**Author details**

Fermented dairy products are vital for bone health because of their unique combination of various nutrients and microorganisms that support and maintain positive bone metabolism [57, 60]. Additionally, dairy matrix and nutrient composition may affect the delivery of

**11. Cardiovascular health, menaquinones, and fermented dairy products**

Coronary artery calcification (CAC) is a predictor of cardiovascular disease (CVD) and mortality. Based on vitamin K's role in activating matrix Gla protein (MGP), a calcification inhibitor, vitamin K is proposed to play a preventive role in CAC and CVD [59, 62]. As recently reviewed, randomized controlled trials that examined the influence of vitamin K on the risk of cardiovascular disease are scarce [63]. The results of observational studies have shown an association between higher dietary menaquinone consumption and less calcification [64], decreased risk of coronary heart disease (CHD), CHD mortality, and all‐cause mortality [65– 67]. The results of a Dutch prospective cohort study suggested that of all MKs the long‐chain menaquinones (MK‐7 to MK‐9) have the most beneficial effects on cardiovascular disease [67]. Although these results are promising, they must be interpreted with caution, because validated

Complex milk fatty acid chemistry and several minerals, such as calcium, magnesium, phosphorus, and potassium provided in relevant concentrations, have been proposed to be involved in the complex mechanism of dairy products and their support to reduce CVD risk [68]. Among the high number of different fatty acids in dairy products, trans‐palmitoleic acid, stearic acid, lauric acid, myristic acid, and oleic acid have been associated with beneficial effects on blood lipids and serum lipoprotein levels [56]. These assumptions are supported by the inverse association observed between CHD risk and the consumption of milk, cheese, and meat

Our knowledge of the consumption of menaquinones should be improved with weighed and extended food records [51] in combination with (multiple) biomarkers in the blood for vitamin K status [52] and the content of the various menaquinones in food items such as cheese, which

As different lactic acid bacteria strains used in cheese production influence the expression of various MKs, analysis of a wide variety of different cheeses may be necessary for a represen‐ tative overview of the vitamin K2 content in this food group. Although results from well‐ designed clinical trials investigating the association between menaquinones and bone health, as well as cardiovascular health, are rare, dairy products seem to be predestined to play a major role in the Western diet because of their nutrient density and matrix properties that improve

menaquinones and improve vitamin K status [61].

76 Vitamin K2 - Vital for Health and Wellbeing

biomarkers for single MK intake are missing [16].

as the richest sources of MKs in the Western diet [6, 67].

contribute most to the supply with this vitamin.

the bioavailability of vitamin K2.

**12. Conclusion**

Barbara Walther\* and Magali Chollet

\*Address all correspondence to<EMAIL_ADDRESS>
Federal Department of Economic Affairs, Education and Research EAER Agroscope, Institute for Food Sciences IFS, Berne, Switzerland

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www.lmreview.com/articles/view/Vitamin-K2-Essential-for-Prevention-of-Age-Associated-Chronic-Disease/[Accessed:2016-05-18]

**Chapter 5**

**Provisional chapter**

**The Impact of Vitamin K2 on Energy Metabolism**

Environmental and behavioral adaptations introduced during the last decades have synergistically enhanced man's lifespan, but also paved the ground for disease states involving impairment of multiple organs, which are both modulating and depending on

Diabetes, obesity, and/or bone brittleness now occur frequently in our society, inducing ailments affecting overall health and well-being. Therefore, an improved comprehension of how organs (e.g. bone and adipose tissue) may provide homeostasis and sound strategies to treat these diseases, thus improving health and life quality of most age categories, should be sought. The steroid and xenobiotic receptor (SXR) (pregnane X receptor = PXR) is a nuclear steroid-like hormone receptor, stimulated by hormones, steroids, drugs, and xenobiotic compounds. SXR exhibits a versatile ligand binding domain, serving as a xenobiotic sensor, regulating xenobiotic clearance from the liver and intestine. However, new and interesting functions of SXR in the regulation of inflammatory processes, cholecalciferol and bone metabolism, lipid and energy homeostasis, and cancer therapy, have emerged. Hence, the discovery and pharmacological development of new PXR modulators, like vitamin K2, represent an interesting and innovative therapeutic approach to combat various diseases, of which glucose and lipid metabolism (i.e., energy metabolism and

**The Impact of Vitamin K2 on Energy Metabolism**

© 2016 The Author(s). Licensee InTech. 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,

© 2017 The Author(s). Licensee InTech. 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.

and reproduction in any medium, provided the original work is properly cited.

In the past years, we have seen a plethora of research reports illuminating the link between bone physiology and energy metabolism, even though the central and sympathetic nervous

Mona Møller, Serena Tonstad, Tone Bathen and

Additional information is available at the end of the chapter

homeostatic calorie "accounting."

adiposity) should be emphasized.

**Keywords:** vitamin K2, energy metabolism, PXR, SXR

Additional information is available at the end of the chapter

Mona Møller, Serena Tonstad, Tone Bathen

Jan Oxholm Gordeladze

and Jan Oxholm Gordeladze

http://dx.doi.org/10.5772/67152

**Abstract**

**1. Introduction**

#### **The Impact of Vitamin K2 on Energy Metabolism The Impact of Vitamin K2 on Energy Metabolism**

Mona Møller, Serena Tonstad, Tone Bathen and Jan Oxholm Gordeladze Mona Møller, Serena Tonstad, Tone Bathen and Jan Oxholm Gordeladze

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67152

#### **Abstract**

www.lmreview.com/articles/view/Vitamin-K2-Essential-for-Prevention-of-Age-

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[67] Gast GCM, de Roos NM, Sluijs I, Bots ML, Beulens JWJ, Geleijnse JM, et al. A high menaquinone intake reduces the incidence of coronary heart disease. Nutrition, Metabolism and Cardiovascular Diseases. 2009;19(7):504–510. DOI: 10.1016/j.numecd.

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disease: the rotterdam study. Journal of Nutrition. 2004;134(11):3100–3105.

rosclerosis. 2009;203(2):489–493. DOI: 10.1016/j.atherosclerosis.2008.07.010

Associated-Chronic-Disease/[Accessed:2016-05-18]

26. DOI: 10.1002/14651858.CD011148.pub2

10.3945/an.111.001644

82 Vitamin K2 - Vital for Health and Wellbeing

2008.10.004

Environmental and behavioral adaptations introduced during the last decades have synergistically enhanced man's lifespan, but also paved the ground for disease states involving impairment of multiple organs, which are both modulating and depending on homeostatic calorie "accounting."

Diabetes, obesity, and/or bone brittleness now occur frequently in our society, inducing ailments affecting overall health and well-being. Therefore, an improved comprehension of how organs (e.g. bone and adipose tissue) may provide homeostasis and sound strategies to treat these diseases, thus improving health and life quality of most age categories, should be sought. The steroid and xenobiotic receptor (SXR) (pregnane X receptor = PXR) is a nuclear steroid-like hormone receptor, stimulated by hormones, steroids, drugs, and xenobiotic compounds. SXR exhibits a versatile ligand binding domain, serving as a xenobiotic sensor, regulating xenobiotic clearance from the liver and intestine. However, new and interesting functions of SXR in the regulation of inflammatory processes, cholecalciferol and bone metabolism, lipid and energy homeostasis, and cancer therapy, have emerged.

Hence, the discovery and pharmacological development of new PXR modulators, like vitamin K2, represent an interesting and innovative therapeutic approach to combat various diseases, of which glucose and lipid metabolism (i.e., energy metabolism and adiposity) should be emphasized.

