*2.5. SR, CR and Combined Extracts Suppressed Hepatic Glucose Output by Inhibiting Gluconeogenesis and Glycogenolysis as Well as Promoting Glycolysis and Glycogenesis*

Gluconeogenesis and glycogenolysis are two major pathways for endogenous glucose production [24,25]. PEPCK, FBPase, G6Pase, and GP are the rate-limiting enzymes controlling gluconeogenesis and glycogenolysis in the liver, respectively. The activities of these enzymes were increased in T2DM rats compared to normal rats but the increased activities were remarkably decreased to a range from 96.4% to 71.1% by SR and CR, especially by their combined extracts (Figure 6).

Glycolysis is regarded as a feeder pathway that prepares glucose for further catabolism and energy production. Besides, glucose storage in the form of glycogen could suppress hepatic glucose output to maintain blood glucose homeostasis. GK, PFK, PK, and GS are the rate-limiting enzymes controlling glycolysis and glycogen synthesis, respectively [26]. However, the activities of GK, PFK, PK, and GS were decreased in T2DM rats. After treatment, the decreased activities were increased to a range from 111.7% to 141.5% by SR and CR, especially their combined extracts (Figure 6).

#### *2.6. Intervention Effects of SR, CR and Combined Extracts on the Metabolic Profiling of T2DM Rats*

In this study, UPLC-Q-TOF-MS/MS was applied to generate metabolic profiles of plasma and urine from normal and T2DM rats before and after oral administration of SR, CR, LSC, and HSC in the negative and positive ESI modes. The metabolomics profiles of N and M groups were separated by PCA analysis and orthogonal partial least squares discriminant analysis (OPLS-DA). To investigate the amelioration of SR, CR, LSC, and HSC for curing T2DM, another PCA model was built. The variations of plasma and urine metabolic profiling of SR, CR, LSC, and HSC were restored to the levels of the normal group (Figures 7 and 8). Furthermore, the relative quantities of nine potential biomarkers in plasma and five in urine were significantly regulated by LSC and HSC. There were no obvious effects on the levels of xanthosine and N-acetyl-L-tyrosine for the SR group. The levels of xanthosine and hippuric acid were not regulated by the CR group. It was interesting to note that the levels of xanthosine were neither affected by SR nor CR, but by their combination. The contents of the potential biomarkers in Table S2 were considered as biomarkers for the effects of treatment.

The metabolic pathway was established by importing the potential metabolites into the web-based database MetPA. The pathway impact value calculated from pathway to topology analysis with MetPA above 0.1 was screened out as the potential target pathway. Here in Figure 9, glycerophospholipid metabolism with the impact values of 0.18 were filtered out as the most important metabolic pathways. Moreover, a correlation network of potential metabolites related to T2DM was exhibited in Figure 10. Our results also indicated that administration of LSC and HSC could more effectively modify these biomarkers and their related metabolic pathways including purine metabolism in which SR and CR could not be involved in T2DM rats.

**Figure 6.** Activities of glucokinase (GK), phosphofructokinase (PFK), pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase (FBPase), glucose 6-phosphatase (G6Pase), glycogen synthase (GS) and glycogen synthase (GP) in livers of the normal group (N), model group (M), and groups gavaged with metformin (P), scutellaria Radix (SR), coptidis Rhizome (CR), low dose of combined extracts group (LSC) and high dose of combined extracts (HSC). The values were shown as mean ± SD. # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 vs. normal group; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. model group. Data were analyzed by One-way-ANOVA.

**Figure 7.** PCA model results between normal (N) and T2DM (M) rats in negative mode ((**A**) 2-D plot of plasma; (**B**) 2-D plot of urine). 3D PLS-DA scores plot of LC–MS spectral data ((**C**) plasma; (**D**) urine). S-plot of OPLS-DA model for M vs. N group ((**E**) plasma; (**F**) urine). PCA analytical results from N and M rats treated with metformin (P), scutellaria Radix (SR), coptidis Rhizome (CR), low dose of combined extracts group (LSC) and high dose of combined extracts (HSC) at negative mode ((**G**) plasma; (**H**) urine).

**)L**

**Figure 8.** PCA model results between normal (N) and T2DM (M) rats in positive mode ((**A**) 2-D plot of plasma; (**B**) 2-D plot of urine). 3D PLS-DA scores plot of LC-MS spectral data ((**C**) plasma; (**D**) urine). S-plot of OPLS-DA model for M vs. N group ((**E**) plasma; (**F**) urine). PCA analytical results from N and M rats treated with metformin (P), scutellaria Radix (SR), coptidis Rhizome (CR), low dose of combined extracts group (LSC) and high dose of combined extracts (HSC) at positive mode ((**G**) plasma; (**H**) urine).

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**Figure 9.** Metabolic pathways involved in potential markers in plasma and urine. (**A**) Scutellaria Radix group (SR); (**B**) Coptidis Rhizome group (CR); (**C**) metformin group (P), Low dose of combined extracts group (LSC) and High dose of combined extracts group (HSC). (**1**) Glycerophospholipid metabolism, (**2**) primary bile acid biosynthesis, (**3**) pyrimidine metabolism, (**4**) linolenic acid metabolism, (**5**) alpha-linolenic acid metabolism, (**6**) arachidonic acid metabolism, (**7**) purine metabolism.

**Figure 10.** Correlation networks of main potential biomarkers in response to T2DM rats. Compared with the normal group, the red arrows represent the decrease of the contents of endogenous metabolites, while the green arrows represent the increase of the contents of endogenous metabolites.

#### **3. Discussion**

SR and CR, especially their combined extract, have been clinically used to treat T2DM for thousands of years; however, their compatibility mechanism remains unknown. Thus, in this study, the amelioration of SR, CR and their combined extracts on T2DM was first investigated, and their compatibility mechanism was further unraveled by the metabolomics and MAPK/PI3K/Akt signaling pathway. Importantly, the induction of the T2DM animal model is widely desired.  Therefore, a suitable and stable T2DM rat model using HFD along with low dose of STZ was successfully established in our study. The levels of FBG, TG, TC, LDL-C, and FFA were remarkably increased, while the level of HDL-C was notably decreased in T2DM rats, which was similar to human T2DM sharing common symptoms (Figure 1).

Additionally, accumulating studies indicated that chronic inflammation was a major contributor to insulin resistance and T2DM [27–30]. Pro-inflammatory cytokines, particularly TNF-α, could enhance the serine phosphorylation of insulin receptor substrate-1, which induced insulin resistance. Some studies have shown glucose lowering effects of specific TNF-α or IL-1β inhibitors [31,32]. As expected, SR and CR could markedly ameliorate inflammation, insulin resistance, hyperglycemia, and hyperlipemia of T2DM rats. Furthermore, their compatibility (LSC and HSC) was more effective than single drug (Figures 1 and 3).

MAPK signaling pathway played a crucial role in inflammation [33]. Especially, NF-κB was a principle factor in regulating inflammatory mediators during the inflammatory process. The stimulation of NF-κB increased the levels of downstream inflammatory factors such as TNF-α, IL-1β, and IL-6, which promoted the insulin resistance in the liver. mRNA expressions of key targets in

MAPK pathway were significantly decreased after treatment. Additionally, protein expression of p-P65 was also decreased. Current data elucidated that LSC and HSC could exert better anti-inflammatory effects via blocking of the MAPK pathway (Figure 4).

The amelioration of inflammation might contribute to activating insulin signaling in liver. Remarkably, LSC and HSC inhibited inflammation in liver, but it was tempting to know whether this action was responsible for improving the insulin signaling pathway. The levels of Glut2, p-PI-3K, and p-Akt are major indexes that reflect the activity of the insulin signaling pathway, play a vital role in insulin-activated glucose uptake in liver, and inhibit glucose release from hepatocytes. As expected, LSC and HSC markedly up-regulated the mRNA expressions of IRS1, PI3K, Akt2, and Glut2, while protein expressions of p-PI3K, p-Akt, and Glut2 were also enhanced. These data suggested that LSC and HSC could overcome the impairment in PI3K/Akt insulin signaling pathway, contributing to the improvement of glucose disposal in T2DM rats (Figure 5).

Subsequently, the influence of activated insulin signaling pathway on glycogenolysis, glycogen synthesis, gluconeogenesis, and glucose metabolism in liver was investigated. The liver is crucial for the maintenance of normal glucose homeostasis—it produces glucose during fasting and stores glucose postprandially. Insulin could inhibit glycogenolysis, stimulate glycogen synthesis, reduce gluconeogenesis, and increase glucose metabolism. However, these hepatic processes were dysregulated in T2DM because the liver became insulin resistant. Gluconeogenesis and glycogenolysis constituted two major pathways for endogenous glucose production. Gluconeogenesis involved the production of glucose from non-carbohydrate precursors such as glycerol, glycogenic amino acid, and lactate during non-availability of dietary glucose, or glycogen stored in the liver was degraded to glucose by glycogenolysis. PEPCK, FBPase, and G6Pase acted irreversibly in the gluconeogenesis pathway. FBPase inhibitors and a decrease in transcription of PEPCK and G6Pase genes could reduce excessive endogenous glucose production in T2DM [34,35]. GP regulated hepatic glycogenolysis and GP inhibitor could lower blood glucose [36]. GS modulated hepatic glycogen synthesis and GSK-3 specific inhibitor could enhance glycogen synthesis by increasing GS activity [37]. GK mainly expressed in hepatocytes had effects on regulating blood glucose level in vivo [38]. Previous study also found that the ability of glucose metabolism in T2DM was reduced when the syntheses of PFK and PK were decreased [39]. In this work, owing to the amelioration of the insulin signaling pathway, PEPCK, FBPase, G6Pase, and GP activities were notably decreased, while prominent increased activities of GK, PFK, PK, and GS were detected after treatment. The above data indicated that the activities of key enzymes related to glycometabolism were modulated to notably lower FBG by SR and CR, especially by their compatibility (Figure 6).

