Molecular mechanism of anti-obesity effect of total lutein oxidized products (LOPs) in diet-induced obese mice

Abstract

The present study explores the molecular mechanism of anti-obesity effect of total lutein oxidized products (LOPs) in high-fat diet-induced obese C57BL/6 mice. Total LOPs (50, 100, 200 mg/kg b.wt.) were intubated for 22 weeks. Lipid profile, peroxides, AST, ALT, GST activities, lipid metabolizing enzymes/molecules, 3T3-L1 cell differentiation, TGF-β1 and p38 MAPK expressions were studied. Total LOPs at 100 mg/kg b.wt. reduced plasma and hepatic cholesterol, triglycerides, phospholipids, ALT and AST activities. FAS and CPT activities were inhibited in the liver homogenate, and epididymal WAT (eWAT). Total LOPs at 200 mg/kg b.wt. reduced HMGCoA:Mevalonate in eWAT, lipid peroxides in eWAT and liver. GST activity was high in the liver and eWAT. Total LOPs (100μg/ml) suppressed adipogenesis and decreased the accumulation of lipid droplets from two to eight days. Adiponectin was upregulated, PPAR γ, lipoprotein lipase, TGF-β1, and p38 MAPK were down-regulated. The results have shown that total LOPs are potent anti-obese molecules.

ABBREVIATIONS: AIN 93G: American institute of nutrition 93 for growth; ALT: alanine transaminase; AST: aspartate transaminase; CEBPα: ccaat/enhancer binding protein α; C-DNA: complementary deoxyribonucleic acid; CPT: carnitine palmitoyl transferase; GST: glutathione-S-transferase; ELISA: enzyme-linked immune sorbent assay; FAS: fatty acid synthase; HDL: high-density lipoprotein; HFD: high-fat diet; HMG CoA: β-Hydroxy β-methylglutaryl-Coenzyme A; LDL: low-density lipoprotein; LOPs: total lutein oxidized products; LPL: lipoprotein lipase; MDA-Malonaldehyde; MTT: 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium bromide; MVA: mevalonate; p38MAPK: p38 mitogen-activated protein kinases; RNA: ribonucleic acid; RTq-PCR: quantitative reverse transcription polymerase chain reaction; PPAR-γ: peroxisome proliferator-activated receptor-gamma; TGF-β1: transforming growth factor beta 1; WAT: white adipose tissue.

KEYWORDS:

• Lutein oxidized products
• C57BL/6 mice
• lipid metabolizing enzymes
• 3T3-L1 cells
• adiponectin
• p38 MAPK
• pre-adipocytes

1. Introduction

Obesity is a growing health condition globally with excessive fat accumulation giving a negative impact on human health. People who are obese are prone to various risk factors like high blood sugar levels/type 2 diabetes, High blood pressure resulting in hypertension, cardiovascular diseases leading to heart failure and stroke, stiffness in bones and joints causing joint pain/osteoarthritis, sleep apnea, and some cancers [1]. Clinical trials and animal experiments suggest that obesity is directly linked with dyslipidemia resulting in elevated blood and hepatic cholesterol, phospholipids, triglycerides, impaired lipid functions, nonalcoholic fatty liver disease (NAFLD), inflammation, and oxidative stress [2].

At the molecular level, obesity is linked to the dysregulation of various signalling molecules, transcription factors, enzymes, and hormones involved in the process of fat accumulation. Obesity imbalances adipogenesis i.e. the process of formation of mature adipocytes from the proliferation and differentiation of adipocyte precursor cells, and results in adipose hypertrophy, hyperplasia, and hypoxia [3]. The peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (CEBPα) are the principal regulators of adipogenesis. Activation of PPARγ and CEBPα, in turn, transcribes various lipid metabolizing enzymes/molecules involved at different stages of lipid metabolism like Lipoprotein lipase (LPL), Fatty acid synthase (FAS), Carnitine palmitoyltransferase (CPT), Fatty acid-binding protein 4 (FABP4), Adiponectin, Hormone-sensitive lipase (HSL), Glucose transporter 4 (GLUT4), and Stearyl-CoA-desaturase(SCD) [4]. Hence regulators/modulators of adipogenesis play a vital role in controlling the progression of obesity.

Orlistat is the only FDA-approved drug available in the market, which reversibly inhibit pancreatic and gastric lipases. Orlistat covalently binds to serine residues of lipases and inactivation of the enzyme occurs, inhibiting the hydrolysis of triglycerides such that free fatty acid is not absorbed in the intestinal wall/gut and is eliminated as faeces. The adverse effects of the long-term usage of orlistat drug include gastrointestinal, hepatotoxicity and renal injury [5].

Currently, plant-derived bioactive molecules showing antiobesity have been an important area of interest to study [6]. Researchers have demonstrated the mechanism of action of various herbs and their molecules with the antiobesity property. The mechanism of action of a plant-based antiobesity molecule can be pancreatic lipase inhibitors, antioxidants, lipid-regulating enzymes, anti-inflammatory, modulators of thermogenesis, and signalling pathways. The methanolic extracts of Vitis vinifera showed IC₅₀ of 14.1 µg/ml [7] and leaves of Cyclocarya paliurus exhibited pancreatic lipase inhibition with IC₅₀ of 9.1 µg/ml [8]; suppressors of adipogenesis- flavonoids from Acer Okamoto down-regulated PPARγ and inhibited adipocyte differentiation in 3T3-L1 cells [9], glycyrrhizic acid obtained from the root of Glcyrrhizia glabra suppressed early adipogenesis through CEBPβ/δ expression in 3T3L-1 cells [10]. The antiobesity Cosmos caudatus kenth leaves ethanol extracts displayed antioxidant properties [11]. Fatty acid synthase, glucose 6-phosphate dehydrogenase, phosphatidate phosphohydrolase were suppressed and activation of fatty acid oxidation was observed in resveratrol (a natural polyphenol found mainly in grape skin) [12]. Formononetin extracted from Astragalus membranaceus increased expression of UCP 1 and thermogenesis binding directly with PPARγ [13], Scutellanein extracted from Erigeron breviscapus showed antiobesity effects associated with antiinflammation based on liver protective effects [14] and Geinstein a soybean derived isoflavone inhibited the phosphorylation of p38 Mitogen-activated protein kinase (MAPK) preventing Janus kinase 2 (JAK 2) expression [15].

