Design and Evaluation of a Complex Phytoceutical Formulation for Circulatory Diseases

Obesity and type 2 diabetes are increasing worldwide problems. In this book we reviewed insulin secretion in both healthy individuals and in patients with type 2 diabetes. Because of the risk associated with progression from insulin resistance to diabetes and cardiovascular complications increases along a continuum, we included several chapters on the damage of endothelial cells in type 2 diabetes and genetic influences on endothelial cell dysfunction. Cardiovascular complications occur at a much lower glucose levels, thus a review on the oral glucose tolerance test compared to other methods was included. The medical conditions associated with type 2 diabetes such as pancreatic cancer, sarcopenia and sleep disordered breathing with diabetes were also discussed. The book concludes with several chapters on the treatments for this disease offering us hope in prevention and successful alleviation of the co-morbidities associated with obesity and type 2 diabetes.

. Functional energy reserve ladder. Energy drop affects the human organism, going from a state of well being with high Energy reserves and low entropy -low disorderthrough various stages and finally to that of death, or state of zero energy and maximun disorder

Example of I, E and O increase by providing Panax ginseng
Panax ginseng active principles are bonded to beta-adrenoceptors in the cellular membrane, triggering a secondary transmitting message system (cyclic AMP) which travels through a transducer pathway to the mitochondria to increase MDH, SDH and CTS activity, enzymes of the glycolisis or tricarboxylic acid cycle. ATP generation is thus increased, raising energy levels using glucose as fuel ( Figure 3a). Thus, either the ATP/ADP ratio increase or the binding of ginsenosides to cell membrane receptors results in the KATP channel closure and insulin secretion (Rotsheyn & Zito, 2004) (Figure 3b). Thus with the increase of energy a larger health triangle is obtained and the system's intelligence has acquired more capacity to organize. Panax ginseng provides an example of a remarkable phytomedicine which is capable of enhancing I, E and O simultaneously in the living system.

Significance of the intelligence (I) within the systemic triangle. What is intelligence?
The importance (and significance) of a system's intelligence is pivotal. How can this controversial and subjective concept be defined accurately? Olalde (2005a) confronted this dilemma by examining its definition in different fields. Table 1 lists different concepts of the term intelligence. Fig. 3. a: (top) Panax intracellular action mechanism results in ATP synthesis stimulation, whose hydrolysis produces energy; b: (bottom) shows the Sodium-potassium ATP pump, illustrating ATP generation by administering ginsenosides in Panax ginseng to the Living System.

Domain
Reference Definition of intelligence Cybernetics Wiener, 1954 That whose core concepts are communication, control and learning, by means of feedback mechanisms. Physics Refers to regulation processes.

Biophysics
Living system's endogenous regulation processes effectively constitute intelligence. Multivarious World Wide Web a) Ability of a system to process general information to react appropriately to specific events; b)The product of communication, resulting from the collection, processing, integration, analysis, evaluation and interpretation of available information; c)Ability to acquire, store, retrieve, process and generate information; d) Ability to learn or understand or to deal with new or challenging situations; e) Accumulation of experiences together with the understanding of how these experiences are connected; f)Capacity to act purposefully, to think rationally, to communicate and to deal effectively with his or her environment. (g) Entity. (h) Emergent.

Intelligence = Informational entity
By analyzing common traits within the definitions given in Table 1, intelligence may be defined as that emergent informational entity, capable of learning, exerting control, emitting and receiving communication, handling energy flows, establishing feedback mechanisms and creating organization for survival. Emergent implies a higher level of intelligence of the whole, stemming from the intelligence of its parts. According to Laszlo, living systems are special third-stage systems, self-creating and self-replicating, that engender order out of chaos (Lazlo, 1987). However, Wiener states 'It is my thesis that the physical functioning of the living individual and the operation of some of the newer communications machines are precisely parallel in their analogous attempts to control entropy through feedback' (Wiener, 1954). The notion of intelligence that  adopts is that of an informational entity, i.e. one which is emergent, can generate, process and exchange informational flows in order to control entropy. The concept of intelligence becomes more objective, and functional, when treated as an informational entity, one dependent on information exchange, which as we know has a thermodynamic interpretation. A change of entropy (S) will produce a change or variation in information availability, and therefore a change in intelligence and www.intechopen.com order in the biological system. Any entity that can exchange informational flows can also generate changes of entropy. Thus, informational flow change in entropy change in intelligence. According to Stonier's proposed theory 'Pure energy can perform no 'useful' work (entropy reducing) without a concomitant input of information (Stonier, 1996). Conversely, all expenditures of energy lead to a reorganization of the universe, hence a change in its information status. Energy and information are inter-convertible'. This theory could provide additional support for the indispensable existence of an intelligent entity to handle information, since information without intelligence is without value. A possible corollary is: intelligence is that entity which can causatively alter entropy; and vice versa, entropy changes will also affect intelligence. Figure 4 shows an intelligent cell, an example of an informational entity capable of modifying entropy.