As T2DM was a metabolic disease, the effects of SR, CR, LSC, and HSC on metabolic disorders in T2DM rats were further investigated to depict more insight into their compatibility mechanism. Metabolite profiles are closely related to the development of T2DM [40–43]. Among the most recent investigations on metabolic dysfunctions and plasma metabolites in T2DM, plasma level of bile acids have been confirmed to be closely associated with T2DM [44,45]. Moreover, metabolites of plasma and urine could reflect the changes of systemic physiology. Therefore, based on the comparative analysis of plasma and urine metabolomics in normal and T2DM rats, potential biomarkers and the correlated pathways could be discovered, which would be helpful to clarify the compatibility mechanism of SR and CR in improving metabolic disturbance of T2DM rats. In this study, plasma metabolomics of T2DM rats suggested that cholic acid and deoxycholic acid (secondary bile salts) levels were obviously increased, while the glycocholic acid (conjugated primary bile salts) level was dramatically decreased. Bile acids are major components of bile formed from cholesterol through various enzymatic reactions in hepatocytes. Primary bile acids are synthesized mainly by conjugation with taurine or glycine through the terminal side-chain carboxylic group presenting in the bile acid structure. Bile salt hydrolases, which are produced by many members of gut microbiota, could remove the conjugated amino acid from the primary bile salt to cholate and deoxycholate. Bile acids could exert effects on glucose metabolism via activation of bile acid receptors and G-protein-coupled membrane

bound receptors (TGR5) [46–51]. TGR5 was expressed in cells of the hematopoietic system such as monocytes and macrophages and conferred anti-inflammatory properties in vitro and in vivo, decreasing cytokine production in monocytes, macrophages and kupffer cells [52,53]. Recent works revealed that the immune-suppressive actions of TGR5 in macrophages contributed to the prevention of the T2DM process [48]. Thus, we speculated that glycocholic acid might be an endogenous TGR5 ligand, while cholic acid and deoxycholic acid were ligand inhibitors. So glycocholic acid could suppress inflammation by TGR5 activation. Both P38 and ERK were significantly stimulated by deoxycholic acid in a prior study [54]. Supplied with deoxycholic acid, serum glucose and triglyceride levels were increased in mice [55]. After treatment, the levels of cholic acid and deoxycholic acid were markedly decreased and glycocholic acid was notably increased. Furthermore, the combination of SR and CR was more effective than the single drug (Table S2).

Additionally, the glycerophospholipid metabolism was the most important metabolic pathway. The decreased levels of glycerophospholipid metabolites including LysoPE(0:0/18:0), LyspPC(16:0), LysoPE(20:2(11Z,14Z)/0:0), LysoPC(18:2(9z,12z)), PC(16:1(9z)/18:1(11z)), and LysoPC(16:1(9z)) indicated a marked perturbation in the glycerophospholipid metabolism of T2DM rats. PCs, LysoPCs, LysoPEs were largely involved in the pathogenesis of inflammatory and metabolic diseases such as diabetes [56]. PCs and LysoPCs could block ERK, P38 activation and NF-ƙB translocation to the nucleus in peritoneal macrophages, which had an intrinsic anti-inflammatory property [57–59]. Many studies also indicated that levels of lysoPCs are closely associated with oxidative stress and inflammation [60,61]. LysoPCs containing an unsaturated fatty acyl group such as C20:4 and C22:6 were reported to show potent anti-inflammatory activity in in vivo and in vitro models [62,63]. Lower levels of LysoPCs were found to be predictors for T2DM in earlier studies [64] and treatment with LysoPCs could result in a significant increase in the level of GLUT4 at the plasma membranes of 3T3-L1 adipocytes [65]. Thus, the modification of SR, CR, LSC, and HSC on LysoPCs, LysoPEs and PCs levels might contribute to the amelioration of inflammation and insulin resistance. SR and CR combined therapy exhibited better efficacy (Table S2).

By the analysis of urine metabolomics, purine metabolism was notably modified after combined therapy, however, the single drug showed no effect; purine metabolism was terribly dysregulated which was associated with T2DM [66]. Xanthosine, a nucleoside derived from the purine base xanthine and ribose, was elevated in T2DM rats and catabolism of xanthosine could ultimately lead to the production of high uric acid which was linked to diabetic complications [67,68]. Interestingly, xanthosine was affected by neither SR nor CR, but by their combination. Additionally, hippuric acid, which could be converted from dietetic aromatic compounds by gut microbes [69], was significantly decreased in urine of T2DM rats. This might be due to disturbed gut microbiota metabolism under diabetic status [70]. As reported, concentrations of hippuric acid were lower in HFD-induced hyperlipidemic rats than in normal rats [71]. D-glucurono-6,3-lactone, participating in ascorbate and aldarate metabolism, was increased in T2DM rats and was downregulated after treatment of SR, CR and their combined extracts. Pyrimidine metabolism was also disturbed and increased metabolite of deoxyuridine in the urine of T2DM rats was identified. After 30 days therapeutic intervention, these changed trends in T2DM rats were reversed by SR and CR, particularly by their combined extracts (Table S2).

### **4. Materials and Methods**

#### *4.1. Chemicals, Reagents, and Materials*

Acetonitrile (HPLC grade) was bought from TEDIA (Fairfield, OH, USA). Formic acid was supplied by Merck KGaA (Darmstadt, Germany). Metformin was purchased from Merck Serono (Shanghai, China). SR and CR were purchased from Nanjing Guo-yao Pharm (Nanjing, Jiangsu, China). Streptozotocin (STZ) was obtained from Sigma chemical (St Louis, MO, USA).


**5.1.1 Methodology**

276 Macro to Nano Spectroscopy

Reagent B – Weight 75g Potassium sodium tartrate (Rochelle salt), add 125mL of destiled

Subsequently, add reagent A over the B, homogenize under heating until dissolve

Get an aliquot 500µg.mL-1 of the sample. Add 1mL of DNS, shake in vortex and let in a boiling bath during 5 minutes, put the material in ice bath until cool, add 3.75mL of destiled

An adaptation of the DNS method to determine the total sugar can be getting from a previous hydrolysis before dosage. The hydrolysis is made with 0.5mL HCl concentrate and incubation in water bath at 60°C for 10 minutes. After then, the solution must be neutralized

The procedure for total sugar quantification follow the same described for reducing sugar

This method is based on determination of reducing sugar before and after the digestion by invertase using acid for p-hydroxy benzoic acid hydrazide – PAHBAH. In this method, with some modifications, there is possible to perform the colorimetric determination of glucose, fructose and sucrose, during this analysis procedure with invertase. The major subject of the methodology described in this chapter is for determination of reducing sugar. The phydroxy benzoic acid hydrazide also has the advantage that glucose and fructose when reacting produce the same intensity of colour, and then all free monossacharides present in

Reagent A: 10g p-hydroxy benzoic acid dissolved in 60mL water, add 10mL hydrochloride

Reagent B: 24.9g trisodium citrate in 500mL water, 2.2g calcium chloride, 40g sodium

Get an aliquot 50µg.mL-1 of the reducing sugar sample, add 5mL p-hydroxy benzoic acid hydrazide reagent (mixed in the same day) shake in vortex, incubate in water bath at 100°C by six minutes, cool until room temperature, and read the absorbance in spectrophotometer

**5.1 Sulphuric acid-Cysteine-Tryptophan method (Messineo & Edward Musarra,1972)**  Fructose is dehydrated in acid medium for formation of furfural derivative, which complexs with cysteine hydrochloride to produce a chromophore at starting of green color that is

In the day of analysis, mix reagents A and B in proportion of 1:10, after keep at 4°C.

completely, and after cool the mix, complete the volume of the solution to 250mL.

water, shake again, and read the absorbance in spectrophotometer at 540nm.

**4.2 Hydrazide method of p-hydroxy-benzoic acid (Blakeney & Mutton, 1980)**

with NaOH 2M and cooled with ice bath until room temperature.

water. Shake under heating until total dissolution.

used above.

the sample can be determined.

acid and complete volume to 200mL.

**5. Fructose determination**

hydroxide, and to complete the volume to 2.000mL.

**4.2.1 Methodology**

Reagents:

at 410nm.

Reagents: sulphuric acid 75%, cysteine hydrochloride 2.5%, tryptophan solution in hydrochloride acid to formation of tryptophan hydrochloride (100µg.mL-1 in HCl 0.1M).

Getting an aliquot 50µg.mL-1 of the sample, add 2.8mL of sulphuric acid 75%, shake in vortex, add 0.1mL cysteine hydrochloride solution 2.5%, shake in vortex again, let in waterbath 45-50°C for 10 minutes, cool at room temperature and add 1mL tryptophan hydrochloride solution, shake again in vortex.