The use of natural products is more beneficial rather than synthetic drugs in the treatment of obesity [16]. Formulating natural products into a drug is challenging due to its instability, high molecular weight, less solubility, low gastrointestinal metabolism, and irreversible binding to cellular DNA and proteins [17]. To overcome these issues, a biomolecule should be studied in multiple dimensions. For the first time, we reported the synthesis of total lutein oxidized products (LOPs) naturally by exposure to direct sunlight and without the use of any synthetic/chemical products.

In our previously published article (https://doi.org/10.1039/D1FO04064B), we explored the pancreatic lipase inhibitory activity of total LOPs. The structural elucidation was carried out on LCMS/MS-TOF, and docking studies on LOP 6 (one of the oxidized products among eight products obtained) concerning PPARγ and pancreatic lipase were reported. ADME (Absorption, digestion, metabolism and excretion) properties were studied revealing good solubility, gastrointestinal tract absorption and other pharmacodynamic parameters of LOP6 in comparison with orlistat proving its drug-likeness property. The pancreatic lipase inhibitory effect of LOP-6 i.e. IC₅₀ was 11.8420 µg/ml whereas, total LOPs were inhibited at a very less IC₅₀ concentration of 1.6953 µg/ml. The synergistic effect of total LOPs is far better than an individual LOP-6. Hence further (in-vivo) studies were conducted with total LOPs instead of LOP-6. [18]. The present investigation aims to understand the mechanism of action of total LOPs in modulating lipid metabolising molecules and enzymes in C57BL/6 mice and key adipogenic factors in 3T3-L1 preadipocytes.

2. Experimental design and analysis

2.1. Chemicals

Porcine pancreatic lipase, 4-Nitrophenyl palmitate, and standard lutein were obtained by Sigma Aldrich Co. USA. Orlistat was a kind gift from GR Pharmaceutical Distributors, Mysuru, Karnataka, India and all other chemicals including hexane, methanol, diethyl ether, ethanol, chloroform, isopropanol were of analytical grade. HiDiff™ 3T3-L1 Differentiation Kit (CCK011), Dulbecco’s Modified Essential Medium, high glucose (AL007A), Dulbecco’s Phosphate Buffered Saline (TL1006), Fetal Bovine Serum (RM1112), Trypsin-EDTA solution(TCL007), Antibiotic solution (A002), EZAssay™ TBARS Estimation kit for lipid peroxidation (CCK023), EZAssay™ GST Activity Estimation Kit (CCK028) were procured from Himedia. Estimation of triglycerides (12011024), HDL (11414003), LDL (11415003), total cholesterol (12011009), AST (11408003), and ALT (11409003) were quantified using LyphoCHEK and LiquiCHEK kit, Agappe Diagnostics Ltd. RNA isolation kit, P³⁸ MAPK (Total) ELISA Kit (KH00061) and TGF-β1 ELISA kit (KAC1688) were procured from INVITROGEN, USA. Verso cDNA Synthesis Kit (Thermo Scientific, USA)

2.2. Animal treatment

Animal experiments were conducted after due clearance from the Institutional Animal Ethical Committee (IAEC approval No. KCC/IAEC/014/2020). 8-week-old male C57BL/6 mice (n = 56) weighing 25 ± 2 g were housed in polycarbonate cages at temperature 23 ± 3⁰C, relative humidity of 40–70% and 12hr light /dark cycle with water ad libitium. Following 7 days of acclimatization, mice were randomly divided into 7 groups with 8 mice in each group. Group-I received a normal diet as prescribed by the American Institute of Nutrition (AIN93G), and Group 2 received a high-fat diet (HFD) as prescribed by AIN-93G. Groups 1 and 2 were orally gavaged with only vehicle i.e. olive oil. Group 3 is the HFD + LOPs-Low dose group which received HFD and intubated with total LOPs of 50 mg/kg.b.wt. dispersed in olive oil. Group 4 was named as HFD + LOPs-Medium dose group, receiving HFD and intubation of total LOPs of concentration 100 mg/kg.b.wt. in olive oil. Group 5 was captioned as HFD + LOPs-High dose which received HFD and orally gavaged with 200 mg/kg.b.wt. The concentration of total LOPs dispersed in olive oil. Group 6 is the HFD + Lutein group fed with HFD and intubated with lutein of concentration 100 mg/kg.b.wt. through olive oil. Group 7 is called HFD + Orlistat group which received HFD and orlistat of concentration 50 mg/kg.b.wt. intubated using olive oil as a vehicle. Throughout the animal experiment, the body weight and food intake of each mouse in all 7 groups were measured once a week. At the end of the experiment (after 22 weeks), animals fasted for 12 h and Carbon-di-oxide (CO₂) was used as an anaesthetic agent for euthanization. The epididymal, renal, and perirenal white adipose tissue and liver were removed, weighed and stored at −80°C until further analysis.

2.3. Extraction of lutein and synthesis of total LOPs

Marigold flowers were purchased from the local market of Mysore District, Karnataka. Petals were separated from the flower and rinsed thoroughly using double distilled water. Cleaned petals were shade dried, powdered and sieved using the mesh size of 260 microns to obtain a fine powder. The powder was then stored in an air-tight container in the dark at room temperature until further use. The extraction of lutein from marigold flower petals was done according to the method described by Frederick Khachik [19]. The synthesis of total LOPs and characterization was done according to our previously published article. Briefly, the extracted lutein was kept on the horizontal surface towards the east direction, so that direct sunlight falls on it. The average solar intensity was calculated as 5.89 kW h m⁻² d⁻¹ (calculated using the solar irradiance calculator available at https://www.solarelectricityhandbook.com) and the temperature was 31 ± 2 °C from day 1–10. After that, there was no further oxidation/ degradation of lutein and 6.5% of lutein remains as rp-HPLC profile shows that a small amount of lutein is present in day 10 sunlight-exposed lutein. Hence the synthesis yield of LOPs was 95.3% [16].

2.4. Lipid profile, peroxides, AST, ALT, and GST activities

The blood (0.4 ml) was collected to the heparinized tubes from retro-orbital venous plexus from overnight fasted animals after CO₂ euthanization. Plasma (0.2 ml) was separated from heparinized blood by centrifugation at 3000 rpm for 15 min. Lipids from the liver were extracted as described by Folch et al. [20]. Total cholesterol (Tch), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and triglycerides (TG) were estimated in plasma and liver using LyphoCHEK and LiquiCHEK kit, Agappe Diagnostics Ltd. Phospholipids were determined colorimetrically with ammonium ferrothiocyanate using dipalmitoyl phosphatidylcholine as a reference as described by Stewart [21]. Additionally, aspartate transaminase (AST) and alanine transaminase (ALT) were quantified in plasma using a LyphoCHEK kit, Agappe Diagnostics Ltd. Lipid peroxides and glutathione-s-transferase (GST) were assessed in the liver and epididymal WAT by EZAssay™ TBARS estimation kit and EZAssay™ GST Activity estimation kit, respectively, as per manufacturer's instructions. Each group consisted of 8 mice i.e. n = 8 and the results were statistically analysed as mentioned in Section 2.10.