**Keywords:** vitamin K2, energy metabolism, PXR, SXR

#### **1. Introduction**

In the past years, we have seen a plethora of research reports illuminating the link between bone physiology and energy metabolism, even though the central and sympathetic nervous

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. 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, © 2017 The Author(s). Licensee InTech. 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.

systems, but also the gastrointestinal and pancreatic axes serve essential functions related to the systemic regulation of energy expenditure. It may be asserted that the fat tissue is more prominent due to its basic implication in storing and dissipating energy. During the last 25 years, a major progress has been communicated within the medical societies, featuring a modern understanding of the origin of fat tissues, their specialized characteristics and functioning, as well as the pathophysiological consequences of their impairment. These advances consequently lead to the discovery that adipose tissue metabolism is heavily coupled to the homeostasis of the skeleton.

has been shown to protect mice from diet-induced obesity and metabolic dysfunction [9]. Furthermore, ablation of beige fat cells by adipocyte-only deletion of the transcriptional modulator PRDM16, yields experimental animals, which become prone to diet-induced

The Impact of Vitamin K2 on Energy Metabolism

http://dx.doi.org/10.5772/67152

85

In line with the cited article published by Lecka-Czernik et al. in Archives of Biochemistry and Biophysics, 2014 [13], we performed the following experiment: Human adipose stem cells and 3T3-L1 preadipocytes, the latter with an activated mutation (ref) in the Gi2α-protein were differentiated into fully mature adipocytes as indicated by coloration with Alizarin Red. Interestingly, both adipocyte species accumulated triglycerides upon differentiation to mature adipocytes; however, exposure to vitamin K2 (MK-7) diminished their ability to turn into fully mature adipocytes. Furthermore, both adipocyte species were tested for expression of various genes differing white adipocytes from beige adipocytes. From the gene expression profile, featuring genes like the beta-adrenergic receptor β3-AR, Foxc2, PGC1α, PPARα, Dio2, UCP1, Adipoq, and Leptin, it was quite obvious that the preadipocytes in question differentiated more in the "direction" of beige, lipid (or fatty acid) metabolizing adipocytes than into white, triglyceride-storing adipocytes. Hence, it could be concluded or hypothesized that vitamin K2 (MK-7), in fact, was able to direct preadipocytes into becoming an energy-dissipating, rather than energy-storing adipocyte phenotype (see **Figure 1**, left and right panels). A putative model featuring this development, referring to a suggested mecha-

Here, we have demonstrated that vitamin K2 affects the differentiation of preadipocytes to mature adipocytes, ensuring that the "end-point" phenotype may be tilted in the direction of the beige, energy "dissipating" phenotype. In their paper from 2014, Lecka-Czernik and coworkers [13] assert that an impairment in fat function correlates with a reduced bone mass and an increased incidence of fractures. But, the question posed by the authors is: does accumulation of bone marrow adipose tissue (BMAT) exert a detrimental effect on bone structure and or mineralization, and will a diminished bone mass stimulate the accumulation of BMAT? Historically, BMAT was construed as a dormant or inert type of fat that accumulated within the bone marrow in order to pose as empty space filling subsequent to involution of hematopoietic tissue. Hence, its relationship with a lower bone mass was construed as circumstantial. However, novel evidence asserted that marrow adipogenesis should be construed as a process, which is tightly bound to the differentiation of osteoblasts, since they "share" common precursor cells, and they are subjected to same modulatory signaling patterns. This yields, however, opposite end results where a positive correlation with an overall fat metabolism

indicates that BMAT, in fact, actively induces bone mass loss and inferior quality.

Both, over and malnutrition represent systemic changes in energy metabolism, affecting bone fat volume and bone mass. Despite the fact that adipose (overweight) individuals present with a higher body weight, the more often than not demonstrate a lower BMD. In obese older men and women, an enhanced percentage of body fat and low amount of lean (muscle) mass predicts a lower BMD and thus an enhanced frailty risk [14]. The enhanced fracture risk incurred by postmenopausal women and older men [15] is mainly due to enhanced circulatory levels of adipose tissue derived in adiponectin and proinflammatory cytokines, with a concomitant

obesity and ensuing insulin resistance [12].

nism, is given in **Figure 2**.

Fat tissue is able to deposit and releases energy rich compounds during feeding and fasting, respectively, and it modulates the energy homeostasis in a versatility of organs via its endocrine potential. Fat cells (adipocytes) may accumulate energy in the form of triglycerides, as well as burning it by degrading fatty acids via so-called β-oxidation. Additionally, adipocytes produce and release so-called adipokines, among which leptin and adiponectin are the most important ones. These hormones regulate both the ingestion of calories, as well as the body's sensitivity to insulin. The functional multiplicity of adipose tissue is sustained by various subtypes of adipocytes in depot storing fat. Mitochondria-sparse white adipose tissue (WAT), characterized as visceral and subcutaneous fat, stores energy as triglycerides for which the level is regulated by the body's sensitivity to insulin and the overall metabolism of glucose in the liver and skeletal muscle cells, respectively. Contrastingly, mitochondria-enriched brown adipose tissue (BAT), which in adults is positioned as discrete entities localized in the neck and other regions of the trunk of the body [1], serves to dissipate energy to sustain adaptive thermogenesis [2]. This process is facilitated by the action of uncoupling protein 1 (UCP1), stimulating a leakage of protons, in order to uncouple respiration from ATP synthesis, thus favoring heat production. The thermogenesis sustained by BAT is mastered via the central nervous system, the impact of both the signaling systems comprised by catecholamines (i.e., β-adrenergic signaling), as well as deiodinase 2 (Dio2)-facilitated thyroid hormone conversion from T4 (thyroxine) to T3 (triiodothyronine). As an extra feature, compared to its role in adaptive thermogenesis, BAT is also protecting against obesity, as well as against insulin resistance and development of diabetes [5–8]. Finally, it is worth noticing that genetic ablation of BAT in small experimental animals is resulting in diet-induced obesity, diabetes mellitus with insulin resistance, as well as enhanced blood lipids [3].

It has been asserted that BAT may originate from two sources. The classical or ordinary, preformed BAT stems from Myf5-positive dermomyotomal progenitor cells which may also yield skin and muscle, as well as functioning in so-called nonshivering thermogenesis [4]. On the other hand, Myf5-negative progenitor cells may differentiate into white adipocytes which play a role in energy storage, or to BAT-like or "beige" fat cells. The latter adipocytes demonstrate both brown and white fat cell characteristics [5]. Of major importance is the fact that BAT-like adipocytes can be transformed into WAT-like adipocytes through a plethora of mechanisms, of which a few deserves mentioning; cold exposure, endocrine action of FGF21 [6] or irisin [7], and via transcriptional regulators including FoxC2 [8], PRDM16 [9], and PPARc [10], which lead to SirT1-mediated deacetylation of the PPARc protein [11].  An overexpression of BAT-specific transcription factors, i.e., either FoxC2 or PRDM16 in WAT adipocytes, has been shown to protect mice from diet-induced obesity and metabolic dysfunction [9]. Furthermore, ablation of beige fat cells by adipocyte-only deletion of the transcriptional modulator PRDM16, yields experimental animals, which become prone to diet-induced obesity and ensuing insulin resistance [12].

systems, but also the gastrointestinal and pancreatic axes serve essential functions related to the systemic regulation of energy expenditure. It may be asserted that the fat tissue is more prominent due to its basic implication in storing and dissipating energy. During the last 25 years, a major progress has been communicated within the medical societies, featuring a modern understanding of the origin of fat tissues, their specialized characteristics and functioning, as well as the pathophysiological consequences of their impairment. These advances consequently lead to the discovery that adipose tissue metabolism is heavily coupled to the

Fat tissue is able to deposit and releases energy rich compounds during feeding and fasting, respectively, and it modulates the energy homeostasis in a versatility of organs via its endocrine potential. Fat cells (adipocytes) may accumulate energy in the form of triglycerides, as well as burning it by degrading fatty acids via so-called β-oxidation. Additionally, adipocytes produce and release so-called adipokines, among which leptin and adiponectin are the most important ones. These hormones regulate both the ingestion of calories, as well as the body's sensitivity to insulin. The functional multiplicity of adipose tissue is sustained by various subtypes of adipocytes in depot storing fat. Mitochondria-sparse white adipose tissue (WAT), characterized as visceral and subcutaneous fat, stores energy as triglycerides for which the level is regulated by the body's sensitivity to insulin and the overall metabolism of glucose in the liver and skeletal muscle cells, respectively. Contrastingly, mitochondria-enriched brown adipose tissue (BAT), which in adults is positioned as discrete entities localized in the neck and other regions of the trunk of the body [1], serves to dissipate energy to sustain adaptive thermogenesis [2]. This process is facilitated by the action of uncoupling protein 1 (UCP1), stimulating a leakage of protons, in order to uncouple respiration from ATP synthesis, thus favoring heat production. The thermogenesis sustained by BAT is mastered via the central nervous system, the impact of both the signaling systems comprised by catecholamines (i.e., β-adrenergic signaling), as well as deiodinase 2 (Dio2)-facilitated thyroid hormone conversion from T4 (thyroxine) to T3 (triiodothyronine). As an extra feature, compared to its role in adaptive thermogenesis, BAT is also protecting against obesity, as well as against insulin resistance and development of diabetes [5–8]. Finally, it is worth noticing that genetic ablation of BAT in small experimental animals is resulting in diet-induced obesity, diabetes mellitus

It has been asserted that BAT may originate from two sources. The classical or ordinary, preformed BAT stems from Myf5-positive dermomyotomal progenitor cells which may also yield skin and muscle, as well as functioning in so-called nonshivering thermogenesis [4]. On the other hand, Myf5-negative progenitor cells may differentiate into white adipocytes which play a role in energy storage, or to BAT-like or "beige" fat cells. The latter adipocytes demonstrate both brown and white fat cell characteristics [5]. Of major importance is the fact that BAT-like adipocytes can be transformed into WAT-like adipocytes through a plethora of mechanisms, of which a few deserves mentioning; cold exposure, endocrine action of FGF21 [6] or irisin [7], and via transcriptional regulators including FoxC2 [8], PRDM16 [9], and PPARc [10], which lead to SirT1-mediated deacetylation of the PPARc protein [11].  An overexpression of BAT-specific transcription factors, i.e., either FoxC2 or PRDM16 in WAT adipocytes,

with insulin resistance, as well as enhanced blood lipids [3].

homeostasis of the skeleton.