This sequence must be followed rigorously during assay because the formation of the final chromophore depends on the initial formation of the first formed chromophore by complexation with cysteine hydrochloride. After that, read the absorbance in spectrophotometer at 518nm.


**7. Supportive therapy**

Supportive care is critical throughout the clinical course of patients with MM because they are particularly susceptible to infections. Diligence in identifying and initiating treatment at the earliest sign is advised [27].

All MM patients with newly diagnosed MM and with adequate renal function should be initiated with monthly bone-modulating therapy at diagnosis with either denosumab, zoledronic acid or pamidronate (high recommendation) [27].

Patients with clinical manifestation of MM but without objective evidence of lytic lesions using the conventional radiography can be managed with zoledronic acid (intermediate recommendations), though its advantages using the more advanced objective measurements like CT and MRI is not yet well established [53].

In patients without clinical manifestations of myeloma, the use of bisphonphonates is not generally advised (high evidence of toxicities) [8]. The continuous use of Zoledronic acid recommended as long as the cycles of treatments are on progress, but sufficient data are lacking supporting it after partial response to therapy is

achieved [54, 55]. Denosumab doses can be administered at home if nursing facility is available or the patient should be trained for self-administration. Long-term discontinuation of denosumab treatment is associated with rebound effect and thus should be circumvented [8].

Sufficient data are available that prohibits the use of bortezomib-based regimens in patients with baseline clinical renal impairments. However, evidences are lacking supporting the discontinuation of therapy in patients who develop drug induced renal impairments [56, 57].

Severely anemic patients who do not respond to the conventional anemia management or deteriorating should be urgently switched to erythropoiesis stimulating agents (ESAs) in order to prevent the need for blood transfusions and frequent hospital visits. At present, the whole blood supplies has been extensively restricted due to the COVID-19 lockdown [8]. There are only few data advocating the use of ESAs in patients with persistent symptomatic anemia (hemoglobin <10 g/dL) where other causes of anemia have ruled out. Hence, ESAs should be discontinued after 6–8 weeks of therapy in patients' who fail to respond adequately to anemia treatment [58].

Immunization against influenza is recommended for specific infections caused by streptococcus pneumonia and hemophilus influenza, but sufficient data are lacking supporting the efficacy of the vaccination due to the fact that suboptimal immune responses are fairly seen after management [59].

If patients are receiving PIs & ASCT, the prophylactic use of antiviral agents such as acyclovir (or valacyclovir) are highly recommended [8, 60]. Acyclovir should be prescribed according to local protocols. Immunoglobulin administration may be given in an individualized basis, depending on the depth of suppression of polyclonal immunoglobulins and patient history of recurrent infections [34].

Antiviral prophylaxis has to be recommended in drugs associated with increased risk of herpes zoster reactivation such as Daratumumab and PIs. The prophylaxis is recommended for at least 3 months after exposure if no contraindications are observed [61].

Routine prophylaxis immunization should be considered with a series of pneumococcal conjugate vaccine 13 and pneumococcal polysaccharide vaccine, as well as annual influenza immunization [62]. Drugs such as IMiDs enhance the risk of venous thromboembolism, so preventive measures should be considered during active therapy [61], and the antithrombotic prophylaxis should be considered according to local or international guidelines. For countries with high incidence of COVID-19, low-molecular-weight heparin (LMWH) has to be preferred over aspirin as thromboprophylaxis in MM patients under IMiD administration, irrespective of their thrombotic risk [8, 63].

Patients with a history of neutropenias and/or recurrent infections should receive prophylactic G-CSF injections. Co-trimoxazole prophylaxis for Pneumocystis jirovecii for all patients and levofloxacin prophylaxis for the first three months of treatment for NDMM patients are also highly recommended [8, 55].

#### **8. Follow-up and monitoring**

Patients should be followed-up and monitored for complete blood counts (CBC), serum and urine electrophoresis with or without the use of serum-FLC determination, and also for serum calcium and creatinine measurements; at least in 2–3 months interval. In patients are complaining of bone pain, skeletal X-ray, MRI or CT scan should be carried out to detect and rule out new bone lesions [64].

**37**

*Treatment Approaches of Multiple Myeloma DOI: http://dx.doi.org/10.5772/intechopen.97390*

**cellular therapies**

**9.1 Venetoclax**

venetoclax [27].

BELLINI trial [27, 68].

undergoing [71].

refractory MM [73].

treatment paradigm [74, 75].

**9.2 BRAF & BRAF/MEK inhibitors**

**9.3 Monoclonal antibodies (CAR T cells and BCMA)**

**9. Emerging role of targeted therapies, monoclonal antibodies, and**

Venetoclax is an orally bioavailable selective B-cell lymphoma 2 (Bcl-2) inhibitor. Bcl-2 and cyclin D1 are over expressed in MM patients with a translocation of (11;14), which is present in approximately 20% of patients with MM [65]. Venetoclax is an antiapoptotic protein and an emerging and effective treatment for relapsed or refractory MM [66, 67], and also being tried for treatment of chronic lymphocytic leukemia (CLL) cells [66] and non-Hodgkin lymphoma (NHL) [65]. Although response rates with venetoclax-dexamethasone are impressive in patients with t(11;14), PIs, which inhibit induced myeloid leukemia cell differentiation protein (Mcl-1), have synergistic activity when combined with

Currently the venetoclax is suspended by the Food and Drug Administration (FDA) because of a report obtained from the BELLINI trial, which stated an increased relative risk of death in the venetoclax group. As more recent data are being collected to have a better understanding of the safety concerns raised by the

BRAF is a proto-oncogene, that its protein product is a serine/threonine-protein kinase B-Raf that make conform the MAP kinase/ERKs signaling pathway, which works during cell division and differentiation [69]. In patients with MM, the incidence of BRAF V600E mutations is about 7–12% [70]. Trials evaluating BRAF and BRAF/MEK inhibitors in patients with MM harboring BRAF mutations are still

Monoclonal antibodies represent an emerging class of agents in MM treatment [72]. Daratumumab-RVd versus RVd, are being evaluated for ASCT-eligible patients [27]. Isatuximab, a humanized immunoglobulin G1 monoclonal antibody, has distinct characteristics in contrast to other anti-CD38 monoclonal antibodies [72]. Isatuximab in combination with IMROZ, isatuximab-RVd in ASCT-ineligible patients with newly diagnosed MM are being studied [72]; and isatuximab-Kd (IKEMA) and isatuximab-Pd (ICARIA) have been tried in patients with relapsed or

B-cell maturation antigen (BCMA) is a significant target communicated on MM cells, with other targets counting GPR5CD, CD138, CD74, CD48, CD38, SLAMF7, and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI). Several strategies targeting BCMA include conjugated antibodies, cellular approaches, and bispecific T-cell engagers. Clinical trials evaluating BCMA-directed Chimeric antigen receptor redirected T cells (CAR-T cells) are furthest in development. Two notable BCMA CAR T-cell products are bb2121 and LCAR-B38M.69–71 Trials are evaluating CAR T cells in patients with relapsed or refractory MM to better understand their role in the MM

Newly introduced therapies that uses CAR T cells and BCMA antibodies bid promises of adding agents to the antimyeloma armamentarium of neoadjuvant mechanisms of actions [49]. Enduring trials will permit for the integration of


**4. Results and discussion**

### **4.1. The geometries of the triscarbene-nickel-hydrido complex and the triscarbenepalladium-hydrido complex**

The geometrical structures of the triscarbene-nickel-hydrido complex (**Pro-Ni**) and the triscarbene-palladium-hydrido complex (**Pro-Pd**) are firstly determined theoretically. The optimized geometries for these two species are computed at the M06-L/Def2-SVP level of theory. As

**Scheme 4.**

shown in **Figure 2**, the M06-L calculations show that the computed M─C bond lengths for both molecules (average 1.924 and 2.089 Å at M06-L) compare favorably with the average M─C bond lengths that are determined from X-ray data (1.907 and 2.057 Å) [43]. Similarly, the average values for the ∠C─M─C and ∠C─M─H angles for these two structures are calculated to be 98.26° and 82.58° (Ni) and 97.41° and 82.23° (Pd), which agrees reasonably well with the experimental data (97.85, 82.00, 95.94, and 84.00°, respectively) [43], as shown in **Figure 2**. Given the agreement between the M06-L method using the Def2-SVP basis set and the available experimental data [43], it is expected that the same relative accuracy is applicable to any discussion of their reactivities and the reaction mechanisms, for which experimental data are still not available.

**Figure 1** shows that the energy of point **2** (left in **Figure 1**), the anchor point for 3

and 16-electron CpML and 14-electron L2

In this qualitative theoretical treatment, the transition-metal fragment L2

is broken. This analysis is used to interpret the results in the following section.