2.5. Lipid metabolizing enzymes/molecules

Quantification of enzymes/metabolites involved in the lipogenic pathway was done in various subcellular fractions of the liver and epididymal WAT. Differential centrifugation was performed according to Hulcher and Oleoson method [22]. Fatty acid synthase (FAS) activity from cytosolic fractions was determined by quantifying consumption of NADPH at 340 nm during the formation of palmitate in the presence of acetyl coenzyme-A (CoA) and Malonyl CoA [23]. Carnitine palmitoyltransferase (CPT) was assessed in mitochondrial fractions by the release of CoA-SH from palmitoyl CoA using a thiol reagent at 412 nm [24]. In cholesterol biosynthesis reduction of HMG-CoA to mevalonate is a rate-limiting step. The ratio of β-Hydroxy β-methylglutaryl-Coenzyme A (HMG-CoA) and mevalonate was measured according to Venugopala Rao and Ramakrishnan protocol [25]. Protein estimation was done using the Bradford method [26]. Each group consisted of 8 mice i.e. n = 8 and the results were statistically analysed as mentioned in Section 2.10.

2.6. Cell culture studies

3T3L1 cell lines with request No. 725/2021-22 and passage no. P^N7 was procured from Cell Repository, National Centre for Cell Science, S.P. Pune University Campus, Pune. Differentiation and oil-red-o staining of 3T3L1 cells treated with total LOPs along with suitable controls were carried out using HiDiff™ 3T3-L1 Differentiation kit (CCK011), Himedia, Mumbai, India; according to manual protocol. The differentiation medium 1 consists of DMEM high glucose medium, 0.5 mm IBMX (3-Isobutyl-1-methylxanthine), insulin 1 µg/ml and dexamethasone 0.25 µM. differentiation medium 2 consists of DMEM high glucose medium and insulin 1 µg/ml. Briefly, cells were cultured in DMEM, high glucose medium supplemented with 10% FBS. The cells were maintained at 37°C and 5% atmospheric CO_2. Once the cell reached 70% confluency, it was sub-cultured (Day 0) and after reaching 70–80% confluency, was treated with differentiation medium 1 (Day 2) and differentiation Medium 2 (Day 4). Complete differentiation was achieved on Day 8 and the lipid droplets were stained using the Oil-red-O staining method. Cells were viewed under Moticam Pro 205 A microscope with 400× magnification.

2.7. Cell viability assay

Briefly, around 1.0 × 10⁴ cells were seeded into each well of 96 well plates which were placed for incubation at 37°C and 5% CO₂ in a CO₂ incubator. After 24 h, the cells were treated with total LOPs at various concentrations from 5 µg to 500 µg/ml. All the mentioned concentrations were tested in triplicates and the results were given as an average of each combination. The cells were again incubated in the CO₂ for either 24 or 48 h under the same incubation condition. After the incubation time, either at 24 or 48 h, the respective 96 well plates were removed from the incubator which was further processed for the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay where, the media in each well were replaced with the media containing MTT reagent (5 mg/ml in media, 100 µL/well) and were further incubated for another 3hrs in the CO₂ incubator. After the incubation time, the 96 well plates were removed and the MTT media in each well was replaced with 100 µl of Dimethyl sulphoxide (DMSO) and were placed for 30 min at room temperature in the dark. Followed by this the plates were subjected to spectrophotometric reading at the λ 570 nm along with the reference λ 540 nm as well as 590 nm using a microplate reader.

2.8. RNA isolation

Total RNA was isolated from the cells treated with 100, 50, 25, and 0 µg concentration of total LOPs using Trizol reagent (Invitrogen, Life Technologies, USA) as per the manufacturer’s protocol. The quantity and quality of the isolated RNA were measured using a Nanodrop spectrophotometer (Thermo Fisher, USA) by estimating the ratio of absorbance at 260/280 and 260/230 nm. The cDNA was synthesized from the 5 µg of total RNA using Verso cDNA Synthesis Kit (Thermo Scientific, USA). The required quantity of cDNA for qPCR (1–2 ng/µl) was prepared by dilution (nuclease-free water) of first-strand cDNA. The quantitative RTq-PCR (quantitative reverse transcription polymerase chain reaction) was carried out in Stratagene Mx3005P (Agilent Technologies) using SYBR (N′N′-dimetyl-N-[4-[(E-(3-methyl-1,3-benzothiazol-2-ylidene) methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine) green PCR pre-mix. The PCR mixture was composed of 1 µl of diluted cDNA (2 ng/µl) and the PCR cycling parameters included an initial polymerase activation step of 95°C for 10 min, followed by 40 cycles of 95°C for the 30s and annealing temperature (Ta) for 60s and 72°C for 60s with actin gene [27] as a constitutive expression control. The data represent the average of each three replicates, and the relative expression value was calculated by the comparative 2−ΔC_T method [28]. The PCR primer sequence used in the study were as follows: PPARγ, 5′-AAC TCT GGG AGA TTC TCC TGT TGA-3′ (forward) and 5′-TGG TAA TTT CTT GTG AAG TGC TCA TA-3′ (reverse); Lipoprotein lipase, 5′-GCA GAC GCG GGA AGA GAT T-3′ (forward) and 5′-TGG CAG TTA GAC ACA GAG TCT GCT A-3′ (reverse); Adiponectin, 5′-CTG GCC ACT TTC TCC TCA TTT C-3’(forward) and 5′-GGC ATG ACT GGG CAG GAT TA-3′ (reverse); β-actin 5′-CAA AAG CCA CCC CCA CTC CTA AGA-3′ (forward) and 5′-GCC CTG GCT GCC TCA ACA CCT C-3′ (reverse). The significance of fold change of gene expression in three concentrations (25, 50 and 100 µg) of total LOPs was statistically analysed by one-sample t-test as mentioned in Section 2.10.