Synergy and informational systems
Synergy is a quality of informational systems. It may be understood as emergent, i.e. the informational participation of each fractal member of the system in order to achieve a higher plateau of self-organization and survival (Haken, 2005). It can also be understood as the resulting effect that is greater than the algebraic sum of the parts. Synergy is an important characteristic of third-stage systems. The increase of energy availability within a third-stage, living, system also decreases its entropy, potentially generating an endogenous tendency for informational flow and a heightened intelligence; this in turn generates organization. By analogy, an increase of information will, on its own, raise intelligence which will positively influence energy and organization. In synergic terms, each of the three elements shown above has the capacity to affect (increase or decrease) the other two. Thus we derive the following synergic bi-conditionals, inherent to any third-stage system: survival potential = health = ↑I ↑E ↑O S and also demise = sickness = I E O S↑.

Informational substantiation of biological intelligence
If there is communication, intelligence exists. Communication is a manifestation of intelligence. The existence of biochemical and bio-photonic communication in cells has been corroborated: biochemical communication, for example, between the neurological and www.intechopen.com immune systems, has been examined, among others, by Blalock (1989), Cavagnaro and Lewis (1989) and more recently Takeda and Okomura (2004) and Cooper (2004). The biophotonic communication concept was discovered by Alexander Gurwitsch in 1923, as an 'ultraweak' photon emission from living systems (Gurwitsch, 1991). About the same time, Frolich, father of the 'coherent' notion of living systems, discovered that nucleated cells are capable of picking up, storing and broadcasting information about the environment. The term biophotons '…denotes a permanent spontaneous photon emission from all living systems…' and explains '… biological phenomena like intracellular and intercellular communication, cell growth and differentiation, interactions among biological systems and microbial infections…' (Popp, 2003). Different scientific groups have confirmed the existence of (and suggested some uses for) this subtle photon emission in: Australia (Tilbary & Quickenden, 1988;Trushin 2003), Japan (Kobayashi et al., 1996;Takeda et al., 1998), Poland (Slawinska & Slawinski, 1987) and Germany (Popp, 2003). Other prominent scientists in the study of biophotons are Professor Voeikov (Voikov, 2003) and Dr. Albrecht-Buehler. This last sustains the thesis that cells are intelligent: 'capable of deriving abstract data and emitting near infrared signals' (Albrecht- Buehler, 1998Buehler, , 1985.

Biological Intelligence (BI)
Intelligence is best measured by its manifestations. In structural terms, the BI is 'omnipresent' in the organism due to the intelligent nature of all cells (Gurwitsch, 1991); however, in functional terms, the BI's common denominator is comprised of the immune intelligence (II), cellular intelligence (CI) and biochemical (or neuroendocrine) intelligence (BI) (Blalock, 1989;Cavagnaro and Lewis, 1989;Cooper, 2004;Takeda and Okomura, 2004). The BI functions as an emergent informational entity, oriented towards survival, capable of auto-regulation, bidirectional communication, generating, processing and manipulating energy flows within the body. It is in charge of establishing, maintaining and restituting the organization. Figure 5 shows the interaction between neuroendocrine, immune and cellular intelligence. CI is the most important of BI's constituents since it regulates genetics and metabolism of each and every organic cell and gives birth to the autonomous II and BI. These three elements also constitute a synergistic trio, since none of them can exist in the absence of another, due to essential feedback and information exchange amongst them (Blalock, 1989;Cavagnaro and Lewis, 1989;Cooper, 2004;Takeda and Okomura, 2004). BI could also be represented schematically as a triangle, since alterations to one side of a triangle always affect the other two. Its healing potential may be defined as the mathematical product of its immune, cellular and neuroendocrine state, i.e. BI (Healing Potential) = I I x C I x B I . In consequence, it is possible to enhance BI by increasing any of its three essential components, for example with immune modulators (Bocharova et al., 2003;Geng et al., 2005;Kidd, 2000;Kohguchi et al., 2004;Kormosh et al., 2004). The opposite also holds true, a collapse of any component will affect the other two.