**4.1. The geometries of the triscarbene-nickel-hydrido complex and the triscarbene-**

The geometrical structures of the triscarbene-nickel-hydrido complex (**Pro-Ni**) and the triscarbene-palladium-hydrido complex (**Pro-Pd**) are firstly determined theoretically. The optimized geometries for these two species are computed at the M06-L/Def2-SVP level of theory. As

the value of ∆*E*st + ∆*Eσσ*\*

Since CH2

**Scheme 4.**

and a″ symmetry.

molecular result of the insertion of the L2

158 Descriptive Inorganic Chemistry Researches of Metal Compounds

**4. Results and discussion**

**palladium-hydrido complex**

the reactant geometry, is governed by the singlet-triplet energy gap for both LnM and C─H; i.e., ∆*E*st (= *E*triplet − *E*singlet for LnM) + ∆*Eσσ\**(= *E*triplet − *E*singlet for C─H). In other words, the smaller

tion [64–68]. If a reactant, LnM, has a singlet ground state with a small triplet excitation energy, there is a greater probability that a triplet LnM contributes to the singlet reaction and the reactions occur readily. Both the order of the singlet and triplet states and the magnitude of the singlet-triplet energy separation also determine the existence and the height of the energy barrier.

orbitals with the same symmetry patterns (**5**), in which each fragment has one orbital of a′

an empty electrophilic orbital (i.e., a', as shown in **5**) that interacts with a filled hydrocarbon fragment orbital. This facilitates a concerted 1,2-hydrogen migration. In other words, the net

is that a new M─C *σ* bond and a new M─H *σ* bond are formed and the C─H *σ* bond of an IC

, the lower is the activation barrier and the more exothermic is the reac-

[LnM]3

M and CpML has

M are isolobal [70], each has two valence

M and CpML complexes into a C─H *σ* bond of an IC

[IC] in

**Figure 2.** Selected geometrical parameters (in Å and deg) for the triscarbene-nickel-hydrido complex (**Pro-Ni**) and the triscarbene-palladium-hydrido complex (**Pro-Pd**), calculated at the M06-L/Def2-SVP level of theory and a comparison with the experimental values [43]. Hydrogens are omitted for clarity.

#### **4.2. The geometries and energetics of the L2 M + 1,2-dimethylimidazolium cation**

The results for four regions on the potential energy surfaces for L2 M (M = Ni, Pd, Pt; L = 1,3-aryl-NHC, aryl = 2,4,6-trimethylphenyl) and 1,2-dimethylimidazolium cation (IC) are shown: 14-electron L2 M plus free IC (**Rea**), a precursor complex (**Pcx**), the transition state (**TS**), and the oxidative addition product (**Pro**). The fully optimized geometries for the key points, calculated at the M06-L/Def2-SVP level, are shown in **Figure 3**. The important geometrical parameters and relative energies and the potential energy profiles at the same level of theory are listed in **Table 1** and **Figure 4**, respectively. Four points are noteworthy.

	- **1.** The reactants, **Rea-Ni, Rea-Pd**, and **Rea-Pt**, are computed as both low-spin (singlet) and high-spin (triplet state) complexes. The M06-L computations demonstrate that these transition metal complexes all adopt the singlet ground state. The computations also show that the singlet-triplet triplet free energy splitting (∆*E*st; kcal/mol) for these fragments are in the order: **Rea-Ni** (23.7) < **Rea-Pd** (50.1) < **Rea-Pt** (63.9). These values are much greater than those for other previously studied L2 M complexes that have various ancillary ligands [51–56]. Therefore, it is possible that the oxidative addition reactions (Eq. (1)) that are studied in this work proceed on the singlet surface. The singlet surface is therefore the focus of this study, from this point.
	- 2. The optimized transition state structures (**TS-Ni, TS-Pd**, and **TS-Pt**) and arrows that indicate the main atomic motion in the transition state eigenvector are shown in **Figure 3**. These model computations show that the oxidative addition reactions that are studied using these model reactants all proceed in a concerted fashion via a threecenter transition state, as shown in **Figure 3**, and all reactions are exothermic. It is noted that for the oxidative addition reactions involving the group 10 transition metals that are studied in this work, the free energies for the transition states are all less than those for the corresponding reactants. It is theoretically predicted that these oxidative addition reactions proceed readily, even at room temperature. Further supporting evidence comes from the fact that the oxidative additions between **Rea-Ni** and **Rea-Pd** species and an imidazolium cation have been experimentally proven to be easy [43].
	- 3. According to the theoretical analysis of the VBSCD model that is discussed in Section 3, the smaller the value of ∆*E*st for L2 M, the lower is the barrier height and the more exothermic is the reaction and the faster is the oxidative addition reaction. The model evidence confirms this prediction. For the M06-L calculations for the model systems that have group 10 transition metals, a plot of the activation barrier (∆*E*‡ ) versus the ∆*E*st is shown, for which the best fit is ∆*E*‡ = 0.518∆*E*st – 11.2. The linear correlation between ∆*E*st and the Gibbs free energy (∆*G*), which is also calculated at the same level of theory, is ∆*G* = 0.566 ∆*E*st − 67.5. The theoretical results definitely show that for the facile oxidative addition of C─H bonds, an understanding of the ∆*E*st of the coordinatively unsaturated 14-electron L2 M is crucial, since it can be used to predict the reactivity of the reactants.

The Mechanisms for the Oxidative Addition of Imidazolium Salts to a Group 9 Transition Metal Atom (Co0, Rh0, and... http://dx.doi.org/10.5772/67567 161

**4.2. The geometries and energetics of the L2**

160 Descriptive Inorganic Chemistry Researches of Metal Compounds

shown: 14-electron L2

─H4

**2.** The C3

The results for four regions on the potential energy surfaces for L2

are listed in **Table 1** and **Figure 4**, respectively. Four points are noteworthy.

are much greater than those for other previously studied L2

3, the smaller the value of ∆*E*st for L2

shown, for which the best fit is ∆*E*‡

14-electron L2

singlet surface is therefore the focus of this study, from this point.

L = 1,3-aryl-NHC, aryl = 2,4,6-trimethylphenyl) and 1,2-dimethylimidazolium cation (IC) are

and the oxidative addition product (**Pro**). The fully optimized geometries for the key points, calculated at the M06-L/Def2-SVP level, are shown in **Figure 3**. The important geometrical parameters and relative energies and the potential energy profiles at the same level of theory

**1.** For the optimized structures, see **Figure 3**. For the relative free energies, see **Figure 4**.

**1.** The reactants, **Rea-Ni, Rea-Pd**, and **Rea-Pt**, are computed as both low-spin (singlet) and high-spin (triplet state) complexes. The M06-L computations demonstrate that these transition metal complexes all adopt the singlet ground state. The computations also show that the singlet-triplet triplet free energy splitting (∆*E*st; kcal/mol) for these fragments are in the order: **Rea-Ni** (23.7) < **Rea-Pd** (50.1) < **Rea-Pt** (63.9). These values

various ancillary ligands [51–56]. Therefore, it is possible that the oxidative addition reactions (Eq. (1)) that are studied in this work proceed on the singlet surface. The

2. The optimized transition state structures (**TS-Ni, TS-Pd**, and **TS-Pt**) and arrows that indicate the main atomic motion in the transition state eigenvector are shown in **Figure 3**. These model computations show that the oxidative addition reactions that are studied using these model reactants all proceed in a concerted fashion via a threecenter transition state, as shown in **Figure 3**, and all reactions are exothermic. It is noted that for the oxidative addition reactions involving the group 10 transition metals that are studied in this work, the free energies for the transition states are all less than those for the corresponding reactants. It is theoretically predicted that these oxidative addition reactions proceed readily, even at room temperature. Further supporting evidence comes from the fact that the oxidative additions between **Rea-Ni** and **Rea-Pd** species and an imidazolium cation have been experimentally proven to be easy [43]. 3. According to the theoretical analysis of the VBSCD model that is discussed in Section

thermic is the reaction and the faster is the oxidative addition reaction. The model evidence confirms this prediction. For the M06-L calculations for the model systems that

and the Gibbs free energy (∆*G*), which is also calculated at the same level of theory, is ∆*G* = 0.566 ∆*E*st − 67.5. The theoretical results definitely show that for the facile oxidative addition of C─H bonds, an understanding of the ∆*E*st of the coordinatively unsaturated

M is crucial, since it can be used to predict the reactivity of the reactants.

have group 10 transition metals, a plot of the activation barrier (∆*E*‡

bond distance in IC (reactant) is calculated to be 1.090 Å.

**M + 1,2-dimethylimidazolium cation**

M plus free IC (**Rea**), a precursor complex (**Pcx**), the transition state (**TS**),

M (M = Ni, Pd, Pt;

M complexes that have

) versus the ∆*E*st is

M, the lower is the barrier height and the more exo-

= 0.518∆*E*st – 11.2. The linear correlation between ∆*E*st

**Figure 3.** M06-L/Def2-SVP optimized geometries for the stationary points for the oxidative addition reactions of **Rea-M** (M = Ni, Pd, and Pt) molecules. For selected geometrical parameters and relative energies for each species, see **Table 1**. The bold arrows denote the main atomic motions in the transition state eigenvector. Some hydrogens are omitted for clarity.

**Table 1.** Selected geometrical parameters (bond distances in Å), relative energies ∆*E* (zero-point corrected; kcal mol-1) and relative Gibbs free energies ∆*G* (kcal mol-1) at 298 K at the M06-L/Def2-SCP level of theory for the optimized stationary points on the oxidative addition reactions (Eq. (1)) [1–30].