2.9. p38MAP kinase and TGF-β1 estimation in 3T3-L1 cells cultured with total LOPs

p38 mitogen-activated protein kinases (p38MAP kinase) and transforming growth factor beta-1 (TGF-β1) are important signalling molecules in the process of adipogenesis. Day-5 3T3-L1 cells (treated with 100 µg/ml of total LOPs) were collected by scraping from culture flasks, lysed using cell extraction buffer for 30 min and centrifuged at 13,000 rpm for 10 min at 4⁰C. Cell lysates were diluted at the ratio of 1:10 using the standard dilution buffer provided in the kit and sandwich ELISA was performed by adding 100 µl of diluted cell lysates with appropriate controls as per manual instructions provided in the kit. The results were compared with their respective control (triplicate values) as mentioned in Section 2.10.

2.10. Statistical analysis

The statistical data has been analysed using Graph pad Prism 9.4.1 software. All the experiments were conducted in triplicate and the data were expressed as mean ± standard deviation (SD). Two-way ANOVA was used for the comparison between each group. Dunnet’s multiple comparison posthoc tests were used to compare different dosed groups with the HFD control group. *P < 0.01, **P < 0.001, ***P < 0.005, ****P < 0.0001 was considered to indicate a statistically significant difference.

3. Results

3.1. Effect of total LOPs on body weight and food intake

Daily intubation with total LOPs (50, 100, and 200 mg/kg.b.wt), Lutein, or Orlistat did not influence the body weight gain significantly up to 6 weeks. However, after 6 weeks up to 22 weeks, the High-fat diet (HFD) + orlistat group reduced its body weight by 16% (P < 0.0001) and the total LOPs (100 mg/kg.b.wt) group body weight was reduced to 14% (P < 0.0001) in comparison with the HFD group, whereas HFD + total LOPs (50 mg/kg.b.wt), HFD + total LOPs (200 mg/kg.b.wt) and the HFD + Lutein group reduced body weight by 9% (P < 0.001) (Figure 1). Food intake was not significantly altered in any group. Concerning organs; a high-fat diet increased the weight of organs resulting in fatty liver and expansion of epididymal WAT by 35–40% in comparison with normal diet mice group.

Figure 1. Effect of total LOPs on body weight gain in mice. Note: The intervention of the above-mentioned groups was done for 22 weeks. HFD, High-Fat Diet; LOP, Lutein Oxidized Product. Data are represented as mean ± standard deviation with n = 8/group. Two-way ANOVA was used for the comparison between each group. Dunnet’s multiple comparison posthoc tests were used to compare different dosed groups with the HFD control group. *P < 0.01, **P < 0.001, ***P < 0.005, ****P <0.0001.

The intervention of total LOPs at 50 mg/kg.b.wt decreased the weight of the liver, epididymal WAT, renal WAT and perirenal WAT by 14% (P < 0.01), 51% (P < 0.0001), 42% and 32%, respectively. Intubation of total LOPs at 100 mg/kg.b.wt reduced the weight of the liver, epididymal WAT, renal WAT and perirenal WAT by 13%, 43% (P < 0.0001), 43% and 30%, respectively. At 200 mg/kg.b.wt total LOPs showed a decrease in the weight of the liver, epididymal WAT, renal WAT and perirenal WAT by 12%, 47% (P < 0.0001), 40% and 32%, respectively. Concerning lutein and orlistat intubated group organ weight decreased by 14% (P < 0.01) and 16% (P < 0.01) in the liver, 49% (P < 0.0001) and 53% (P < 0.0001) in epididymal WAT, 39% and 41% in renal WAT, 30% and 32% in perirenal WAT (Table 1). Hence intubation of total LOPs ranging from low dose to high dose had a significant impact on the reduction of body and organ weights in comparison to the high-fat diet-fed group.

Table 1. Effect of total LOPs on organ weights in the high-fat diet-fed mice.

Groups	Liver (g)	Epi-WAT(g)	Renal-WAT(g)	Perirenal-WAT(g)
Normal Diet (Control)	2.99 ± 0.56*	0.585 ± 0.14****	0.095 ± 0.05	0.201 ± 0.03*
HFD Control	3.47 ± 1.29	1.927 ± 0.58	0.344 ± 0.11	0.705 ± 0.24
HFD + total LOPs (50 mg/kg/b.wt)	2.98 ± 0.51*	0.950 ± 0.05****	0.199 ± 0.02	0.476 ± 0.11
HFD + total LOPs(100 mg/kg/b.wt)	3.02 ± 0.14	1.097 ± 0.21****	0.195 ± 0.02	0.497 ± 0.15
HFD + total LOPs(200 mg/kg/b.wt)	3.07 ± 0.37	1.016 ± 0.11****	0.206 ± 0.02	0.482 ± 0.08
HFD + Lutein	2.99 ± 0.65*	0.982 ± 0.13****	0.210 ± 0.18	0.492 ± 0.08
HFD + Orlistat	2.93 ± 0.24*	0.914 ± 0.13****	0.204 ± 0.01	0.474 ± 0.16

Notes: The intervention of the above-mentioned groups was done for 22 weeks. HFD, High-Fat Diet; LOP, Lutein Oxidized Product; Epi, Epididymal; WAT, White Adipose Tissue. Data are represented as mean ± standard deviation with n = 8/group. Two-way ANOVA was used for the comparison between each group. Dunnet’s multiple comparison posthoc tests were used to compare different dosed groups with the HFD control group. *P < 0.01, **P < 0.001, ***P < 0.005, ****P < 0.0001.

3.2. Lipid profile and marker enzyme activities

HFD control group fed mice elevated the levels of plasma and hepatic Tch, TG, and PL by 35–40% in comparison with the normal diet fed control group. Mice intervened with total LOPs (50, 100 and 200 mg/kg.b.wt) reduced plasma Tch [22%, 39%, 35%] where P < 0.0001 in all the three concentrations, LDL [9%, 21% (P < 0.005)], [11% (P < 0.01)], TG [8% (P < 0.01), 36% (P < 0.0001), 33% (P < 0.0001)] and PL [(9%, 38%, 35%)], respectively, where P < 0.0001 in all the three concentrations in comparison to HFD group. HDL was increased by 31% (P < 0.01), 18% and 16% in all three groups of total LOPs. Lutein and orlistat intubated group showed a reduced plasma Tch [32%, 26%], where P < 0.0001 in both groups, LDL [22% (P < 0.0001), 14% (P < 0.001)], TG [30%, 25%], where P < 0.0001, and PL [31%, 26%], respectively, where P < 0.0001 in both groups. HDL was increased by 16% and 30%.