84 Vitamin K2 - Vital for Health and Wellbeing

In line with the cited article published by Lecka-Czernik et al. in Archives of Biochemistry and Biophysics, 2014 [13], we performed the following experiment: Human adipose stem cells and 3T3-L1 preadipocytes, the latter with an activated mutation (ref) in the Gi2α-protein were differentiated into fully mature adipocytes as indicated by coloration with Alizarin Red. Interestingly, both adipocyte species accumulated triglycerides upon differentiation to mature adipocytes; however, exposure to vitamin K2 (MK-7) diminished their ability to turn into fully mature adipocytes. Furthermore, both adipocyte species were tested for expression of various genes differing white adipocytes from beige adipocytes. From the gene expression profile, featuring genes like the beta-adrenergic receptor β3-AR, Foxc2, PGC1α, PPARα, Dio2, UCP1, Adipoq, and Leptin, it was quite obvious that the preadipocytes in question differentiated more in the "direction" of beige, lipid (or fatty acid) metabolizing adipocytes than into white, triglyceride-storing adipocytes. Hence, it could be concluded or hypothesized that vitamin K2 (MK-7), in fact, was able to direct preadipocytes into becoming an energy-dissipating, rather than energy-storing adipocyte phenotype (see **Figure 1**, left and right panels). A putative model featuring this development, referring to a suggested mechanism, is given in **Figure 2**.

Here, we have demonstrated that vitamin K2 affects the differentiation of preadipocytes to mature adipocytes, ensuring that the "end-point" phenotype may be tilted in the direction of the beige, energy "dissipating" phenotype. In their paper from 2014, Lecka-Czernik and coworkers [13] assert that an impairment in fat function correlates with a reduced bone mass and an increased incidence of fractures. But, the question posed by the authors is: does accumulation of bone marrow adipose tissue (BMAT) exert a detrimental effect on bone structure and or mineralization, and will a diminished bone mass stimulate the accumulation of BMAT? Historically, BMAT was construed as a dormant or inert type of fat that accumulated within the bone marrow in order to pose as empty space filling subsequent to involution of hematopoietic tissue. Hence, its relationship with a lower bone mass was construed as circumstantial. However, novel evidence asserted that marrow adipogenesis should be construed as a process, which is tightly bound to the differentiation of osteoblasts, since they "share" common precursor cells, and they are subjected to same modulatory signaling patterns. This yields, however, opposite end results where a positive correlation with an overall fat metabolism indicates that BMAT, in fact, actively induces bone mass loss and inferior quality.

Both, over and malnutrition represent systemic changes in energy metabolism, affecting bone fat volume and bone mass. Despite the fact that adipose (overweight) individuals present with a higher body weight, the more often than not demonstrate a lower BMD. In obese older men and women, an enhanced percentage of body fat and low amount of lean (muscle) mass predicts a lower BMD and thus an enhanced frailty risk [14]. The enhanced fracture risk incurred by postmenopausal women and older men [15] is mainly due to enhanced circulatory levels of adipose tissue derived in adiponectin and proinflammatory cytokines, with a concomitant lower secretion of leptin and IGF-1. Furthermore, one will often detect lowered blood levels of 25-hydroxyvitamin D and higher serum parathyroid hormone levels in these "patients" [15].

**2. Vitamin K2 and its mechanism of action – beyond xenobiotic**

The steroid and xenobiotic receptor (SXR), which is synonymous with the pregnane X receptor = PXR, is characterized as a nuclear hormone receptor that is stimulated by a plethora of hormones, dietary steroids, pharmaceutically active agents, as well as xenobiotic compounds. SXR exhibits a binding domain, which diverges across mammalian species. SXR is construed as a xenobiotic sensor, facilitating xenobiotic clearance in the liver and intestine. However, newly published experiments unravel novel roles for SXR in modulating phenomena like inflammation, bone turnover, metabolism of vitamin D, lipid, and energy homeostasis, as well as cancer. The characterization of SXR as conveyer of hormone-like signals has now been recognized as a key instrument for the study of novel mechanisms, through which diet may ultimately affect health and disease. The discovery and pharmacological development of new PXR modulators, like vitamin K2, might represent an interest-

**Figure 2.** Putative model system featuring the mechanism behind the impact of vitamin K2 (MK-7). Based on previously published consensus material, our new findings of the effect of vitamin K2 (MK-7) shows that K2, via binding to the transcription factor PXR/SXR affects several signaling molecules and/or pathways, via the β3-adrenergic system, impinging on signaling molecules like PKA, PGC1α, C/EBPβ, and Dio2, which eventually affects the activity of the uncoupling protein UCP1, which produces heat (and not ATP) from fatty acids. Not mentioned in the text is the reciprocal regulatory loop consisting of the microRNA species hsa-mir-155 and the transcription factor C/EBPβ, which can be manipulated to reinforce the "beige" phenotype of the adipocyte after differentiation from the stem cells or preadipocytes.

The Impact of Vitamin K2 on Energy Metabolism

http://dx.doi.org/10.5772/67152

87

ing and innovative therapeutic approach to combat various ailments and diseases.

**metabolism**

**Figure 1.** Human adipose stem cells and mutated 3T3-L1 preadipocytes were differentiated towards mature adipocytes. *Top panel*: Control cells are not manipulated with differentiating medium show lack of ability to store lipids (no Alizarin Red accumulation). Cells are differentiated in the presence of insulin, as well as IBMX (an inhibitor of phosphodiesterase) accumulate lipids, while cells are also exposed to vitamin K2 (MK-7) and lose their ability to produce and store triglycerides. *Bottom panel*: Percentage modulation relative to the "control" stage seen with exposure of human adipocytes and mutated 3T3-L1 cells to differentiating medium versus exposure to vitamin K2 (MK-7). Cells treated with vitamin K2 exhibit significant increments in genes characterizing beige adipocytes, where the alterations in PGC1α, PPARα, PPARγ, Dio2, and UCP1 expression were more prominent.

lower secretion of leptin and IGF-1. Furthermore, one will often detect lowered blood levels of 25-hydroxyvitamin D and higher serum parathyroid hormone levels in these "patients" [15].

86 Vitamin K2 - Vital for Health and Wellbeing

**Figure 1.** Human adipose stem cells and mutated 3T3-L1 preadipocytes were differentiated towards mature adipocytes. *Top panel*: Control cells are not manipulated with differentiating medium show lack of ability to store lipids (no Alizarin Red accumulation). Cells are differentiated in the presence of insulin, as well as IBMX (an inhibitor of phosphodiesterase) accumulate lipids, while cells are also exposed to vitamin K2 (MK-7) and lose their ability to produce and store triglycerides. *Bottom panel*: Percentage modulation relative to the "control" stage seen with exposure of human adipocytes and mutated 3T3-L1 cells to differentiating medium versus exposure to vitamin K2 (MK-7). Cells treated with vitamin K2 exhibit significant increments in genes characterizing beige adipocytes, where the alterations in PGC1α,

PPARα, PPARγ, Dio2, and UCP1 expression were more prominent.