**Figure 4.** The reaction energy profile (in kcal/mol) for the oxidative addition reactions: L2 M + 1,2-dimethylimidazolium cation (M = Ni, Pd, and Pt; L = 1,3-aryl-NHC, aryl = 2,4,6-trimethylphenyl). All of the energies are calculated at the M06-L/Def2-SVP level. See also **Table 1** and **Figure 3**.

#### **4.3. The geometries and energetics of the CpM′+ 1,2-dimethylimidazolium cation**

Similarly to the study of the L2 M system, the M06-L/Def2-SVP level is also used to study the mechanisms for the oxidative addition reactions for CpM′L (M′ = Co, Rh, Ir; L = 1,3-aryl-NHC, aryl = 2,4,6-trimethylphenyl), as shown in **Figure 5**. The relative M06-L energies and the key geometrical parameters for the stationary points are also listed in **Table 2**. The corresponding potential energy profiles are given in **Figure 6**. Three interesting conclusions can be drawn from these figures and the table.

	- **1.** The M06-L calculations in **Table 2** show that the ground states for the CpCoL and CpIrL fragments are triplets, but the CpRhL complex is a singlet. The M06-L results also show that the ∆*E*st value for the CpCoR and CpIrR fragments are respectively computed to be− 8.3 and − 0.88 kcal/mol and the ∆*E*st of CpRhR was predicted to be 10.2 kcal/mol. It is worthy to note that whenever a reactant contains a heavy atom that is not necessarily directly included in the reaction, a strong spin-orbit coupling (SOC) can occur [71–75]. That is, because of the presence of the heavy atom, a triplet reactant can cause a spin-inversion process. It transfers to the singlet reactant and then forces the singlet reaction. Since these theoretical calculations show that both the CpCoR and the CpIrR species have a small value for ∆*E*st and a heavier transition metal is involved, the SOC is expected to be substantial for the oxidative additions and these would eventually proceed in the singlet chemical reactions.
	- 2. **Figure 6** shows that, similar to the case for L2 M molecules, the energy of the transition state for Co, Rh, and Ir is less than that for the reactants, which demonstrates that the CpM′L (M′ = Co, Rh, and Ir) complexes readily overcome the energy barrier and then undergo oxidative addition into the C─H bond of IC in a concerted fashion, even at room temperature. The model computations show that the oxidative addition of a CpM′L fragment that has a group 9 metal (M′) decreases in the order: CpCoL > CpIrL > CpRhL. For the reverse process (right to left in **Figure 6**), the barriers to reductive elimination for the Co, Rh, and Ir systems have much higher energies than those for the corresponding oxidative addition. The theoretical evidence demonstrates that these CpM′L complexes undergo oxidative additions more easily than reductive eliminations, as noted in the introduction. That is to say, because the attached NHC groups readily donate electrons, the electrons in the metal, M′, are abundant and the CpM′L readily allows oxidative additions with the incoming molecules. The theoretical studies suggest that the CpM′L molecules prefer to undergo oxidative addition reactions with the imidazolium cation, even at room temperature.
	- 3. According to the VBSCD model, the smaller the ∆*E*st value for CpM′L (if ∆*Eσσ*\* is a constant), the lower is the barrier height, the more exothermic is the reaction and the faster is the oxidative addition reaction [64–68]. The M06-L/Def2-SVP results support this prediction. The M06-L calculations show that value for ∆*E*st (kcal/mol) increases in the order: CpCoL (−8.3) < CpIrL (−0.77) < CpRhL (+8.0). As shown in **Table 2**, both the

**Figure 4.** The reaction energy profile (in kcal/mol) for the oxidative addition reactions: L2

M06-L/Def2-SVP level. See also **Table 1** and **Figure 3**.

cation (M = Ni, Pd, and Pt; L = 1,3-aryl-NHC, aryl = 2,4,6-trimethylphenyl). All of the energies are calculated at the

**Geometrical structures Energies**

**Table 1.** Selected geometrical parameters (bond distances in Å), relative energies ∆*E* (zero-point corrected; kcal mol-1) and relative Gibbs free energies ∆*G* (kcal mol-1) at 298 K at the M06-L/Def2-SCP level of theory for the optimized

**Rea-Ni** 1.836 1.836 – – – 0.0 0.0 **Pcx-Ni** 1.938 1.961 – 1.107 2.056 −50.24 −33.65 **TS-Ni** 1.981 1.947 1.886 1.482 1.527 −49.38 −32.26 **Pro-Ni** 1.911 1.904 – 1.985 1.394 −69.31 −54.36 **Rea-Pd** 2.015 2.015 – – – 0.0 0.0 **Pcx-Pd** 2.050 2.053 – 1.115 2.105 −39.69 −23.67 **TS-Pd** 2.159 2.138 2.039 1.437 1.679 −25.99 −8.417 **Pro-Pd** 2.057 2.067 – 2.144 1.572 −55.36 −38.23 **Rea-Pt** 2.003 2.003 – – – 0.0 0.0 **Pcx-Pt** 2.022 2.019 – 1.119 2.134 −41.35 −22.46 **TS-Pt** 2.128 2.064 2.067 1.333 1.797 −16.49 −1.384 **Pro-Pt** 2.054 2.060 – 2.139 1.621 −47.52 −31.98

**-H4 M-H4 ∆***E* **∆***G*

**Systems M-C1 M-C2 M-C3 C3**

162 Descriptive Inorganic Chemistry Researches of Metal Compounds

stationary points on the oxidative addition reactions (Eq. (1)) [1–30].

M + 1,2-dimethylimidazolium

**Figure 5.** M06-L/Def2-SVP optimized geometries for the stationary points for the oxidative addition reactions of **Rea-M**′ (M′ = Co, Rh, and Ir) molecules. For the selected geometrical parameters and relative energies for each species, see **Table 2**. The bold arrows denote the main atomic motions in the transition state eigenvector. Some hydrogens are omitted for clarity.

The Mechanisms for the Oxidative Addition of Imidazolium Salts to a Group 9 Transition Metal Atom (Co0, Rh0, and... http://dx.doi.org/10.5772/67567 165

**Table 2.** Key geometrical parameters (bond distances in Å), relative energies ∆*E* (zero-point corrected; kcal mol-1) and relative Gibbs free energies ∆*G* (kcal mol-1) at 298 K, calculated at the M06-L/Def2-SCP level of theory, for the optimized stationary points on the oxidative addition reactions (Eq. (2)) [1–30].

**Figure 6.** The reaction energy profile (in kcal/mol) for the oxidative addition reactions: CpML + 1,2-dimethylimidazolium cation (M = Co, Rh, and Ir; L = 1,3-aryl-NHC, aryl = 2,4,6-trimethylphenyl). All of the energies are calculated at the M06-L/ Def2-SVP level. See also **Table 1** and **Figure 3**.

**Figure 5.** M06-L/Def2-SVP optimized geometries for the stationary points for the oxidative addition reactions of **Rea-M**′ (M′ = Co, Rh, and Ir) molecules. For the selected geometrical parameters and relative energies for each species, see **Table 2**. The bold arrows denote the main atomic motions in the transition state eigenvector. Some hydrogens are omitted for clarity.

164 Descriptive Inorganic Chemistry Researches of Metal Compounds

activation energies (∆*E*‡ ) and the Gibbs free energies (∆*G*) follow the same order as the ∆*E*st value (kcal/mol): Co (+9.28, −62.4) < Ir (+10.8, −46.2 kcal/mol) < Rh (+11.0, −39.1). In order to determine a good model for the facile oxidative addition of 16-electron CpM′L to a C-H bond of an imidazolium cation, an understanding of the ∆*E*st of the coordinatively unsaturated CpM′L is important.


**1. Introduction**

Excessive body weight is a growing health problem worldwide. It is a well-known risk factor in cardiovascular disease, diabetes, hypertension, and cancers, among other conditions [1]. According to World Health Organization, obesity has been considered as one of the leading threats to future public health.

Obesity is a complex health issue resulting from a combination of causes and individual factors such as behavior and genetics [2]. Behaviors can include physical activity, inactivity, dietary patterns, medication use, and other exposures. Although there are genetic, behavioral, metabolic and hormonal influences on body weight, obesity occurs when you take in more calories than you burn through exercise and normal daily activities that is when energy intake exceeds energy expenditure. Even though the cause of obesity is complicated, dietary habit or lifestyle plays an important role in developing obese conditions [3].

The increasing westernization, urbanization and mechanization occurring in most countries around the world is associated with changes in the diet towards one of high fat, high energy-dense foods and a sedentary lifestyle [4]. Nutrition transition in developing countries leads to dietary intakes of micronutrient – poor, energy – dense foods, which may be important determinants of overweight/obesity [5].

There is also a direct relationship between the amount of dietary fat and the degree of obesity. High-fat diets induce greater food intake and weight gain than highcarbohydrate diets as indicated by animal studies [6]. Contributing factors are caloric density, satiety properties and post-absorptive processing. The satiating effects after meals with a high fat:carbohydrate ratio is less than for meals with a lower ratio.

Mediterranean pattern of nutrition, has been described as containing high proportion of mono-unsaturated fat acids versus saturated fats and contributes in the preservation of body weight, while expands longevity and life expectancy. This pattern of nutrition has been adopted by many countries and it is diversified according to the cultural and socio-economic features of each country. Several research studies have commented on the beneficial effects of the Mediterranean diet in the preservation or decrease of body weight, in the primary or secondary prevention of coronary disease, in the maintenance of high density cholesterol (HDL) and triglycerides within normal rates, as well as in the significant reduction in mortality rates [7–9].