In the liver, total LOPs at 50, 100 and 200 mg/kg.b.wt reduced Tch [23% (P < 0.001), 40% (P < 0.0001), 36% (P < 0.0001)], TG [9% (P < 0.005), 38% (P < 0.0001), 35% (P < 0.0001)] and PL [25% (P < 0.01), 28% (P < 0.01), 23%)]. Lutein and orlistat intubated mice group also decreased the Tch [34% (P < 0.0001), 24% (P < 0.001)], TG [31%, 26%], where (P < 0.0001) in both group and PL [28% (P < 0.01), 18%] (Table 2). The liver function test marker enzymes AST and ALT were dysregulated in HFD control group by elevation upto 86% plasma AST and 33% plasma ALT in comparison with Normal control group. The intervention of total LOPs decreased AST and ALT by 43% and 41% at 50 mg/kg.b.wt, 46% and 41% at 100 mg/kg.b.wt, 42% and 34% at 200 mg/kg.b.wt, 38% and27% with lutein and orlistat showed a decrease in enzyme activities by 45% and 28%, respectively, in comparison with the high-fat diet-fed group where P < 0.0001 in all the cases (Table 3).

Table 2. Effect of total LOPs on plasma and hepatic lipid profile in high-fat diet-fed mice.

Plasma lipid profile	Total cholesterol (mg/dl)	HDL (mg/dl)	LDL (mg/dl)	TG (mg/dl)	PL (mg/dl)
Groups
Normal Diet (Control)	97.28 ± 2.66 ****	31.81 ± 0.47***	50.35 ± 0.61***	64.730 ± 3.53****	30.42 ± 1.76****
HFD Control	134.56 ± 9.56	21.05 ± 4.93	63.67 ± 3.53	87.70 ± 7.54	41.90 ± 3.01
HFD + total LOPs (50 mg/kg/b.wt)	104.42 ± 9.61****	27.61 ± 5.47*	57.65 ± 4.68	80.39 ± 7.18*	38.25 ± 2.87****
HFD + total LOPs (100 mg/kg/b.wt)	82.52 ± 8.44****	24.81 ± 2.76	50.11 ± 6.83***	55.89 ± 7.01****	26.00 ± 2.80****
HFD + total LOPs (200 mg/kg/b.wt)	87.87 ± 6.50****	24.32 ± 1.90	56.51 ± 6.78*	58.64 ± 5.16****	27.37 ± 2.07****
HFD + Lutein	90.64 ± 2.79****	23.41 ± 0.95	49.74 ± 0.82****	61.43 ± 5.83****	28.77 ± 2.33****
HFD + Orlistat	99.78 ± 4.18****	25.68 ± 0.95	55.05 ± 1.20**	65.71 ± 7.19****	30.90 ± 2.88****
Hepatic lipid profile	Total cholesterol (mg/g)			TG (mg/g)	PL (mg/g)
Groups
Normal Diet (Control)	9.65 ± 0.27***	–	–	30.55 ± 1.76****	7.05 ± 0.02*
HFD Control	13.51 ± 0.96	–	–	42.03 ± 3.77	9.94 ± 0.37
HFD + total LOPs (50 mg/kg/b.wt)	10.39 ± 0.96**	–	–	38.38 ± 3.59***	7.47 ± 0.47*
HFD + total LOPs (100 mg/kg/b.wt)	8.12 ± 0.84****	–	–	26.13 ± 3.51****	7.11 ± 0.46*
HFD + total LOPs (200 mg/kg/b.wt)	8.68 ± 0.65****	–	–	27.51 ± 2.58****	7.63 ± 0.42
HFD + Lutein	8.96 ± 0.28****	–	–	28.90 ± 2.92****	7.22 ± 0.53*
HFD + Orlistat	9.91 ± 0.42**	–	–	31.04 ± 3.60****	8.20 ± 0.50

Notes: The intervention of the above-mentioned groups was done for 22 weeks. HFD, High-Fat Diet; LOP, Lutein Oxidized Product; HDL, High-Density Lipoprotein; LDL, Low-Density Lipoprotein; TG, Triglycerides; PL, Phospholipids. Data are represented as mean ± standard deviation with n = 8/group. Two-way ANOVA was used for the comparison between each group. Dunnet’s multiple comparison posthoc tests were used to compare different dosed groups with the HFD control group. *P < 0.01, **P< 0.001, ***P < 0.005, ****P < 0.0001.

Table 3. Effect of total LOPs on plasma AST and ALT in high-fat diet-fed mice.

Groups	AST U/L	ALT U/L
Normal Diet (Control)	53.04 ± 1.96****	46.33 ± 3.10****
HFD Control	98.67 ± 3.71	61.49 ± 2.62
HFD + total LOPs (50 mg/kg/b.wt)	56.07 ± 4.69****	36.51 ± 2.87****
HFD + total LOPs (100 mg/kg/b.wt)	53.41 ± 4.57****	36.14 ± 3.21****
HFD + total LOPs (200 mg/kg/b.wt)	57.29 ± 4.20****	40.85 ± 3.69****
HFD + Lutein	61.44 ± 0.42****	44.89 ± 5.55****
HFD + Orlistat	54.29 ± 5.29****	44.57 ± 4.51****

Notes: The intervention of the above-mentioned groups was done for 22 weeks. HFD, High-Fat Diet; LOP, Lutein Oxidized Product; AST, Aspartate Transaminase; ALT, Alanine Transaminase. Data are represented as mean ± standard deviation with n = 8/group. Two-way ANOVA was used for the comparison between each group. Dunnet’s multiple comparison posthoc tests were used to compare different dosed groups with the HFD control group. *P < 0.01, **P < 0.001, ***P < 0.005, ****P < 0.0001.

3.3. Effect of total LOPs on MDA level and GST activity in the liver and epididymal WAT

The HFD-fed mice group increased the malonaldehyde (MDA) levels by 40–80% and decreased the GST levels by 60–90% in the liver and epididymal WAT homogenate when compared with the normal diet mice group. The intervention of total LOPs decreased MDA levels in liver and epididymal WAT by 35% and 13% at 50 mg/kg.b.wt, 24% and 17% at 100 mg/kg.b.wt, 44% and 22% at 200 mg/kg.b.wt. Only an 8% decrease of MDA was observed in the liver and epididymal WAT with lutein and 35% and 27% with orlistat treated group where P < 0.0001 in all the cases except lutein P<0.005. The GST level in the liver and epididymal WAT increased up to 16% and 53% at 100 mg/kgb.wt, 11% and 81% at 200 mg/kgb.wt, 21% and 48% in lutein and 29% and 53% in orlistat, further there was no increase at 50 mg/kgb.wt in the liver, but in epididymal WAT, GST level was increased up to 69% in comparison to HFD group (Table 4).