From Systemic theory to Systemic medicine
At the beginning of this section a question was posed. Was it possible to set up a system -or periodic table-where plants and other natural remedies could, according to their superior medicinal properties, be arranged to produce specific formulae that provide well-being for a given pathology? The Systemic Theory was set forth to provide an answer to this crucial question. Systemic Theory postulates that Health (H) is directly proportional to the integrity of a living system's Energy (E), Bio-Intelligence (I) and Organization (O). Systemic Theory also established a common denominator to all sickness and ascertains the cause of all diseases to be an entropy increase: 'disorder augmenting within the biologically open system, stemming from energo-informational and organizational impacts, either of external or internal nature' (Olalde, 2005c). Systemic Therapeutics -or Medicine-should then include a negentropy supply to enhance the system's: energy-work capacity or E (i.e. physiological mechanisms associated with ATP synthesis, such as oxidative phosphorolysis, Krebbs cycle, β-oxidation etc.), its informational potential intelligence or I (i.e. the entity responsible for regulating neuroendocrine, biochemical, immune and cellular processes) and finally structure and functional organization or O. Systemic Medicine's (SM) treatment strategy is based on identifying and prescribing superior herbs-tonic or adaptogenic-or any nutraceuticals or medicine with potential to s t r e n g t h e n E , I , O b y p r o v i d i n g e n e r g o , informational and organizational aid to the overall network of intelligent cells and cell systems that constitute the body. The main premise proposes that when all three factors are brought back to ideal levels patients' conditions begin recovery to normal health. Table 2 provides a list of some E, I and O ceuticals -or superior medicines-whose capacity to enhance E, I and O have been studied and referenced.   Table 2. Examples of ceuticals -or superior medicines-whose capacity to enhance E, I and O has been studied and referenced

The case for a systemic complex herbal formulation: Circulat
Thus a composition was formulated taking into account the precepts established in both the Systemic Theory and Medicine. Circulat is a systemic standardized HPLC fingerprinted plant extract combination which consists of: 1) Energy plants (E) associated with ATP synthesis (such as tricarboxylic acid cycle, oxidative phosphorylation, etc) which boost the system's energy-work capacity: Eleutherococcus senticosus, Leuzea carthamoides, Panax ginseng, Panax quinquefolius, Pfaffia paniculata, and Rhodiola rosea; 2) Bio-Intelligence plants (I) which modulate the neuroendocrine and immunological systems and cellular processes enhancing the system's informational potential intelligence (specifically in this formulation they increase insulin production, insulin receptor sensitivity, improve intracellular glucose uptake, contribute antimicrobial properties, improve inflammation as well as humoral and unspecific cellular immunity): Echinacea angustifolia, Echinacea purpurea, Ganoderma lucidum, Grifola frondosa, Hydrastis canadensis, Petiveria alliacea, Sutherlandia frutescens, and Uncaria tomentosa; and finally 3) Organizational plants (O) which enhance the structure and functional organization of specific organs supporting overall health (in Circulat's case, among others, promoting vasodilatation, tissular perfusion improvement, regeneration and skin scarring): Angelica sinensis, Crataegus oxyacantha, Croton lechleri, Ginkgo biloba, Hydrocotyle asiatica, Ruscus aculeatus, Vaccinium myrthillus, and Tabebuia avellanedae. Although some of these plants act predominantly over one of the factors that influence individual's overall health (E, I, and O), some act over more than one factor (i.e. Panax and Ganoderma). Please, see Table 3.