Current recommendations for weight management emphasize the importance of healthy eating patterns that include a variety of nutrient-dense foods, limit portions of energy-dense foods, and reduce overall energy density [10].

A unifying factor for weight loss across dietary patterns is energy density [11]. When a diet's energy density is reduced; it allows individuals to consume satisfying amounts of food for fewer calories. The goal of dietary therapy in the management of obesity is to reduce the total number of calories consumed.

## **2. Physiological causes and effects of obesity**

Obesity is a complex disease that involves an excessive amount of body fat. It is a medical problem that increases your risk of other diseases and health problems, such as heart disease, diabetes, high blood pressure and certain cancers. Although there are genetic, behavioral, metabolic and hormonal influences on body weight, obesity occurs when you take in more calories than you burn through exercise and normal daily activities that is when energy intake exceeds energy expenditure.

Even though the cause of obesity is complicated, dietary habit or lifestyle plays an important role in developing obese conditions [3, 12]. Lifestyle changes and nutritional habits, such as irregular meal patterns; mainly skipping breakfast, consumption of foods and beverages of low nutritional value and sub-optimal intake of dairy products, fruits and vegetables, intake of refined carbohydrates such as sugar sweetened soft drinks are all contributing factors to the development of obesity [12–15].

Obesity as a complex disorder has multiple etiological factors. The primary factor which is considered as a driver of obesity is obesogenic environment and unhealthy eating behavior. Secondary factors such as genetic and neuroendocrine factors, and diseases such as hypothyroidism and polycystic ovary syndrome are also related to excessive weight [16].

In a study carried out among undergraduates in Nigeria observed a significant relationship between vegetable consumption and Body Mass Index (X2 = 16.031, p-value = 0.001) and there exist no significant relationship between cereals consumption and body mass index (X2 = 8.916, p-value = 0.710) [17].

Obesity has been shown to be a predisposing factor in the rising prevalence of morbidity and mortality associated with non – communicable diseases like type-2 diabetes mellitus, hypertension, cancer, stroke among adults [18].

Overweight and obesity increases the likelihood of various diseases, particularly heart diseases, type2 diabetes, breathing difficulties during sleep, certain types of cancer and osteoarthritis [19].

#### **3. Dietary fat and obesity**

Fat are of various types, some are more beneficial to health than others. Those found in foods are known as dietary fats, the body needs fat to function fully as it

#### *Diet and Obesity DOI: http://dx.doi.org/10.5772/intechopen.98326*

has a lot of functions in the body and is essential to health. For instance, fat soluble vitamins (Vitamins A, D, E and K) cannot be transported in the absence of fat. However, a diet with too much fat can increase body weight and also increase the risk of cardiovascular disease.

Dietary fats are believed to play an important role in the development of heart disease [20]. The National health institutions has recommended to reduce the intake of dietary fat to prevent CVDs [21].

#### **3.1 Healthy versus unhealthy fat**

The healthy fat are primarily unsaturated fats:

**Monounsaturated fat**: This type of fat is found in a variety of foods and oils. Eating foods rich in monounsaturated fats improves blood cholesterol levels, which in turn decrease the risk of heart disease and may also help decrease the risk of type 2 diabetes.

**Polyunsaturated Fat:** This fat is found mostly in plant-based foods and oils. Omega-3 fatty acids, which is a polyunsaturated fat has been attributed to low rate of heart disease [22]. Also, it helps to raise HDL cholesterol level [23].

Oils such as canola oil, olive oil, corn oil, sunflower oil and peanut oil are made up of monounsaturated and polyunsaturated fats and are liquid at room temperature. Fish such as tuna, salmon, trout, mackerel, herring and sardines are high in omega-3 fatty acids. Plant sources of omega-3 fatty acids are soybean oil, flaxseed oil, and nuts (walnuts, butternuts).

#### **3.2 Unhealthy fats**

Saturated fats and trans fat are often termed unhealthy fats, this is due to the fact that both raise LDL cholesterol levels. High level of LDL cholesterol in the blood increases the risk of heart disease and stroke. A larger amount of saturated fats is from animal sources, including meat and dairy products. Foods high in saturated fat include fatty beef, pork, butter, lard and cream, poultry with skin. Plant-based oils, such as coconut oil, palm oil also contain saturated fats, but do not contain cholesterol.

Trans fat is a product of a process that adds hydrogen to liquid vegetable oils to make them more solid and also refer to as partially hydrogenated oils. These partially hydrogenated oils increase LDL cholesterol and lowers HDL cholesterol which in turn increase the risk of cardiovascular disease. Foods high in trans fat include fried foods, such as French fries, stick margarines and shortenings, pastries, packaged foods, baked goods and pizza dough.

Fatty foods are energy dense and give 9calories per gram compared to carbohydrate and protein that gives 4calories per gram. Also, if physical activity is inadequate, excess consumption of fat can results into weight gain. Also, it does not take as much energy (about 3%), to convert and store dietary fat as it does to convert and store glucose or protein. Fats are quickly and easily stored by the body.

Obesity is a multifactorial and complex affectation that is characterized by a longterm excess energy intake(EI) above energy expenditure (EE) and epidemiological evidence have suggested that a high-fat diet promotes the development of obesity [24].

There is also a direct relationship between the amount of dietary fat and the degree of obesity. An overview of animal studies had indicated that high-fat diets induce greater food intake and weight gain than high-carbohydrate diets [25]. Contributing factors are caloric density, satiety properties and post-absorptive processing. The satiating effects after meals with a high fat:carbohydrate ratio is less than for meals with a lower ratio. It has been reported that the most important variable influencing meal size is the nutrient content of the range of foods consumed and not the level of hunger.  Several mechanisms have been proposed to explain why high fat intake might lead to greater body fat [26]. Dietary fat is the most energy dense macronutrient in the diet, providing 9 kcal/g as opposed to 4 kcal/g for carbohydrate or protein; this could lead to overconsumption of energy if food volume is regulated. Fat adds greater flavor and palatability to foods, which could thus increase their consumption.

Fat has a lower thermogenic effect than carbohydrate and protein and this inhibit energy expenditure. A positive energy balance can be the result of overconsumption of energy, perhaps because a high-fat diet has a lower satiating effect per joule than a low-fat diet. A series of studies has produced robust evidence that the fattening effect of dietary fat is linked mainly to the higher energy density of fatty foods compared with carbohydrate and protein rich foods.

In addition, when studied under careful metabolic conditions for short periods, carbohydrate produces a greater thermogenic effect than fat, suggesting that dietary fat may be utilized more efficiently and accumulate as body fat [27]. Carbohydrate intake, but not fat intake, is regulated; thus, individuals on a high-fat diet would tend to consume more total energy to gain the required amount of carbohydrate than would someone on a low-fat diet [28]. Although these mechanisms may seem compelling at face value, foods are not eaten in isolation, and the energy for weight of foods is more determined by the water and fiber content.

Different types of fat contain the same amount of energy, although there are differences in their respective influence on energy balance, energy expenditure and satiety. Low-fat diets and in weight-maintenance diets, have been shown to be affected by the quality of dietary fat.

Monounsaturated was found to induce a lower level of postingestive satiety and a larger subsequent energy intake than polyunsaturated and saturated fat in a study on the effect in lean subjects of high-fat meals, differing in fatty acid composition [30].

#### **4. Dietary patterns and obesity**

Dietary patterns that have inverse relationship with obesity include vegan diet, Mediterranean diet and prudent diet. Western diet is directly associated with obesity.

#### **4.1 The vegan diet**

This is a form of vegetarian diet that eliminates meat and animal product, this diet is beneficial to health because it reduces the intake of cholesterol and saturated fat that is predominant in animal product. It has been proven that those who practice a vegan diet minimize their overall risk of coronary heart disease, obesity and high blood pressure.

#### **4.2 The Mediterranean diet**

This diet recommends the use of plant based oil as alternative to butter, it emphasizes adding vegetables to each meal. Avoidance of meat is recommended, though not eliminated. This diet has been proven to help with depression, in addition to controlling blood sugar levels and helping with weight loss. Whole grains, nuts and herbs are also used in larger amounts.


4.3.4. Interpretation

The results are obtained considering the descriptions presented in the preceding sections. As stated above, the ReCiPe method will be used to carry out the environmental impact assessment of the bridge. For this purpose, by means of the midpoint approach, 18 impact categories will be shown with the associated uncertainty. In addition, the contribution of the different processes of the bridge life-cycle for the most popular impact categories will be represented. In the endpoint approach, the three damage categories are studied. Both approaches allow a higher level of interpretation.


**Preface**

Energy storage and conversion are crucial research and industry topics, especially within the context of sustainable development. Scientific and technological progress in these fields have the potential to improve energy use efficiency across traditional, renewable and unconventional sources.

Energy storage technologies, including batteries, fuel cells, supercapacitors (ultracapacitors) and superconducting magnetic energy storage (SMES), combined with decreasing costs are creating new scenarios and opportunities in the development and market of energy generation, grids, industrial plants, complex systems and consumer electronics.

This reprint collects some of the latest developments and applications in these interdisciplinary fields, offering a common framework for authors from different research areas.