**Figure 2.** Putative model system featuring the mechanism behind the impact of vitamin K2 (MK-7). Based on previously published consensus material, our new findings of the effect of vitamin K2 (MK-7) shows that K2, via binding to the transcription factor PXR/SXR affects several signaling molecules and/or pathways, via the β3-adrenergic system, impinging on signaling molecules like PKA, PGC1α, C/EBPβ, and Dio2, which eventually affects the activity of the uncoupling protein UCP1, which produces heat (and not ATP) from fatty acids. Not mentioned in the text is the reciprocal regulatory loop consisting of the microRNA species hsa-mir-155 and the transcription factor C/EBPβ, which can be manipulated to reinforce the "beige" phenotype of the adipocyte after differentiation from the stem cells or preadipocytes.


*In Vitro* Assessment of Antioxidant Capacity, Potential to Induce Autophagy/Mitophagy, and Mitochondrial Turnover of Natural Compounds for Cytotoxicity Studies

*Madhulika Tripathi, Manish Kumar Verma and Brijesh Kumar Singh*

## **Abstract**

The dynamic process of intracellular reduction-oxidation status (redox homeostasis) is influenced by various factors, with mitochondria being one of the most significant contributors. Mitochondria play a crucial role in the bioenergetic pathway, fulfilling the metabolic energy demands of cells. To maintain increased energy requirements, mitochondrial biogenesis and fusion are employed, while decreased energy demands or damaged mitochondria are addressed through fission and autophagic removal, known as mitophagy. Any disruption in these adaptive responses can compromise redox homeostasis and cellular function, and make cells more vulnerable to oxidative stress, resulting in oxidative DNA damage, inflammatory responses, and apoptotic/anti-apoptotic reactions. Such dysregulation contributes to the development of "free radical diseases" like metabolic disorders and cancer. Traditional medicines and herbs (possessing antioxidant and autophagic properties) have been utilized for centuries in the treatment of various diseases; however, it is only recently that researchers have begun to investigate their molecular, cellular, and tissue-level modes of action. Nevertheless, concerns about their cytotoxicity have also arisen. This manuscript focuses on the current technological advancements in assessing the properties of plant-based natural compounds. Both cell-free and cell-based methods are employed to evaluate the therapeutic potential of these compounds, allowing for their scientific evaluation and validation.

**Keywords:** antioxidant potential, autophagy, mitophagy, cytotoxicity, natural compound

## **1. Introduction**

Past decades have perceived growing interest of studies investigating the role of Reactive Oxygen Species (ROS) and its amelioration with natural products [1–4]. There are increasing evidences that suggest increased oxidative stress from ROS plays critical role in several pathophysiologies and causes Endoplasmic Reticulum stress and mitochondrial dysfunction. The intracellular redox balance (reduction–oxidation status) is a dynamic system which maintains the cellular homeostasis and may change via many factors [5–7]. Mitochondria, being one of the most important organelles, play a crucial role in maintaining intracellular source of energy. Any alteration in mitochondrial dynamics can cause an overproduction of ROS, which urges the induction of oxidative DNA damage and up- or down-regulation of phosphatases, proliferative/anti-proliferative factors, apoptotic/anti-apoptotic factors, etc. [5]. However, physiological levels of ROS are crucial for a proper cell function (i.e. intracellular signaling, inflammation, and immune function) [8]. Moreover, mitochondrial dysfunction and redox imbalance can contribute to a wide range of pathologies under a wide group termed as "free radical diseases" (e.g. cancer, neurodegeneration, atherosclerosis, inflammation, etc.) [5]. Altered cellular homeostasis by oncogenes, mitochondrial dysfunction, and/or oxidative stress leads to an increased genomic instability and the target cells can adapt to the changing environment [9].

Oxygen is vital for most unicellular and multicellular organisms, paradoxically it is also a key in generating Reactive Oxygen and Nitrogen Species (ROS/RNS). Endogenously, ROS are normal by-products of mitochondrial metabolism and energy production [5]. Exogenous sources include smoking, pollution, physical exercise etc. In metabolic disorders or cancer, ROS/RNS homeostasis compromises leading to oxidative stress [10, 11]. To maintain the fine balance and to counteract the harmful effects taking place in the cell, biological system has evolved itself with some strategies like prevention of oxidative damage, repair mechanism, finally the most important is the antioxidant defense mechanisms. Cells are protected from oxidative stress by several antioxidant mechanisms both inside and outside the mitochondria. These include manganese superoxide dismutase (Mn-SOD) in the mitochondria, copper/zinc-SOD in the cytosol, and extracellular-SOD in the interstitium [12]. Classically antioxidants were defined as molecules that inhibit or prevent oxidation of a substrate and are required to control any excess in these ROS/RNS and to maintain Redox homeostasis.

Plant-based natural products have been basis of treatment of many human diseases. The extracts of several plants have been used as therapeutic agents. Spices and herbs that are sources of natural antioxidants, can protect against oxidative stress and thus play preventive role in many diseases [13]. The medicinal properties of folk plants are mainly attributed to the presence of flavonoids, other organic, and inorganic compounds such as coumarins, phenolic acids, and antioxidant micronutrients and are able to counter various metabolic disorders, cancers, and COVID-19 [14–17].

#### **1.1 Role of mitochondrial oxidative stress in metabolic disorders and cancer**

Metabolism refers to breaking down of food, in a highly-regulated manner, into simpler units of proteins, carbohydrates, and fats in order to generate energy for the organism. Metabolic syndrome is considered to be a progressive pathophysiological state which is clinically manifested by a cluster of interrelated risk factors such as abdominal obesity, atherogenic dyslipidemia, increased blood pressure, insulin resistance, and pro-inflammatory response [18, 19]. "Cancer metabolism" refers to the alterations in

#### In Vitro *Assessment of Antioxidant Capacity, Potential to Induce Autophagy/Mitophagy… DOI: http://dx.doi.org/10.5772/intechopen.112278*

cellular metabolic pathways that are evident in cancer cells compared with most normal tissue cells. Major metabolic changes in cancer cells are increased glycolysis and reduced oxidative phosphorylation coupled with increased biosynthetic intermediates needed for cell growth and proliferation [20]. Likewise, the most common cellular patho-physiologies of metabolic disorders are increased oxidative stress, impaired mitochondrial functions, and cellular repair mechanism (autophagy and apoptosis), etc. [21–23].

Mitochondria are importantly placed in the history of cancer and have been attributed to play a crucial role in metabolic disorders [24, 25]. Although, mitochondria are the center for bioenergetics and biosynthesis pathways, they have also become a critical source of oxidative stress by generating ampule amount of superoxide in ETC [5]. Hence, mitochondrial redox control is utmost important for oxidative phosphorylation, ATP synthesis, and ROS production. The production of ROS in mitochondria is tightly regulated by the mitochondrial superoxide dismutase (SOD2) and glutathione-peroxidase (GPx), as well as by catalase and non-enzymatic antioxidants [5]. Excessive mitochondrial ROS and mitochondrial dysfunctions can promote metabolic−/age-related disorders (NAFLD, diabetes, atherosclerosis, neurodegeneration, etc.) and cancer through suppression of complex I, induction of oxidative mitochondrial DNA damage, increased excessive calcium ion influx, alterations in autophagy [5, 26–28]. Therefore, Phyto molecules/plant-based compounds having antioxidant potential and/or autophagic induction may provide beneficial effects in above-mentioned diseases that are associated with ROS and oxidative stress.

#### **1.2 Therapeutic potential of natural compounds in metabolic disorders and cancer**

Natural products have always been of immense importance in health and disease treatment throughout the human evolution [1–4]. Traditionally several natural products have been used to treat different types of human illnesses including general injuries, wound healing, and pain [29, 30]. Natural products have been shown to have pharmacological properties to regulate cell signaling pathways that cause mitogenic and cytotoxic reactions leading to disease pathologies. Usage of modern technological tools such as genomics, proteomics, and metabolomics paved wider use of these natural products. Nowadays, mostly natural products are processed and have been developed as potential pharmacological agents with effective anti-oxidative, antimitotic, anti-microbial, anti-inflammatory, anti-angiogenic, and anti-carcinogenic properties [13, 31]. Different civilizations have been relying on plants or plant-based products to improve health. This also holds remarkable therapeutic alternatives to synthetic drugs. Millions of Europeans, Africans, Asians, Australians, and North Americans use plant-based medicine system as health supplements for their general health wellbeing. However, in developing countries, ayurveda and TCM, the traditional Indian and Chinese medicine is often the primary source of health care [32].