Energy plants Panax ginseng and Panax quinquefolius
Increases ATP synthesis by stimulating activities of enzymes related to tricarboxylic acid cycle and oxidative-phosphorylation, such as succinate dehydrogenase, malate dehydrogenase, citrate synthetase, cytochrome oxidase, and phosphorylase .

Eleutherococcus senticosus
Increases ATP synthesis by stimulating activities of enzymes related to tricarboxylic acid cycle, such as succinate dehydrogenase and malate dehydrogenase (Sugimura et al., 1989).

Leuzea carthamoides and Pfaffia paniculata
Increase ATP synthesis, stimulates activities of enzymes related to tricarboxylic acid cycle, such as succinate dehydrogenase. Also, normalize NADH dehydrogenase activity, enzyme related to the oxidative phosphorylation processes, contributing to buildup the electrochemical potential used to produce ATP (Tashmukhamedova et al., 1986).

Ganoderma lucidum
Promotes phagocytosis and cytotoxicity of macrophages .

Hydrastis canadensis
Antiinflamatory due to a prostaglandin E2 production reduction as a result of AP-1 binding inhibition (Kuo et al., 2004).

Ganoderma lucidum
Stimulates glucose uptake, stimulating the activity of phosphatidylinositol 3kinase, Protein kinase B, AMP-activated protein kinase which are regulatory molecules in the glucose uptake pathway (Jung et al., 2006). Lowers glucose levels through insulin-releasing activity due to facilitation of Ca2+ inflow to pancreatic beta cells (Zhang and Lin,200 4).

Petiveria alliacea
Decreases: a) blood glucose levels (Lores and Cires Puyol, 1990); and b) fasting glucose, post-prandial glucose levels, and hemoglobin A1c in type 2 diabetic patients, by acting downstream in the insulin signaling pathway .

Sutherlandia frutescens
Normalizes insulin levels and increases glucose uptake (Chadwick et al., 2007). Decreases fasting glucose, post-prandial glucose levels, and hemoglobin A1c in type 2 diabetic patients, by acting downstream in the insulin signaling pathway .

Petiveria alliacea
Broad spectrum of antimicrobial activity .

Hydrocotyle asiatica
Promotes fibroblast proliferation and extracullar matrix synthesis in wound healing because it upregulates 54 genes with known functions for cell proliferation, cell-cycle progression and synthesis of the extracellular matrix (Lu et al., 2004). Table 3. Circulat´s E, I and O -referenced-components and action mechanisms. www.intechopen.com

Assessment of Circulat´s capability to modulate diabetes related gene expression levels in cultured human fibroblasts
Circulat had provided an auspicious early clinical proof of its effectiveness  in the treatment of Diabetic Foot -a circulatory disease. However, a pending assignment was to confirm its molecular activity. One way to test this characteristic was to identify the modulating and/or synergistic roles that such formulation could have in diseases such as Diabetes Mellitus (I and II). The study called 'Analysis of the Effects of the Herbal preparation Circulat on Gene Expression Levels in Cultured Human Fibroblasts' was carried out in cooperation with the Pennsylvania State University, Department of Genetics (Antoshechkin et al., 2007).

Materials and methods
Circulat whole composition -a lyophilized ethanol/water extract of 22 known medical plants in different ratios-and its three E, I and O fractions (or components 1, 2 and 3) identical and in the same proportion as in the formula were tested using microarray analysis using the Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays that provide full genomic coverage and contain probes for more than 47 000 unique transcripts corresponding to more than 38 500 human genes. This allowed monitoring simultaneously the expression levels of practically all annotated genes of the human genome in an unbiased manner. Following hybridization and scanning, raw data in the form of image files were converted to gene expression files using the Affimetrix GeneChip Operating Software (GCOS) which utilizes MAS 5.0 algorithm for data normalization, background subtraction, and the estimation of nonspecific binding, calculation of detection p-values and generation of presence calls. Two-tailed Taylor Student's t-test assuming unequal sample variance was used to identify genes that displayed significant changes in the mean expression levels between control and each of the treated samples with the t-test-p value less than 0.05 and the mean fold change of at least 2. By comparing up and down regulated genes in each individual fraction and whole Circulat, additional genes were identified that were regulated between 1.5 and 2 fold in the whole preparation and followed the same trend as in individual fractions, where they were up -or downregulated by at least 2 fold.

