ratio (mg/dl) |
0.20±0.01 |
1.35±0.1 *** |
0.68±0.02 b
|
0.83±0.01 a
|
0.66±0.01 b
|
Table 6.
Compounds tentatively identified in fraction 2 with their (m/z) peak and Retro Diels-Alder fragmentation (ESI- MS-MS) analysis.
Table 6.
Compounds tentatively identified in fraction 2 with their (m/z) peak and Retro Diels-Alder fragmentation (ESI- MS-MS) analysis.
Compounds |
Molecular mass |
Found ion (m/z+) |
Found ion (m/z-) |
Found ion [m/z- H2O] |
RF value |
β-sitosterol |
414 |
|
|
MS1-397;MS2-265:243:203;MS3-187:147:135 and MS4-144:130:109 |
0.71 |
Stigmasterol |
412 |
|
|
MS1-395; MS2-297:267:255;MS3-241:201:187:149;MS4-173 and MS5-158 |
0.58 |
Thiamine |
265 |
MS1-266 MS2 -156:144:121:1090. |
|
|
0.39 |
Quercetin |
302 |
|
MS1-301; MS2-179:151 and MS3-150 |
|
0.24 |
Table 7.
Tabular representation of binding affinities of the ligands with the selected proteins(receptors) under study.
Table 7.
Tabular representation of binding affinities of the ligands with the selected proteins(receptors) under study.
|
Pancreatic lipase (PDB ID- 1LPB) |
Alpha amylase (PDB ID- 4W93) |
Alpha glucosidase (PDB ID- 7K9N) |
Compounds |
Glide score (Kcal/mol) |
Glide Energy |
Glide score (Kcal/mol) |
Glide Energy |
Glide score (Kcal/mol) |
Glide Energy |
Quercetin |
-4.96 |
-29.26 |
-8.15 |
-40.55 |
-3.83 |
-25.13 |
Stigmasterol |
-4.86 |
-20.95 |
-4.29 |
-29.83 |
-5.42 |
-15.30 |
Sitosterol |
-3.80 |
-19.62 |
-4.05 |
-27.64 |
-5.78 |
-22.08 |
Thiamine |
-3.80 |
-27.99 |
-4.76 |
53 |
-7.17 |
-31.53 |