Table 4. Effect of total LOPs on antioxidant and lipid metabolizing molecules/enzymes in the high-fat diet-fed mice.

Liver	Lipid peroxides (mmol/min/mg of protein)	GST (mmol/min/mg protein)	FAS (nmol/min/mg protein)	CPT (nmol/min/mg protein)	HMGCOA: Mevalonate ratio
Groups
Normal Diet (Control)	25.8 ± 1.02****	1.45 ± 0.21	9.51 ± 0.72****	28.35 ± 2.56****	4.83 ± 1.64****
HFD Control	47.14 ± 2.8	0.14 ± 0.02	15.43 ± 0.66	33.78 ± 2.41	1.5 ± 0.03
HFD + total LOPs (50 mg/kg/b.wt)	30.619 ± 9.6****	1.122 ± 0.22	11.40 ± 0.76****	33.54 ± 3.23	1.17 ± 0.04
HFD + total LOPs(100 mg/kg/b.wt)	36.05 ± 5.23****	1.638 ± 0.016	8.11 ± 0.84****	32.73 ± 4.62	1.03 ± 0.15
HFD + total LOPs(200 mg/kg/b.wt)	26.38 ± 4.45****	1.569 ± 0.075	9.69 ± 0.65****	33.42 ± 3.69	2.89 ± 0.75*
HFD + Lutein	43.5 ± 2.61*	0.700 ± 0.13	14.52 ± 0.88	33.97 ± 3.81	0.65 ± 0.06
HFD + Orlistat	30.69 ± 5.23****	0.841 ± 0.18	8.91 ± 0.92****	31.92 ± 2.59	0.81 ± 0.07
Epididymal WAT
Groups
Normal Diet (Control)	34.2 ± 0.612****	0.726 ± 0.199	9.65 ± 0.27****	30.55 ± 1.76****	5.80 ± 0.21****
HFD Control	50.5 ± 1.23	0.236 ± 0.04	13.51 ± 0.96	42.03 ± 3.77	1.9 ± 0.01
HFD + total LOPs (50 mg/kg/b.wt)	44 ± 0.196****	0.398 ± 0.02	10.39 ± 0.96***	38.38 ± 3.59****	1.94 ± 0.18
HFD + total LOPs(100 mg/kg/b.wt)	41.87 ± 6.02****	0.36 ± 0.03	8.12 ± 0.84****	26.13 ± 3.51****	1.49 ± 0.07
HFD + total LOPs(200 mg/kg/b.wt)	39.3 ± 0.63****	0.427 ± 0.11	8.68 ± 0.65****	27.51 ± 2.58****	3.45 ± 0.09*
HFD + Lutein	46.7 ± 1.45***	0.35 ± 0.038	8.96 ± 0.28****	28.90 ± 2.92****	0.7 ± 0.21
HFD + Orlistat	36.8 ± 0.698****	0.56 ± 0.04	9.91 ± 0.42****	31.04 ± 3.60****	0.4 ± 0.46

Notes: The intervention of the above-mentioned groups was done for 22 weeks. HFD, High-Fat Diet; LOP, Lutein Oxidized Product; WAT, White Adipose Tissue; GST, Glutathione-S-transferase; FAS, Fatty Acid Synthase; CPT, Carnitine Palmitoyl Transferase; HMG CoA, β-Hydroxy β-methylglutaryl-Coenzyme A. Data are represented as mean standard deviation with n = 8/group. Two-way ANOVA was used for the comparison between each group. Dunnet’s multiple comparison posthoc tests were used to compare different dosed groups with the HFD control group.*P < 0.01,**P < 0.001,***P < 0.005,****P < 0.0001.

3.4. Effect of total LOPs on lipid metabolizing enzymes/molecules

The activities of FAS and CPT were elevated in the high-fat diet-fed group by 40–65% and 19–38% in comparison with normal diet fed group. The intervention of total LOPs decreased the enzyme activity. At 50 mg/kgb.wt FAS and CPT was reduced by 26% (P < 0.0001) and 1% in the liver and 23% (P < 0.005) and 9% (P < 0.0001) in epididymal WAT. At 100 mg/kgb.wt the enzyme activities were inhibited by 47% (P < 0.0001) and 3% in the liver and 40% (P < 0.0001) and 38% (P < 0.0001) in epididymal WAT. 200 mg/kgb.wt intubated group decreased enzyme FAS and CPT activities by 37% (P < 0.0001) and 1% in the liver and 36% (P < 0.0001) and 35% (P < 0.0001) in epididymal WAT. Concerning lutein, FAS was reduced by 6% (P < 0.0001) but CPT did not show any inhibition in the liver whereas, in epididymal WAT, the FAS and CPT activities were decreased by 34% (P < 0.0001) and 31% (P < 0.0001), respectively. Orlistat inhibited FAS and CPT activities by 42% (P < 0.0001) and 6% in liver and 27% (P < 0.0001) and 26% (P < 0.0001) in comparison with the high-fat diet-fed group. The accumulation of cholesterol decreases with an increase in the ratio of HMGCoA: Mevalonate. The intervention of total LOPs significantly increased the ratio to 26% (P < 0.0001) in the liver and 29% (P < 0.0001) in epididymal WAT at 200 mg/kgb.wt. At 50 and 100 mg/kgb.wt, lutein and orlistat there was no significant increase in the ratio (Table 4).

3.5. Effect of total LOPs on 3T3-L1 differentiation

The MTT analysis of total LOPs with 3T3-L1 cells with a concentration ranging from 5 to 500 µg/ml did not show any possible cytotoxicity towards the tested cells even after the incubation period of 48hrs. Total LOPs at concentrations of 5 µg to 100 µg/ml exhibited 100% viability whereas total LOPs of concentrations ranging from 100 µg to 500 µg/ml showed viability up to 80%. Therefore, further experiments were carried out with total LOPs concentrations of 25, 50 and 100 µg/ml. The 3T3-L1 preadipocytes were differentiated into mature adipocytes using differentiation mediums 1 and 2. Total LOPs of concentrations 25 µg, 50 µg, and 100 µg/ml were treated with 3T3-L1 cells along with two controls i.e. cell control (without total LOPs and differentiation media) and standard control (with differentiation media and without total LOPs). The formation of lipid droplets initiated from day 4 after adding differentiation medium 2 in both standard control and total LOPs added media. On day 8, cells were stained with oil-red-o-staining. Total LOPs (100 µg/ml) added differentiation media significantly decreased the accumulation of lipid droplets both in number as well as in size in comparison with standard control (Figure 2).