Results
The Affymetrix GeneChip represents state of the art in microarray design and features both perfectly matched and off-by-one probes that together with sophisticated processing algorithm allow distinguishing precisely between specific and non-specific hybridization signals. It has been proven to produce highly reliable data, which in combination with the high quality of starting RNA and sufficient number of replicates virtually eliminates falsepositives. Significant changes in the mean expression levels between control and treated samples resulted in a list of 87, 96, 24 and 187 genes (probes) that were significantly upor down-regulated upon treatment with components 1, 2, 3 and whole Circulat, respectively. The genes regulated by the individual components and the whole Circulat formula showed a significant overlap, as expected. More than 80% of the genes affected by the individual fractions were also affected by the whole preparation. Analysis of the data for each of the three components also identifies a sizable number of genes (23) that www.intechopen.com did not show up in the whole Circulat analysis. On the other hand, 55 genes the expression of which changed significantly after Circulat treatment were not observed in any of the three fractions. The regulation of these 32 genes by Circulat is more likely due to the interaction between active ingredients of the three components that produce a synergistic effect on gene regulation. Taken together the data demonstrates that: 1) Treatment of human fibroblast cells with either Circulat or its components result in marked changes in gene expression patterns; 2) Significant interactions between the active ingredients of Circulat exist resulting in a more complex pattern of gene expression in the complete preparation when compared with those of the isolated components which can be understood to be synergistic. Figure 6. Fig. 6. Circulat -as a whole-modulates 187 genes, 32 genes more than the sum of the E, I and O fractions that make it up.

Analysis of processes and pathway affected by circulat
Examination of the biological process subset of Gene Ontology (GO) terms (Ashburner et al., 2000) associated with each gene and their distribution revealed that genes affected by Circulat are involved in a variety of cellular processes including protein, nucleic acid, lipid and carbohydrate metabolism, regulation of transcription, response to endogenous and external stimuli and stress, signal transduction and cell communication, cell growth and proliferation, development, protein modification and biosynthesis, generation of precursor metabolites and energy, etc. The broad spectrum of processes potentially regulated by Circulat (Figure 7) is consistent with the established ability of Circulat to prevent the development of severe manifestations of type 2 diabetes, which is a complex syndrome www.intechopen.com involving many intracellular signaling cascades and pathways as well as cell-cell interactions. Fig. 7. Number of genes involved in a particular process that were affected by treatment is indicated. Numbers correspond to the number of probes that are annotated within a particular process.
Many of those pathways are involved in energy generation and are regulated on both transcriptional and post-transcriptional levels in response to endogenous or exogenous stimuli. A high proportion of genes identified in the experiments are implicated in the regulation of transcription (e.g.ATF3, Kruppel-like factor 4 Zinc finger protein 117, SATB2), energy production and glucose metabolism (thioredoxin domain containing 11, ATP5F1, PGK1) and signal transduction corroborating the hypothesis that Circulat is capable of normalizing molecular processes perturbed in the course of type 2 diabetes. Specially, Circulat proves to be effective in the treatment of diabetic gangrene, which is often resistant to common treatments. Previous studies have suggested that impaired fibroblast function such as proliferation, cell migration, growth factor production and collagen synthesis may be part of the mechanism of diabetic necrotic damage. Fibroblasts are central to the processes of extracellular matrix deposition and remodeling that take place during tissue repair process. It functions both as a synthetic cell, depositing a collagen-rich matrix, and a signaling cell, secreting the growth factors important for cellcell communication during the tissue repair process. Many Circulat-regulated genes play roles in signal transduction and cell communication (ADAM32, TRIO, RAB7), response to endogenous and exogenous stimuli, an essential component in wound healing (FANCB, SLC19A2, C4A), growth factor-mediated signaling and cell motility (IL6, CXCL2, VIM), cell proliferation and biosynthesis (PBEF1, ABP1, PTGS2) corroborating Circulat's effectiveness in tissue repair. Furthermore, tissue remodeling involves both the generation of new cell types as well as regulated cell death, and several genes important for cell differentiation and apoptosis were identified in the experiment (KLF4, RTN1, www.intechopen.com PDCD5). It was also observed that significantly down-regulated genes are enriched for members of signaling cascades known to regulate transcription and progression through the mitotic cycle such as FOSB, SNF1-like kinase and PRKAR1A (cAMP-dependent protein kinase regulatory subunit), cyclin L2. Up-regulated genes also contain a high number of genes involved in progression through the cell cycle as well as in DNA damage response, including cyclin M. GTSE1, FANCB and BR1P1 (BRCA1 interacting protein C-terminal helicase 1), cyclin-dependent kinase inhibitor C2 (CDKN2C). A number of transcription factors and members of protein degradation machinery (ubiquitin-conjugating enzyme E2E 3, Ring finger and WD repeat domain 2) are also present among those genes. Data suggest that one of the reasons for Circulat's therapeutic effects is derived from its ability to stimulate cellular activities that respond to internal and external stress by slowing or arresting the cell cycle to allow repair of cellular components that could be damaged in the course of the disease, such as DNA and proteins, to be carried out by repair enzymes.