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[67] Okuhara M, Kuroda Y, Goto T, Okamoto M, Terano H, Kohsaka M, et al. Studies on a new phosphonic acid antibiotic III. Isolation and characterisation of FRFR-31564, FR-32863 and FR-33289. Journal of Antibiotics. 1980;**33**:24-28. DOI: 10.7164/antibiotics.33.24

[68] Edwards RL, Brothers RC, Wang X, Maron MI, Ziniel PD, Tsang PS, et al. MEPicides: Potent antimalarial prodrugs targeting isoprenoid biosynthesis. Scientific Reports. 2017;**7**:art.8400

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[74] Blodgett JAV, Zhang JK, Yu X, Metcalf WW. Conserved biosynthetic pathways for phosalacine, bialaphos and newly discovered phosphonic acid natural products. The Journal of Antibiotics. 2016;**69**:15-25. DOI: 10.1038/ja.2015.77

[75] Wild A, Ziegler C. The effect of bialaphos on ammonium-assimilation and photosynthesis I. Effect on the enzymes of ammonium-assimilation. Zeitschrift für Naturforschung. 1984;**44**:97-102. DOI: 10.1515/ znc-1989-1-217

[76] Devkota P, Johnson WG. Glufosinate efficacy as influenced by carrier water pH, hardness, foliar fertilizer, and ammonium sulfate. Weed Technology. 2016;**30**:848-859. DOI: 10.1614/ WT-D-16-00053.1

[77] Tachibana T, Kaneko T. Development of an herbicide, bialaphos. Journal of Pest Science. 1986;**11**:297-304. DOI: 10.1584/ jpestics.11.297

[78] Kato H, Nagayama K, Abe H, Kobayashi R, Ishihara E. Isolation, structure and biological activity of trialaphos. Agricultural and Biological Chemistry. 1991;**55**:1133-1134. DOI: 10.1080/00021369.1991.10870694

[79] Omura S, Murata M, Hanaki H, Hinotozawa K, Oiwa R, Tanaka H. Phosalacine, a new herbicidal antibiotic containing phosphinothricin. Fermentation, isolation, biological activity and mechanism of action. The Journal of Antibiotics. 1984;**37**:829-835. DOI: 10.7164/antibiotics.37.829

[80] Rapp C, Jung G, Kugler M, Loeffler W. Rhizocticins—New phosphonooligopeptides with antifungal activity. European Journal of Organic Chemistry. 1988:655-661. DOI: 10.1002/ jlac.198819880707

[81] Gahungu M, Arguelles-Arias A, Fickers P, Zervosen A, Joris B, Damblon C, et al. Synthesis and biological evaluation of potential threonine synthase inhibitors: Rhizocticin A and Plumbemycin A. Bioorganic & Medicinal Chemistry. 2013;**21**:4958-4967. DOI: 10.1016/j. bmc.2013.06.064

[82] Park BK, Hirota A, Sakai H. Studies on new antimetabolite produced by microorganism. III. Structure of plumbemycin A and B, antagonists of L-threonine from *Streptomyces plumbeus*. Agricultural and Biological Chemistry. 1977;**41**:573579. DOI: 10.1271/ bbb1961.41.573

[83] Borisova SA, Circello BT, Zhang JK, van der Donk WW, Metcalf WW. Biosynthesis of rhizocticins, antifungal phosphonate oligopeptides produced by *Bacillus subtilis* ATCC6633. Chemistry & Biology. 2010;**17**:28-37. DOI: 10.1016/j.chembiol.2009.11.017

[84] Evans BS, Zhao C, Gao J, Evans CM, Ju K-S, Doroghazi JR, et al. Discovery of the antibiotic phosacetamycin via a new mass spectrometry-based method for phosphonic acid detection. ACS

Chemical Biology. 2013;**8**:908-913. DOI: 10.1021/cb400102t

[85] Kugler M, Loeffler W, Rapp C, Kern A, Jung G. Rhizocticin A, an antifungal phosphono-oligopeptide of *Bacillus subtilis* ATCC 6633: Biological properties. Archives of Microbiology. 1990;**153**:276-281. DOI: 10.1007/ BF00249082

[86] Johnson RD, Kastner RM, Larsen SH, Ose EE. Antibiotic A53868 and process for production thereof. U.S. patent 4,482,488; 1988

[87] Circello BT, Miller CG, Lee J-H, van der Donk WA, Metcalf WW. The antibiotic dehydrophos is converted to a toxic pyruvate analog by peptide bond cleavage in *Salmonella enterica*. Antimicrobial Agents and Chemotherapy. 2011;**55**:3357-3362. DOI: 10.1128/AAC.01483-10

[88] Jiménez-Andreu MM, Quintana AL, Aínsa JA, Sayago FJ, Cativiela C. Synthesis and biological activity of dehydrophos derivatives. Organic & Biomolecular Chemistry. 2019;**5**:1097- 1112. DOI: 10.1039/c8ob03079k

[89] Bougioukou DJ, Ting CP, Peck SC, Mukherjee S, van der Donk WA. Use of the dehydrophos biosynthetic enzymes to prepare antimicrobial analogs of alaphosphin. Organic & Biomolecular Chemistry. 2019;**4**:822-829. DOI: 10.1039/C8OB02860E

[90] Bougioukou DJ, Mukherjee S, van der Donk WA. Revisiting the biosynthesis of dehydrophos reveals a tRNA-dependent pathway. Proceedings of the National Academy of Sciences of the United States of America. 2013;**110**:10952-11057. DOI: 10.1073/ pnas.1303568110

[91] Freestone TS, Ju K-S, Wang B, Zhao H. Discovery of a phosphonoacetic acid derived natural product by pathway refactoring. ACS Synthetic

Biology. 2017;**6**:217-223. DOI: 10.1021/ acssynbio.6b00299

[92] Gunji S, Arima K, Beppu T. Screening of antifungal antibiotics according to activities inducing morphological abnormalities. Agricultural and Biological Chemistry. 1983;**41**:2016-2069. DOI: 10.1080/00021369.1983.10865911

[93] Gao J, Ju K-S, Yu X, Velásques JE, Mukherjee S, Lee J, et al. Use of phosphonate methyltransferase in the identification of the fosfazinomycin biosynthetic gene cluster. Angewandte Chemie, International Edition. 2014;**53**:1334-1337. DOI: 10.1002/ anie.201308363

[94] Schneemann I, Nagel K, Labes A, Wiese J, Imhoff JF. Comprehensivei of marine *Actinobacteria* associated with the sponge *Halichondria panacea*. Applied and Environmental Microbiology. 2010;**76**:3702-3714. DOI: 10.1128/AEM.00780-10

[95] Goettge MN, Cioni JP, Ju K-S, Pallitsch K, Metcalf WW. PcxL and HpxL are flavin-dependent, oxime-forming N-oxidases in phosphonocystoximic acid biosynthesis in *Streptomyces*. The Journal of Biological Chemistry. 2018;**293**:6859-6868. DOI: 10.1074/jbc.RA118.001721

[96] Pallitsch K, Kalina T, Stanković T. Synthetic phosphonic acids as potent tools to study phosphonate enzymology. Synlett. 2019;**30**:770-776. DOI: 10.1055/s-0037-1611460

[97] Yamato M, Koguchi T, Okachi R, Yamada K, Nakayama K, Kase H, et al. K-26, a novel inhibitor of angiotensin I converting enzyme produced cy an *Actinomycete* K-26. The Journal of Antibiotics. 1986;**39**:44-52. DOI: 10.7164/antibiotics.39.44

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[99] Kido Y, Hamakado T, Anno M, Miyagawa E, Motoki Y, Wakamiya T, et al. Isolation and characterization of I5B2, a new phosphorus containing inhibitor of angiotensin I converting enzyme produced by *Actinomadura* sp. The Journal of Antibiotics. 1984;**37**:965- 969. DOI: 10.7164/antibiotics.37.965

[100] Kramer GJ, Mohd A, Schwager LSU, Masuyer G, Acharya KR, Sturrock ED, et al. Interkingdom pharmacology of angiotensin-I converting enzyme inhibitor phosphonates produced by *Actinomycetes*. ACS Medicinal Chemistry Letters. 2014;**5**:346-351. DOI: 10.1021/

[101] Masuyer G, Cozier GE, Kramer GJ, Bachmann BO, Acharya KR. Crystal structure of a peptidyl-dipeptidase K-26-DCP from *Actinomycetein* complex with its natural inhibitor. The FEBS Journal. 2016;**283**:4357-4369. DOI:

[102] Ntai I, Phelan VV, Bachmann BO. Phosphonopeptide K-26 biosynthetic intermediates in

*Astrosporangium hypotensionis*. Chemical Communications. 2006:4518-4520. DOI:

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ml4004588

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inhibitor of angiotensin I converting enzyme—A tripeptide containing phosphonic acid. International Journal of Peptide and Protein Research. 1991;**38**:20-24. DOI: 10.1111/j.1399- 3011.1991.tb01404.x

*Contemporary Topics about Phosphorus in Biology and Materials*

Biology. 2017;**6**:217-223. DOI: 10.1021/

T. Screening of antifungal antibiotics according to activities inducing morphological abnormalities. Agricultural and Biological

Chemistry. 1983;**41**:2016-2069. DOI: 10.1080/00021369.1983.10865911

[93] Gao J, Ju K-S, Yu X, Velásques JE, Mukherjee S, Lee J, et al. Use of phosphonate methyltransferase in the identification of the fosfazinomycin biosynthetic gene cluster. Angewandte

Chemie, International Edition. 2014;**53**:1334-1337. DOI: 10.1002/

[94] Schneemann I, Nagel K, Labes A, Wiese J, Imhoff JF. Comprehensivei of marine *Actinobacteria* associated with the sponge *Halichondria*

*panacea*. Applied and Environmental Microbiology. 2010;**76**:3702-3714. DOI:

[95] Goettge MN, Cioni JP, Ju K-S, Pallitsch K, Metcalf WW. PcxL and HpxL are flavin-dependent, oxime-forming N-oxidases in

phosphonocystoximic acid biosynthesis in *Streptomyces*. The Journal of Biological Chemistry. 2018;**293**:6859-6868. DOI:

[96] Pallitsch K, Kalina T, Stanković T. Synthetic phosphonic acids as potent tools to study phosphonate enzymology.