Figure 2. Photograph of different stages of differentiation of 3T3-L1 pre-adipocytes to mature adipocytes. Note: Differentiation and oil-red-o staining of 3T3L1 cells treated with total LOPs were carried out using HiDiff™ 3T3-L1 Differentiation kit (CCK011), Himedia, Mumbai, India; according to manual protocol. Cells were cultured in DMEM, high glucose medium supplemented with 10% FBS. The cells were maintained at 37°C and 5% atmospheric CO_2. On Day 0 the cells were sub-cultured and after reaching 70-80% confluency, were treated with differentiation medium 1 on Day 2 and differentiation Medium 2 on Day 4. Complete differentiation was achieved on Day 8 and the lipid droplets were stained using the Oil-red-O staining method. Cells were viewed under Moticam Pro 205 A microscope with 400× magnification. Group 1 3T3-L1 cells are grown only in DMEM media without differentiation media and total LOPs. Group 2 3T3-L1 cells are grown with differentiation media but without treatment with total LOPs and Group 3 3T3-L1 cells are grown with differentiation media and total LOPs of concentration (100 µg/ml).

3.6. Effect of total LOPs on adipogenesis marker genes

Total LOPs (25, 50 and 100 µg/ml) down-regulated the mRNA expression of PPARγ [17%, 48% (P < 0.01), 58% (P < 0.001)] and LPL [34%, 39%, 54% (P < 0.01)] (Figure 3(A,B)), whereas, adiponectin mRNA expression was increased to 55% (P < 0.001), 60% (P < 0.001), and 70% (P < 0.0001) at concentrations of 25, 50, and 100 µg/ml, respectively (Figure 3(C)).

Figure 3. Effect of total LOPs on the expression of differentiation-associated marker genes in 3T3-L1 cells. Note: Cells were treated with differentiating media 1 and 2 and total RNA was extracted on day 5 and subjected to RT-qPCR to analyse the expression of genes PPARγ, LPL and adiponectin on 3T3-L1 cells treated with total LOPs of concentration. The y-axis indicates fold change in expression among the samples treated with different concentrations of total LOPs using the results from RT-qPCR. LOPs-Lutein Oxidized Products. Data are represented as mean ± standard deviation with triplicate values. One sample t-test was performed to analyse the significance *P < 0.01, **P < 0.001, ***P < 0.005, ****P < 0.0001.

3.7. Effect of p38MAP kinase and TGF-β1 signalling molecules on 3T3-L1 cells cultured with total LOPs

The activities of p38MAP kinase and TGF-β1 were detected in day-5 pre-adipocyte control cells. TGF-β1 expression was decreased by 38% (P < 0.01) and p38MAP kinase activity was reduced by 22% (P < 0.01) in total LOPs treated cells (Figure 4). Hence total LOPs treated 3T3-L1 cells decreased the formation of lipid droplets by lowering the effects of p38MAP kinase and TGF-β1 signalling molecules.

Figure 4. Effect of total LOPs on the expression of p38MAP kinase and TGF-β1 in 3T3-L1 cells. Note: Day-5 differentiated 3T3-L1 cells (treated with 100 µg/ml of total LOPs) were collected by scraping from culture flasks, lysed using cell extraction buffer for 30 min and centrifuged at 13,000 rpm for 10 min at 4°C. Cell lysates were diluted at the ratio of 1:10 using the standard dilution buffer provided in the kit and sandwich ELISA was performed by adding 100 µl of diluted cell lysates with appropriate controls as per manual instructions provided in the kit. Data are represented as mean ± standard deviation, with triplicate values. LOPs-Lutein Oxidized Products. TGF-β1-transforming growth factor beta 1, p38MAPK-p38 mitogen-activated protein. Two-way ANOVA was used for the comparison between each group. *P < 0.01, **P < 0.001, ***P < 0.005, ****P <0.0001.

4. Discussion

The efficacy of total LOPs with stated anti-obese properties was studied by intervening in high-fat diet-induced C57BL/6 mice. The C57BL/6 mice were chosen because they exhibited abnormalities like HFD-induced obesity, hyperinsulinemia, and insulin resistance similar to human metabolic syndrome [29]. In a short-term toxicity study and sub-chronic toxicity study, lutein and its ester forms were administered orally from 4 to 400 mg/kg.b.wt did not show any mortality, change in body weight, or food consumption pattern, organ weight and any other adverse conditions [30]. An acute toxicity study of lutein from Tagetes erecta in lutein-deficient male mice was carried and the LD₅₀ exceeded the highest dose of 10,000 mg/kgb.wt. and in sub-acute toxicity study mice were gavaged with 0–1000 mg/kg/b.wt/day for 4 weeks. In both the study, there were no toxicologically significant effects of lutein in clinical observation, ophthalmic examinations, body and organ weights, haematological, histopathological, and other clinical chemistry parameters [31]. Kruger et al. experimented by feeding lutein up to 639 mg /kg.b.wt/per day for 4 weeks in mice and concluded that there was no exposure-related toxicity or adverse effects in lutein [32]. Some researchers have chemically synthesized oxidized products of lutein by using Azo-mediated products [33], lutein oxidative degradation derivatives mediated through UV-irradiation was also tested with rats to study its anti-oxidant, anti-inflammatory property [34]. In both cases, there was no observed toxicity effect on the subject. In the same way, total LOPs an oxidized product of lutein did not show any mortality/adverse effects on C57BL/6 mice during 22 weeks of experimentation.

Total LOPs at 100 mg/kg b.wt. showed significant ameliorative effects on the gain in body weight, liver and adipose tissue weight gain, total cholesterol, triglycerides, phospholipids, AST, ALT, lipid peroxides, and MDA levels in HFD-fed mice better than that of orlistat and lutein intubated group.

In our previous article (https://doi.org/10.1039/D1FO04064B), total LOPs exhibited significant pancreatic lipase inhibitory activity whereas, the parent molecule lutein did not show any lipase inhibition in-vitro [18]. In the present in-vivo studies total LOPs along with lutein showed an antiobesity effect by reducing the gain in body weight and also displaying lipid-lowering activities. Similarly, Balogun et al. have shown that bulbs of Allium sativum exhibit an antiobesity effect by decreasing adipogenesis and upregulating AMK-activated protein in 3T3-L1 adipogenesis [35]. There are reports which have shown few molecules with antiobesity properties without pancreatic lipase inhibition [17]. Hence compounds without lipase inhibition could possess anti-obesity properties, and therefore it is important to study the mechanism through in-vivo and cell lines to understand the antiobesity nature of a biomolecule.