Many medicinal plants and natural products are considered by the public as a safe, natural, and cost-effective alternative to synthetic drugs without explicit proof by randomized controlled clinical trials [32]. Recently, there is an added attention in the validation of the efficacy and safety of natural products [32].

Several preclinical studies and non-scientific data accumulated over decades of research confirm the use of medicinal plants and plant-based products in the treatment or prevention of metabolic disorders. These products have gained popularity in western societies; however, their expected health benefits are often not backed by rigorous clinical trials. Therefore, it is essential to provide robust scientific evidence on their clinical efficacy and safety.

## **2.** *In vitro* **tools and techniques for the assessments of natural compounds**

## **2.1 Assessment of antioxidant potential of plant extract/natural compound in a cell free system**

## *2.1.1 Total antioxidant capacity by ABTS decolorization assay*

Principle: The assay is based on the ability of antioxidant molecules to quench the stable ABTS (2,2´-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)) radical [33]. A blue-green solution of ABTS cation radical (ABTS•+) is formed by the loss of an electron from stable ABTS radical and absorbs at 743 nm. In the assay, addition of Trolox decolorizes the solution resulted by the quenching the hydrogen atom by nitrogen atom.

Methodology (for 96-well plate format):

## *2.1.2 Nitric oxide (NO) quenching capacity*

Principle: Nitric oxide (NO) interacted with oxygen to produce stable products, nitrite and nitrate that are key to generate RNS. Scavenger of NO competed with oxygen, leading to a reduced production of nitrite. The concentration of nitrite assays by the method of Feelisch and Noack [34] by reacting nitrite with griess reagent with which it forms a stable product which is measured at 542 nm on a spectrophotometer.

Methodology (for 96-well plate format):

In Vitro *Assessment of Antioxidant Capacity, Potential to Induce Autophagy/Mitophagy… DOI: http://dx.doi.org/10.5772/intechopen.112278*

vi.Capacity of extract to inhibit this stable product is taken as NO quenching capacity/mg extract.

Application: Oxidative stress plays a key role on pathophysiology and aging [35–37]. Many phytochemicals/active compounds (polyphenols, flavonoids, terpenes, etc.) have antioxidant properties that are responsible for therapeutic effects of plants/herbs [14, 32]. Evaluating antioxidant potential of herbal/plant extract is the easy and very useful way to validate extract's therapeutic efficacy toward oxidative stress.

#### **2.2 Assessment of autophagy, lipophagy, and mitophagy using** *in vitro* **cell system**

#### *2.2.1 Analysis of autophagy by Western blotting*

Introduction and principle: Autophagy is a catabolic cellular multistep process [38]. In autophagy, most commonly known as macroautophagy, targeted cytoplasmic constituents (such as damaged macromolecules and organelles) are isolated from the rest of the cell within a double-membraned vesicle called autophagosome. These autophagosome eventually fuses with lysosomes forming autolysosome and the contents inside are degraded under acidic environment and get recycled. Non-selective autophagy or macroautophagy is dissociated with other forms of specific autophagy processes such mitophagy (autophagic degradation of mitochondria), lipophagy (autophagic degradation of lipids), ERphagy (autophagic degradation of ER), etc.

Autophagy is executed by several autophagy-related (Atg) genes initially identified in Saccharomyces cerevisiae and later validated in mammalian systems [39]. During initiation of autophagy, two ubiquitin-like proteins Atg12 to Atg5 covalently conjugate and then bind to Atg16L1 to form an E3-like complex which functions as part of the second ubiquitin-like conjugation system. This complex binds and activates Atg3, which covalently attaches mammalian homologs of the ubiquitin-like protein Atg8/LC3B to the lipid phosphatidylethanolamine (PE) on the surface of autophagosomes after conversion from LC3B-I to LC3B-II. This lipidated LC3B (LC3B-II) closes the autophagosomes and enables docking of cargospecific adaptor proteins such as Sequestosome-1/p62. The completed autophagosome then fuses with a lysosome through the actions of multiple proteins. After autophagosome-lysosome fusion, LC3B that is retained on the outer side is cleaved off and recycled whereas inside the vesicle along with Sequestosome-1/p62 gets degraded. Hence, Western blot analysis of the rate of accumulation of LC3B-II (autophagic flux) under lysosomal inhibition (using inhibitors such as chloroquine, Bafilomycin A1 etc.) is the most appropriate way to study autophagy and take as autophagic marker [40].

Methodology:

iii.Treat groups iii and iv with extract or natural compound for 24–72 h.

Analysis of LC3B-II western blot: As shown in the (**Figure 1A**), there will be 3 cases of LC3B-II western blot image.

Step-I: Take normalized densitometric ratios for basal autophagy (yellow box): i.e. lanes 2:1.

Step-II: Take normalized densitometric ratios for autophagy under treatment (green box): i.e. lanes 4:3.

Step-III: Compare the ratios from basal autophagy (step-I) vs. autophagy under treatment (step-II) for significance.

Interpretation of LC3B-II western blot:

As mentioned earlier that autophagy is a kinetic process. However, during the analysis, the expression of cellular LC3B-II at a given time point is a static and unable to represent the rate of autophagosome synthesis and degradation after lysosomal fusion. i.e. autophagy flux. Thus, to better analyze this, use of lysosomal inhibitors is important. This enables us to analyze any increase in the LC3B-II in the presence of a lysosomal inhibitor representing autophagy induction or autophagosome biogenesis before lysosomal activity was blocked. Hence, as shown in the **Figure 1A**, there will be 3 cases (**Figure 1B-D**) of autophagy flux according to LC3B-II expression before and after the treatment of lysosomal inhibitors.

• Case-1: Activation of autophagic flux by treatment X.

If relative LC3B-II protein expression is higher under treatment when compared to untreated group (as shown in **Figure 1B** by red dotted line), and it is further increased after lysosomal inhibition (as shown in Figure B by double-head arrows) in treated group compared to untreated group.

• Case-2: Inhibition of autophagic flux by treatment X at autophagosome formation step (i.e. early block).

If relative LC3B-II protein expression is lower under treatment when compared to untreated group (as shown in **Figure 1B** by red dotted line), and it is further decreased or unchanged after lysosomal inhibition (as shown in **Figure 2B** by doublehead arrows) in treated group compared to untreated group.

In Vitro *Assessment of Antioxidant Capacity, Potential to Induce Autophagy/Mitophagy… DOI: http://dx.doi.org/10.5772/intechopen.112278*

### *2.2.2 Analysis of autophagy and mitophagy by fluorescence microscopy*

Introduction and principle: To analyze the progression of autophagy/mitophagy from autophagome/mitophagosomes to lysosomes, recently developed reporter

**Figure 2.**

*RFP-GFP fusion plasmids to study autophagic/mitophagic flux under fluorescent microscope.*

plasmids (**Figure 2**), that are tandem-tagged RFP-EGFP protein fused and cloned with LC3B (to analyze autophagy) or mitochondrial localization signal (MLS) typically COX-IV (also called mitoRFP-GFP plasmid; to analyze mitophagy), have been used, which exploit the differential stabilities of RFP and GFP in an acidic environment [40]. Green GFP signals are quenched in acidic pH, whereas RFP signals can be visualized in both autophagosome and acidic autophagolysosome, thus an increase in RFP fluorescence in the lysosomes indicates completion of the mitophagy process. However, at basal, yellow signals indicate merged images of GFP and RFP signals for only autophagosome containing mitochondria. Therefore, the formation of red puncta in treatment groups will indicate increased autophagic/mitophagic flux over untreated cells which will show yellow puncta.

Methodology:

i.Seed the cells in appropriate density in chambered slides and incubate at 370C and 5% CO2: 95% Air environment inside an incubator.

In Vitro *Assessment of Antioxidant Capacity, Potential to Induce Autophagy/Mitophagy… DOI: http://dx.doi.org/10.5772/intechopen.112278*

ii.Plasmid transfection: At 70–80% confluency, make two groups as i. untreated and ii. Treatment X (extract or natural compound), then use 0.5 μg plasmid and transfection reagent (usually Lipofectamin P3000 from Invitrogen, USA) to transfect DNA into the cells from both the groups, and incubate for 24 h at 370C and 5% CO2: 95% Air environment inside an incubator.

iii.Treat group ii with extract or natural compound for further 24–48 h.

iv.After the treatment is over, fix the cells in 4% paraformaldehyde for 15 min.

v.Stain cells with Hoechst 33342 or DAPI (Sigma) to stain nucleus.