[97] Yamato M, Koguchi T, Okachi R, Yamada K, Nakayama K, Kase H, et al. K-26, a novel inhibitor of angiotensin I converting enzyme produced cy an *Actinomycete* K-26. The Journal of Antibiotics. 1986;**39**:44-52. DOI:

[98] Hirayama N, Kasai M, Hirata K. Structure and conformation of a novel

Synlett. 2019;**30**:770-776. DOI: 10.1055/s-0037-1611460

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10.1074/jbc.RA118.001721

anie.201308363

[92] Gunji S, Arima K, Beppu

acssynbio.6b00299

Chemical Biology. 2013;**8**:908-913. DOI:

[85] Kugler M, Loeffler W, Rapp C, Kern A, Jung G. Rhizocticin A, an antifungal phosphono-oligopeptide of *Bacillus subtilis* ATCC 6633: Biological properties. Archives of Microbiology. 1990;**153**:276-281. DOI: 10.1007/

[86] Johnson RD, Kastner RM, Larsen SH, Ose EE. Antibiotic A53868 and process for production thereof. U.S.

[87] Circello BT, Miller CG, Lee J-H, van der Donk WA, Metcalf WW. The antibiotic dehydrophos is converted to a toxic pyruvate analog by peptide bond cleavage in *Salmonella enterica*. Antimicrobial Agents and Chemotherapy. 2011;**55**:3357-3362. DOI:

[88] Jiménez-Andreu MM, Quintana AL,

[89] Bougioukou DJ, Ting CP, Peck SC, Mukherjee S, van der Donk WA. Use of the dehydrophos biosynthetic enzymes to prepare antimicrobial analogs of alaphosphin. Organic & Biomolecular Chemistry. 2019;**4**:822-829. DOI:

[90] Bougioukou DJ, Mukherjee S, van der Donk WA. Revisiting the biosynthesis of dehydrophos reveals a tRNA-dependent pathway. Proceedings of the National Academy of Sciences of the United States of America. 2013;**110**:10952-11057. DOI: 10.1073/

[91] Freestone TS, Ju K-S, Wang B, Zhao H. Discovery of a phosphonoacetic acid derived natural product by pathway refactoring. ACS Synthetic

Aínsa JA, Sayago FJ, Cativiela C. Synthesis and biological activity of dehydrophos derivatives. Organic & Biomolecular Chemistry. 2019;**5**:1097- 1112. DOI: 10.1039/c8ob03079k

10.1021/cb400102t

BF00249082

patent 4,482,488; 1988

10.1128/AAC.01483-10

10.1039/C8OB02860E

pnas.1303568110

**112**

[99] Kido Y, Hamakado T, Anno M, Miyagawa E, Motoki Y, Wakamiya T, et al. Isolation and characterization of I5B2, a new phosphorus containing inhibitor of angiotensin I converting enzyme produced by *Actinomadura* sp. The Journal of Antibiotics. 1984;**37**:965- 969. DOI: 10.7164/antibiotics.37.965

[100] Kramer GJ, Mohd A, Schwager LSU, Masuyer G, Acharya KR, Sturrock ED, et al. Interkingdom pharmacology of angiotensin-I converting enzyme inhibitor phosphonates produced by *Actinomycetes*. ACS Medicinal Chemistry Letters. 2014;**5**:346-351. DOI: 10.1021/ ml4004588

[101] Masuyer G, Cozier GE, Kramer GJ, Bachmann BO, Acharya KR. Crystal structure of a peptidyl-dipeptidase K-26-DCP from *Actinomycetein* complex with its natural inhibitor. The FEBS Journal. 2016;**283**:4357-4369. DOI: 10.1111/febs.13928

[102] Ntai I, Phelan VV, Bachmann BO. Phosphonopeptide K-26 biosynthetic intermediates in *Astrosporangium hypotensionis*. Chemical Communications. 2006:4518-4520. DOI: 10.1039/b611768f

**115**

Section 3

Phosphates in Biomaterials

Section 3


**6. Conclusion**

270

to be 22 mHz. A recent experiment extends this concept to Yb-fiber laser and achieves 1.39-

As reported in Ref. [74], a commercial available pump current supply can provide a minimum resolution of pump power as 1.5 μW and thus achieve a controlling accuracy of 0.05 Hz, which is more than two orders of magnitude than PZT-based method. Therefore, an interesting experiment worth to do is to use RIC-method to achieve high-precision *f*rep stabilization. So far, the RIC method has been fully investigated in NPR mode-locked lasers, which applied non-PM fibers and components [73–75], and the locking accuracy limited to ~mHz. Considering the environmental perturbation on non-PM fiber, a straightforward idea is to implement RIC method in a PM fiber laser. Therefore, the following part will discuss high-precision repetition

The laser setup shown in **Figure 13** is same as **Figure 4(a)**, except the net dispersion of laser cavity. In the current experiment setup for all-optical repetition stabilization, a 56-cm-long Er3+-doped fiber (EDF2) is spliced asymmetrically in the NALM to act as a frequency controller, while the LD3, which is controlled by the error signal from frequency mixer, provides the feedback modulating pump power via WDM3 on EDF2. Besides, a segment of DCF38 is used to compensate the anomalous dispersion of PM1550 fiber. The dispersion of linear loop and

dispersion for the whole cavity. Self-started mode-locking in multiple-pulse regime can be achieved by over-pumping method, and stable single-pulse operation can be obtained by decreasing the pump power of LD1 and LD2. At fundamental repetition rate of 11.9 MHz, the

The repetition rate was detected by PD3 and successively compared with standard reference (Rb clock) in a frequency mixer to produce the error signal. Subsequently, the error signal was filtered and amplified by low-noise voltage preamplifier with frequency cutoff at 1 MHz and

The long-term stabilization was depicted in **Figure 14**. As low as 27-μHz accuracy is achieved within 16-h measurement. The inset of **Figure 14** magnifies the measured dates from 30,000 to 31,000 s and shows fluctuation range within ±0.1 mHz. Typically, thermal effect, Kerr nonlinear effect, pump-induced nonlinear effect, and random acoustic perturbations contribute to the precision of *f*rep stabilization. For our experiment, a temperature-controlled incubator with a ripple of 0.2°C was used to take the laser cavity to isolate environmental perturbation. As for Kerr-nonlinearity, the RIC is proportional to the traveling power of resonant laser. Assuming 5-mW traveling power in NALM, the Kerr-induced RIC is estimated as 1.2 × 10–7/mW, having the same order of magnitude of the pump-induced RIC (2.1 × 10−7/mW). However, when the pump power of LD3 increased from 30 to 205 mW, only 1.6% of output power change was observed, which means little change on the dynamic process of pulse evolution in NALM. Thus, the Kerr-induced RIC is near ~1% of the RIC by pump-induced nonlinearity. Therefore, we postulate that the nonlinearity on the RIC of fibers owes to pump-induced nonlinear effect

and further processed by a proportional-integral-derivative

, producing −0.183 ps2 net

mHz SD of residual fluctuation in an hour measurement [73].

rate stabilization by using RIC method in a PM figure-eight laser cavity.

the NALM was estimated numerically to be −0.208 and 0.025 ps2

figure-eight laser cavity delivers 1.5-mW average power via CP2.

a maximum voltage gain of 5 × 104

and thermal effect rather than Kerr effect.

(PID) controller.

In this chapter, we first present several types of mode-locked fiber lasers, as well as their derivatives for SC generation. Second, an effective method named DPA was applied in Erdoped fiber laser system allowing simultaneous pulse amplification and compression so that additional pulse compressor is no longer needed. With ×8 replicas in DPA, as high as 500-mW average power was achieved and the highest SHG conversion efficiency was measured to be 56.3%. Third, an all-optical method, named as pump-induced nonlinearity, is applied to stabilize the repetition rate of a figure-eight Er-doped fiber laser, achieving as low as 27-μHz accuracy within 16-h measurement.


**Author details**

Ronaldo Luis Thomasini Faculty of Medicine of Diamantina, Federal University of Jequitinhonha and Mucuri Valleys, Diamantina, Brazil

\*Address all correspondence to<EMAIL_ADDRESS>
© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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**2. Method**

The description of the method divided into three sections. The first two sections describe the two theories underpinning the analysis, and the third section outlines the application of the methods in six steps.