FAS is the key enzyme that regulates the synthesis of long chain fatty acids such as myristate, palmitate, and stearate from acetyl-CoA and malonyl-CoA in the presence of NADPH [36]. FAS along with CPT and ACC are known to convert excess food intake into lipids for storage in adipose tissue leading to obesity [37]. A study reports FAS inhibitors reduce food intake and body weight by directly acting on appetite centres in the brain in BALB/c mice [38]. Bioactive molecules resveratrol and lovastatin exerted an anti-obesity effect in apoE-deficient mice (0.02% w/w) by decreasing the activity of FAS and various other lipid metabolism-modulating enzymes such as glucose-6-phosphate dehydrogenase and phosphatidate phosphohydrolase (PAP) activity in the liver and adipose tissue but did not show any effects on CPT [39]. In our study, the HFD + lutein group did not significantly inhibit the activity of FAS and CPT in the liver but concerning epididymal WAT homogenate activity of both the enzyme was downregulated significantly. Concerning the HFD + Orlistat group, FAS activity in the liver was attenuated whereas did not have any significant role with CPT. Further, in epididymal WAT, enzyme FAS and CPT were significantly inhibited. total LOPs at 100 mg/kg b.wt. significantly attenuated the activities of both FAS and CPT in the liver and adipose tissue homogenates proving to be the best anti-obese molecule.

Anti-obese mechanism of total LOPs is better understood by unravelling regulatory molecules involved in the process of adipogenesis. Adipogenesis involves four stages such as growth arrest, mitotic clonal expansion, early differentiation, and terminal differentiation [40]. PPARγ and CEBPs are the early key regulators of adipogenesis whereas FAS and adiponectin are important regulators during the formation of mature adipocytes [4]. Total LOPs suppressed the expression of PPARγ and LPL genes and increased the expression of adiponectin and decreased hypertrophy and hyperplasia in mature adipocytes. Similarly, in a study bioactive fraction of Aronia melanocarpa fruit of concentrations 0.025 and 0.05 mg/ml significantly inhibited adipogenic differentiation and decreased mRNA and protein expression in PPARγ, C/EBPα, FABP2, FAS, and LPL in 3T3-L1 cells [41]. Adiponectin is the most abundant peptide secreted by adipocytes, which decreases with obesity and increases with loss of weight and shows health-beneficial effects [42].

A deeper understanding of adipocyte formation involves pathways of various signalling molecules. Mesenchymal stem cells (MSCs) are the precursor cells that lead the differentiation towards osteogenic or adipogenic MSC differentiation, where overexpression of one factor inhibits the expression of others [43]. Oxysterols, an anti-adipogenic bioactive molecule has shown to inhibit adipogenic differentiation of MSCs through extracellular signal-regulated kinases (ERK) [44]. p38 MAP kinase also called stress kinases is one of the cell-signalling MAP kinases which are involved in inflammatory and profibrotic responses and are found to be associated with cell differentiation, proliferation, and apoptosis. The levels of p38 are greatly elevated in the pathogenesis of obesity, diabetes, and cardiovascular diseases [45]. Several studies have shown that p38 inhibitions can suppress adipogenesis. Engelmann et al. proved that specific inhibitors of p38, SB203580 and SB202190 block 3T3-L1 adipogenesis [46]. In another study Genistein, a bioactive isoflavone decreased lipid accumulation in 3T3-L1 cells by inhibiting the phosphorylation of p38 MAPK, FAS, Janus kinase 2 (JAK2)-mediated C-EBP alpha expression [15]. In our study, total LOPs of concentration 100 µg/ml down-regulated p38 MAPK activity and TGF-β1 expression in 3T3-L1 pre-adipocytes. Increased expression of TGF-β1 is known to be directly correlated with an increase in body mass index in animal and human models of obesity [47]. Alessi et al. explain that, in morbidly obese patients, the increase in adipose tissue plasminogen activator inhibitor 1 is due to an increase in TGF-β1 expression [48]. Hence this study suggests that total LOPs possess novel anti-obesity effects accompanied by lipid-lowering, antioxidants and suppression of differentiation of pre-adipocytes. Our results advance the understanding of multiple mechanisms of action of total LOPs and provide a crucial role in controlling the progression of obesity.

To conclude, all three concentrations of total LOPs (50,100 and 200 mg/kg.b.wt) displayed anti-obese properties with multiple targets but total LOPs at 100 mg/kg.b.wt exhibited the best anti-obesity effect. Further clinical trials at different phases and with different species will help total LOPs to emerge as promising nutraceuticals. Extrapolation of doses from one species to another depends on various parameters like the difference in body surface area and body weight of the species. According to Food and Drug Administration (FDA) and the Center for Drug Evaluation and Research (CDER); conversion of animal doses to human equivalent doses is given by the formula: HED = animal dose in mg/kg × (animal weight in kg/human weight in kg)^0.33. Where HED is the human equivalent dose and 0.33 is the correction factor (https://www.fda.gov/media/72309/download) [49].

In the said experimental case, the oral-gavaged dosage of total LOPs in C57BL/6 mice is 50, 100 and 200 mg/kg b.wt. If we extrapolate it to human beings for an obese person weighing 100 kg it will be 0.3, 0.6 and 1.2 g, respectively, to carry out further clinical trials. Till now the dosage has not been set for the consumption of total LOPs as it is a novel product. Further clinical trials will help us to set the dosages.

Acknowledgements

The authors thank Mr Krishna Murthy, KCC Biolabs, Tumakuru for providing an animal house facility and Mr Rukmangada M, Karyome Private Limited, Mysuru for performing RT-qPCR. NS carried out experiments and analysed data. NS and CKM participated in the experiment design and data analysis. CKM designed and directed the project. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All animal studies were conducted under a protocol approved by the Institutional Animal Ethical Committee (IAEC approval No. KCC/IAEC/014/2020).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The datasets generated during and/or analysed during the current study are available from the corresponding author on request.

Additional information

Funding

The authors gratefully acknowledge the financial support from the Indian Council of Medical Research, New Delhi, GoI with grant number 45/13/2019-BIO/BMS.