Analysis of fluorescent images: Both RFP and GFP fluoresces on autophagosomes as LC3B and represents as yellow puncta in overlay under basal condition. However, in autolysosome, under acidic/low pH GFP (green) fluorescence get quenched and only RFP fluorescence can be visualized as red puncta (in overlay) of autolysosome suggesting increase in autophagy flux. Similarly, both RFP and GFP fluoresces on mitochondria and whole mitochondrial network can be visualized under basal condition. However, in mitophagolysosome, under acidic/low pH GFP (green) fluorescence get quenched and only RFP fluorescence will be visualized as red puncta (in overlay) of mitophagolysosome suggesting increase in mitophagy flux.

*2.2.3 Analysis of lipophagy by fluorescence microscopy*

Introduction and principle: To analyze lipophagy (autophagic degradation of lipids), cellular lipid droplets/triglycerides stain using BODIPY™ 493/503 dye (Invitrogen) and endogenous LC3B proteins stain using anti-LC3B antibody. Fluorescence microscopy analysis is performed to analyze co-localization of lipids/ BODIPY with LC3B.

Methodology:

iii.After the treatment is over, fix the cells in 4% paraformaldehyde for 15 min.

iv.Wash cells with 1x phosphate buffer saline (PBS) three times 5 min each.

Analysis of fluorescent images: Both BODIPY™ fluorescence is visualized using GFP/Annexin filter to observe green fluorescence of lipid droplets, however, RFP filter can be used to visualize LC3B red fluorescence. Lipophagy will be seen in overlay as yellow puncta formed by BODIPY-LC3B co-localization.

### *2.2.4 Analysis of autophagy, lipophagy, and mitophagy by electron microscopy*

Introduction and principle: Electron microscopy is one of the best methods for the detection of autophagy and quantification of autophagic accumulation compared to fluorescence microscopy that has lower resolution than transmission electron microscopy [41]. During autophagy induction, the number of degradative compartments increases and the quantification of the number of autophagosome, autolysosomes, lysosomes, and amphisomes per cell section provides a simple morphological readout. But, it is very difficult to distinguish between autolysosomes, lysosomes, and amphisomes, hence, it is much simpler to classify all these organelles together, as degradative compartments or autophagic vesicles. However, autophagosome and autolysosome can be distinguished by observing their double membrane vs. single membrane walls respectively.

Methodology:

In Vitro *Assessment of Antioxidant Capacity, Potential to Induce Autophagy/Mitophagy… DOI: http://dx.doi.org/10.5772/intechopen.112278*

Application: Autophagy plays a key role in the cellular maintenance and development of disease [38, 43]. Impairment in autophagic removal of damaged mitochondria (mitophagy) and fatty acids/lipids (lipophagy) has been shown to be important in various pathological states including neurodegenerative diseases and metabolic disorders [38, 43].  Hence, analyzing autophagy flux and their potential to remove damaged mitochondria and lipids can be an easy way to evaluate plant's therapeutic efficacy.

## **2.3 Assessment of mitochondrial turnover (synthesis of new and removal of damaged mitochondria) using MitoTimer**

Introduction and principle: Fluorescent Timer, or DsRed, is a mutant of the red fluorescent protein, dsRed, in which fluorescence shifts over time from green to red as the protein matures or undergoes oxidation during the course of aging.

This molecular clock gives temporal and spatial information on protein turnover [48]. To monitor mitochondrial turnover, this Timer is engineered and tagged with mitochondrial-targeting sequence (called as "MitoTimer") and cloned in a plasmid. When cells get MitoTimer, it localizes in the mitochondria and disappearance of red fluorescence (for removal of damaged mitochondria) and appearance of green mitochondrial (for synthesis of new mitochondria) i.e. turnover, can be visualized. Methodology:

Analysis of fluorescent images: As shown in the [49], both mitoRFP and mitoGFP fluoresces on mitochondria and represented as yellow mitochondrial network in overlay under basal condition, suggesting mitochondrial homeostasis. However, when there is an increase in mitochondrial turnover by mitophagy, mitoGFP (green) fluorescence increases and mitoRFP fluorescence decreases under (A) treatment X suggesting increase in new mitochondria via biogenesis and removal of old/damaged mitochondria by mitophagy. Similarly, when turnover is decreased, both mitoRFP increases and mitoGFP decreases suggesting decreased mitochondrial biogenesis and accumulation of damaged/aged mitochondria under (B) treatment X.

Application: Mitochondrial turnover is utmost important to maintain mitochondrial homeostasis that is required for cellular health [5, 50]. Dysregulated mitochondrial turnover leads to cause oxidative stress and cell death which is a major cause for metabolic disorders and cancer [24, 26, 51]. Study of mitochondrial turnover using MitoTIMER assay is a convenient method to evaluate phytochemicals efficacy.

#### **2.4 Assessment of cell viability/cytotoxicity** *in vitro*

## *2.4.1 MTT tetrazolium assay*

Principle: Tetrazolium salts are widely used for detecting redox potential of cells for viability, cytotoxicity, and proliferation assays. This is a colorimetric assay that

In Vitro *Assessment of Antioxidant Capacity, Potential to Induce Autophagy/Mitophagy… DOI: http://dx.doi.org/10.5772/intechopen.112278*

measures the reduction of yellow 3-(4,5-dimethythiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase, which demonstrates functional mitochondria [52]. The MTT is cell permeable and goes into the mitochondria and gets reduced to purple colored formazan crystals. These purple crystals are then solubilized using organic solvent. Lastly, this purple solution is measured spectrophotometrically. Notably, reduction of MTT only occurs in metabolically active cells, thus, the level of purple color is a measure of the viable cells.

Methodology:

#### *2.4.2 MTS assay*

Principle: Colorimetric method for sensitive quantification of viable cells in proliferation and cytotoxicity assay. This assay is also based on the same principle as MTT assay, where we read colored formazan product by metabolically active cells is soluble in cell culture media. The MTS assay offers a number of advantages over the MTT assay. First, it eliminates the need for washing or solubilization steps, making it a more efficient and streamlined process. This high-throughput assay can be performed directly in the cell culture media, and can be conducted using a 96-well microtiter plate. The MTS assay is versatile and can be used to measure cell proliferation in response to a variety of stimuli, such as growth factors, cytokines, mitogens, and nutrients. Additionally, it is useful for analyzing the cytotoxic effects of compounds such as anticancer drugs, as well as other toxic agents, and pharmaceuticals.

Methodology:

for further 24–48 h, and incubate at 370C and 5% CO2: 95% Air environment inside an incubator.

#### *2.4.3 Luciferase-based assay for real-time cytotoxicity analysis*

Principle: The latest innovation in viable cell number measurement is a real-time approach using a pro-substrate and luciferase. This method involves adding the reagent directly to the culture medium, where viable cells with active metabolism reduce the pro-substrate into a Luc-substrate. The Luc-substrate then diffuses into the culture medium, and luciferase utilizes it to generate a luminescent signal.

The pro-substrate is non-reactive with luciferase and is well-tolerated by cells, remaining stable in complete cell culture medium at 37°C for at least 72 hours. This long-term stability allows for multiple measurements from the same sample over a period of days without the need for pro-substrate replenishment.

The assay can be performed in both continuous-read and endpoint formats. In continuous-read mode, the luminescent signal is recorded repeatedly from the same sample wells over an extended period to measure the number of viable cells in real time. This new approach provides a reliable and efficient way to monitor viable cell numbers and metabolic activity in a variety of research applications [53].

### Methodology:

RealTime-GloTM MT Cell Viability Assay (Promega) for continuous read mode:

In Vitro *Assessment of Antioxidant Capacity, Potential to Induce Autophagy/Mitophagy… DOI: http://dx.doi.org/10.5772/intechopen.112278*

Application: Analysis of cell viability plays a diverse role in establishing the relationship between drug safety and its toxicity. High-throughput methods are very well embraced in the scientific community for drug's safety evaluation tastings. Hence, above-mentioned cytotoxicity/cell viability assays are best fit to titrate the safe dosage of phytochemicals.

## **3. Discussion**

One of the important and attractive uses of the *in vitro* methods is to evaluate therapeutic efficacy and safety of active compounds/phytochemicals and/or crude extracts in a high-throughput manner, and that also limits the use of animals which can be expensive, labor-intensive, and time taking. The most common assays (as mentioned above in Section 2.1) are to evaluate antioxidant potential of herbal extracts or active compounds in a cell-free system or by using animal cell culture system. Prof. Y. Osumi has received Noble prize for re-stating the role of autophagy in cell health and disease [54]. Technological advancements in scientific field further made it possible to evaluate autophagic flux and related processes, such as mitophagy and lipophagy, in a high-throughput way by using fusion proteins (as discussed above in Sections 2.2 and 2.4) in animal cell culture system. Mitochondrial dysfunction and de-regulation of mitochondrial turnover and homeostasis play key roles in may diseases including cancer. Analysis of OCR that is an indicator of mitochondrial respiration and mitochondrial activity is utmost important for safety evaluation plant-based therapeutic compounds. Recently developed engineered mitochondrialocalized fluorescent proteins [48], that aged or oxidized in time-dependent manner and fluoresce differentially (as discussed above in Section 2.3) are boon for new age research, can be used to evaluate mitochondrial turnover in a high-throughput way. Cytotoxicity is desirable in the cancer-targeted drugs; however, it can be detrimental for normal cells, so analysis of cytotoxicity is important for successful use of plant-based chemo-drugs or to target metabolic diseases. Although, there are several high-throughput assays available for assessing the potential cell viability and cytotoxicity of plant extracts and phytochemicals, this chapter (in Section 2.5) provides a summary of both commonly used and recently developed cell-free and cell-based assays. These assays serve as important tools for evaluating the safety and therapeutic effectiveness of plant-based drugs and compounds.

## **Acknowledgements**

The authors are thankful to the Ministry of Health (MOH), and National Medical Research Council (NMRC), Singapore, for financial assistance grant number NMRC/ OFYIRG/0002/2016 and MOH-000319 (MOH-OFIRG19may-0002) to BKS; NMRC/ OFYIRG/077/2018 to MT; and CSAI19may-0002 to PMY.

## **Conflict of interest**

The authors declare no conflict of interest.

## **Notes/thanks/other declarations**

None.

## **Author details**

Madhulika Tripathi1 \*, Manish Kumar Verma2 and Brijesh Kumar Singh1 \*

1 Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School, Singapore

2 Raghuveer Singh Government (P.G.) College, Lalitpur, Uttar Pradesh, India

\*Address all correspondence to<EMAIL_ADDRESS>and<EMAIL_ADDRESS>
© 2023 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.

In Vitro *Assessment of Antioxidant Capacity, Potential to Induce Autophagy/Mitophagy… DOI: http://dx.doi.org/10.5772/intechopen.112278*

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Section 3


*2.1.2 Washing of soil*

Sediment washing is a simpler technique that is performed *ex-situ*. In this technique, a solution is utilized to wash the contaminated sediment for the transfer of pollutants from sediment to an aqueous solution. This is achieved by mixing the soil with an aqueous solution of alkalis, acids, and surfactants [14]. Washing includes (i) excavation of highly contaminated sediment from the bulk soil; (ii) washing of sediment is processed with the help of aqueous mixtures; (iii) the solubilized contaminants are removed from aqueous solution through various chemical processes. For performing this method more efficiently additives are added to the aqueous solution, depending upon the physicochemical nature of contaminated sediment. These additives should have high treatment efficiency and environmental compatibility. Common additives used are inorganic acids (sulfuric acid, nitric acid), organic acid (oxalic acid, ascorbic acids), and surfactants (sophorolipids

#### **Figure 3.**

*Capping technique for isolating contaminated sediment [18].*

and rhamnolipids) [16]. EDTA has been reported as the additive for the removal of heavy metals, due to its versatile chelating nature, however, the toxic effect of EDTA on the environment and its low biodegradability has reduced its application widely [19]. After washing, the sediment is considered contaminant depleted instead of contaminant free. Therefore, to make this technique successful, the number of contaminants treated should be quantified to be equivalent to the site-specific action limit. This technique is suitable for the contaminants which are weakly associated with sediments, and in coarse-grained sediments [14].

## *2.1.3 Excavation of soil*

This technique includes physical removal of majorly contaminated soil from the bulk soil.  It can be divided into three methodologies (i) substitution of polluted sediment by removing the soil and putting it in another soil. This method is more suitable for land contaminated in small areas; (ii) the deep excavation of contaminated sediment for natural degradation of heavy metals; (iii) importing new soil and mixed with contaminated soil for dilution of heavy metals. This technique is expensive and is efficiently applicable only on land with small areas of contamination [20].

#### **2.2 Chemical remediation techniques**

This technique includes the utilization of chemical reagents, reactions, and principles for the removal of contaminants. Major methodologies used under this technique are solidification, immobilization, vitrification, and electro kinetics.

#### *2.2.1 Immobilization*

This methodology is used to stabilize heavy metals, can be applied *ex-situ* and *insitu*. It often uses organic and inorganic reagents for the reduction of heavy metals mobility, toxicity, and bioavailability in the soil. The primary objective of this technique is to alter the bioavailable phases of metals into more geo-chemically stable phases, with the immobilization of chemicals. It is achieved through combined mechanisms of adsorption, complexation, and precipitation. The stabilizing effect of amendments is dependent upon the physical, chemical, and biological characteristics of sediment, heavy metal type, remediation time, remediation method, and evaluation method. The most common inorganic reagents used for immobilization are silico-calcium reagents, phosphates, iron-containing materials, aluminum salts, and mineral-based amendments. Organic reagents for immobilization of heavy metals include manure, biochar, biosolids, bark, wood chips, sawdust, sewage sludge, and turf. A complex formulation of inorganic and organic amendments can also be applied to the contaminated sediments for more efficient stabilization [21].

#### *2.2.2 Solidification*

It is a technique applied by mixing contaminated sediments with materials that impart physical stability to encapsulate contaminants in a solid product. Solidification is the physical encapsulation of contaminants in a solid matrix, which are formed by cement, bitumen, asphalt, fly ash and thermoplastic binders. During *In-Situ* remediation, a binding agent is added to contaminated sediment which is followed by an auger spin mixing to transform the soil into a solid matrix [15]. The stabilization of heavy metals includes chemical reactions which reduce their mobility in the environment. The entrapped toxic metals are not leachable as

#### *Conventional and Contemporary Techniques for Removal of Heavy Metals from Soil DOI: http://dx.doi.org/10.5772/intechopen.98569*

the solid block is impermeable to water. A mixture of various salts can be used for the solidification or stabilization of contaminants in soil *ex-situ* or *in-situ*. Several economically effective and environmentally friendly waste resources have been reported for their application in contaminated sediment. These waste resources can also improve the quality of polluted soil, such as lime-based agents, calcined oyster shells, eggshells, waste mussel shells, and calcined cockle shells [20]. However, the process does not extract the pollutant. So, over the long term, if the integrity of solid matrix is deteriorated due to natural weathering or any uncontrolled physical disaster the contaminants which are trapped can mobilize into the environment. Therefore, this methodology is applied as a last option for remediation of soil. This technology is dependent on the concentration of contaminants present in the sediment, amount of water, and ambient temperature. These factors affect the binding reaction of contaminants to the solid material, it inhibits the binding and decreases the stability of the solid matrix [14].

## *2.2.3 Vitrification*

This methodology of remediation is a type of stabilization/solidification technique. It requires high thermal energy in contaminated soil, at least 1400°C - 2000°C, for the removal of organic or volatile substances. It is achieved by mixing the contaminated sediments with glass-forming precursors, heating the mixture till its liquid solution is formed. The steam produced by introducing high thermal energy and the products of pyrolysis are collected from exhaust gas [21]. On the cooling of this solution, an amorphous homogenous glass is obtained. The contaminants can be stabilized by two ways of interactions with solid glass matrix, that is chemical bonding and encapsulation. For *in-situ* remediation, electrodes can be inserted directly into the contaminated sediments. This technique is efficient but expensive and complex to perform [20].

#### *2.2.4 Electrokinetic remediation*

In this technique, the electric field is applied to the wet contaminated sediments for the movement of ionized metals towards the cathode or anode. The pollutants are migrated towards electrodes through electro-migration (charged chemical movements), electro-osmotic flow (fluid movements), electrophoresis (charged particle movements), and electrolysis (chemical reaction due to electric field) procedures [21]. On the completion of the remediation process, the contaminant concentrated electrodes can be treated through several techniques for treating the heavy metals. This technique performs more efficiently in fine-grained clayey soil, where heavy metals are present as soluble ions, because of high electric conductivity and strong electric field [16]. To enhance the efficiency of this technique application of chelating agents can be performed, such as EDTA, nitrilinoacetic acid, succinic acid, citric acid. A schematic representation of this technique has been represented in (**Figure 4**).

#### **2.3 Biological remediation**

Biological remediation or bioremediation is a technique of transforming the heavy metals present in the contaminated soil, into a less toxic element. This technique uses biological phenomena that are intrinsic to plants and microorganisms, for the destruction, removal, or immobilization of hazardous contaminants from the polluted environment. Bioremediation is an eco-friendly and economically effective technique for heavy metal removal compared with the conventional chemical and physical methods, which are usually expensive and ineffective especially for sediments contaminated

#### **Figure 4.**

*A schematic representation of* in-situ *electrokinetic installation; B schematic representation of detailed electrokinetic remediation technique [22].*

with low metal concentrations, in addition to producing significant amounts of toxic sludge [20]. The main objective of the bioremediation technique is to stimulate a favorable condition for microflora or plants at the contaminated site by providing suitable growth conditions. So, they can grow at their full potential and produce enzymes as secondary metabolites for immobilizing the toxic metals. During the bioremediation process of the contaminant, chemical bonds are broken, and energy is released, which is further utilized by the microorganisms for their growth. Various investigations show that the total transformation percentage of various heavy metals by microbes are Cr (27%), Co (20%), Cd (31%), Pb (22%) [23]. Bioremediation technology is aided with several methodologies, such as bioventing, bioleaching, and land farming, bioreactor, composting, and bioaugmentation, rhizo-filtration, and biostimulation. Therefore diverse metabolic activity inherent to microbes can be exploited for degradation, removal, or transformation of heavy metals in contaminated soil [24]. Mostly bioremediation can be performed by utilizing microorganisms (algae, fungi, and bacteria), and plants (phytoremediation), or with the combinations of both.

#### *2.3.1 Phytoremediation*

This technique involves the use of various native, imported, or genetically modified plant species for the reduction, and removal of contaminants from soil,

#### *Conventional and Contemporary Techniques for Removal of Heavy Metals from Soil DOI: http://dx.doi.org/10.5772/intechopen.98569*

sludge, wastewater, sediments, and groundwater. This technique is best applicable when the contaminants are present around the rhizosphere and in a wide area of land. The basic principle in phytoremediation involves the disintegration through secondary metabolites or absorption by roots, and storing them in leaves of plants, of contaminants present in soil [20]. Hyperaccumulation and hyper tolerance are very important characteristic for a plant for their utilization in phytoremediation. Phytoremediation technique includes phytoextraction, Phytofiltration, Phytostabilization, Phytovolatilization, and Phytodegradation [19].

Phytoextraction/Photoabsorption/Phytosequestration/Phytoaccumulation refers to a biochemical process where the assimilation of heavy metal contaminants from the sediment or water is processed through roots and translocated to any harvestable part of the plant, based on the mechanism of hyperaccumulation (**Figure 5**). Hyperaccumulators can concentrate 100 to 1000 times higher than those found in non-hyperaccumulators without suffering any apparent phytotoxic effect. This method includes three steps (i) cultivation of suitable plant species in the contaminated land; (ii) harvesting of biomass concentrated with metal; (iii) post-harvest treatment for obtaining economic value [25]. The most used hyperaccumulators are from the family *Fabaceae, Brassicaceae, Lamiaceae, Cryophylaceae, Violaceae, Asteraceae, Cyperaceae, and Poaceae* [24].

Phytofiltration is the cleanup method for a contaminated environment with the use of plant roots. It could be performed in three forms of rhizofiltration (plant roots), blastofiltration (seedlings), caulofiltration (excited plant shoots) [19].

Phytostimulation enhances the conditions of the rhizosphere for the efficient growth of microbes. It is performed for the removal of organic pollutants in the sediment.

Phytostabilization aims to the reduction of mobility and bioavailability of heavy metals in the environment by stabilizing the contaminants in the rhizosphere of plant species. It is performed by reducing the accessibility and mobility of heavy metals through precipitation, root sorption, metal valence reduction, and complexation. The efficiency of this technique can be enhanced by changing the pH and organic matter content in the sediment [25].

Phytodegradation is a technique utilized for degrading organic matter into non-hazardous chemicals through secondary metabolites or enzymes secreted by plants. Enzymes like nitroreductase and dehalogenases are used by plants for the degradation of organic matter. These enzymes are used only in optimal conditions (temperature, pH). This process can be performed more efficiently with the introduction of microorganisms in the contaminated soil, this technique is called Rhizodegradation [26].

Rhizofiltration is the process in which plants absorb and precipitate organic and inorganic contaminants through roots from contaminated wastewater, groundwater, and surface water. Major characteristic features of plants are hypoxia tolerant, and large absorption surface area for a suitable application of this technique. Terrestrial plants are more efficient for this purpose than aquatic plants [27].

#### *2.3.2 Microbial remediation*

Microorganisms can absorb or adsorb the heavy metals present in the soil to transform its chemical nature and reduce its mobility, bioavailability, and solubility. This remediation technique by microbes can be carried out in two ways, through mobilization or immobilization. These processes are accomplished by mechanisms, like bio-precipitation, biosorption, bioaccumulation, bio-assimilation, bioleaching, biodegradation, and biotransformation (**Figure 6**). Commonly microbial species used for remediation methodology are *Bacillus, Arthrobacter, Pseudomonas, Enterobacter, Aspergillus, Penicillium, Rhizopus, Rhodotorula, Candida utilis* [23].

Biosorption is a mechanism where microbes either absorb or adsorb the inorganic contaminants on the cell surface or into the cell. While adsorption is performed on the surface of the cell, absorption involves an entire volume of material. Several mechanisms involved in biosorption are precipitation, the formation of stable complexes with organic ligands, and redox reaction. The process of adsorption involves forming a complex of the heavy metals and functional groups on the cell surface, from where they can be absorbed into the cell. Adsorption is executed by binding heavy metals to the cell surface through electrostatic interaction, complexation, and ion exchange. According to Jin et al. [28], microbes perform adsorption predominantly in comparison to absorption.

Bioleaching is the mobilization of heavy metals from contaminated soil through biological dissolution, complexation, or bio-oxidation by microbial activity. The best-known microbes for bioleaching are *Thiobacillus* and *Leptospirillum ferrooxidans*. Various mechanisms of microbial metabolism produce several secretions, like low molecular organic acids. These organic acids have shown to effectively dissolve heavy metals and soil particles containing toxic heavy metals [28].

Bioaccumulation includes the agglomeration of contaminants into the microbe where it is concentrated, where metal is sequestered.

Bio-assimilation of heavy metals includes the active transport of microbial cell's siderophore for the chelation of toxic metals. Siderophores are biomolecules that are produced when microbes are present in iron-deficient media/environment. These

*Conventional and Contemporary Techniques for Removal of Heavy Metals from Soil DOI: http://dx.doi.org/10.5772/intechopen.98569*

#### **Figure 6.**

*Schematic representation of various mechanisms involved in microbial remediation of heavy metal contaminated soil [3].*

#### **Table 1.**

*Mechanisms, advantages and disadvantages of the available remediation techniques for heavy metal contaminated soil [19].*

*Conventional and Contemporary Techniques for Removal of Heavy Metals from Soil DOI: http://dx.doi.org/10.5772/intechopen.98569*

biomolecules are specifically iron (Fe III) chelators which are finally transported into microbes by various uptake proteins. Many reports have suggested that if siderophores are bonded with other metals, they can still be recognized by uptake protein for its transportation into the microbial cell [16, 24].

Bioprecipitation is a method that uses the mechanism of immobilization for the reduction of mobility and bioavailability of heavy metals in soil. It involves converting soluble heavy metals into insoluble hydroxides, carbonates, sulfides, and phosphates.

Biotransformation changes the chemical nature of heavy metals, altering their toxicity, mobility, and bioavailability. This methodology includes methylation, reduction, dealkylation, and oxidation of heavy metals for altering their soluble form into an insoluble form [16].

The applicability of these individual techniques in any specific soil remediation project is determined primarily by contamination site geography, characteristics of contaminants, the goal of remediation, cost-effectiveness, financial budget, readiness in implementing the technique, the time provided, and public acceptability (**Table 1**). Integration of more than one technique has been experimentally proved to be more efficient, such as application of chemical remediation in highly heavy metal contaminated sediment, which can be followed by phytoremediation for further removal of remaining contaminants [15].