Discussion
It was shown that four genes, IL6, HMGA1, SLC19A2 and C4A that are known to be involved in the development and progression of diabetes are strongly regulated by Circulat. This not only validated the experimental approach, but also allowed for the first time to suggest an explanation for the clinical effectiveness of Circulat at the molecular level. The role of interleukin-6 (1L6) in diabetes type 1 and 2 is thoroughly documented (for a review see Kristiansen and Mandrup-Poulsen, 2005). Low-grade inflammation has been proposed to be involved in the pathogenic processes causing type 2 diabetes and inflammatory mechanisms are known to play a key role in the pathogenesis of type 1 diabetes. As a mediator of inflammation, IL6 is thought to be involved in events causing both types of the disease when present at elevated levels. In addition, IL6 can also regulate glucose homeostasis and metabolism directly and indirectly by both increasing the destruction of insulin producing β-cells by promoting apoptosis and playing a role in mounting insulin resistance in skeletal muscle, adipocytes and other tissues. Since elevated levels of IL6 are the known predisposition factors for development of diabetes, reduction of IL6 concentration should have the opposite therapeutic effect. The results demonstrate that Circulat treatment reduces IL6 expression more than two fold. (0.42, p =0.002) providing a solid link between the molecular events and the clinical manifestations taking place upon Circulat treatment. HMGA1 expression levels were elevated by more than two fold (2.07, p = 0.02) in samples treated with Circulat.
Mutations in this small nuclear protein that acts as an architectural transcription factor have been detected in individuals suffering from type 2 diabetes (Foti et al., 2005). This correlated with the insulin receptor's expression reduction and consequent development of insulin resistance. Deletion of HMGA1 gene in mice resulted in almost a complete loss of insulin receptor expression, development of insulin resistance and type 2 diabetes-like symptoms. HMGA1 thus plays a crucial role in glucose homeostasis and its increased expression promoted by Circulat may counteract deleterious effects caused by the loss of insulin receptor observed in type 2 diabetic patients. Two other genes, SLC19A2 and C4A, that are affected by Circulat (0.43, p = 0.03 and 0.48, p = 0.02, respectively) have also been unequivocally linked to diabetes. SLC19A2 encodes a thiamine transporter protein and causes thiamine-responsive megaloblastic anemia (TRMA) also known as Rogers syndrome, when mutated (Labay et al., 1999). Diabetes (both type 1 and 2) is the primary disease that defines the syndrome. C4A encodes the acidic form of complement factor 4, part of the classical activation pathway. Deficiency of this protein is associated with systemic lupus erythematosus and type 1 diabetes mellitus (Palsdottir et al., 1983). While no direct link between C4A and diabetes type 2 has been found thus far, it has been suggested that the two types of the disease share many of the underlying processes thus making C4A involvement in type 2 diabetes a real possibility. Precise molecular mechanisms of SLC19A2 and C4A involvement in the disease development are not established as well as for IL6 and HMGA1. Nevertheless, their link to diabetes is indisputable and the ability of Circulat to influence their expression suggests additional possible Circulat action mechanisms. Analysis of genes affected by Circulat also reveals that 26 of them have been implicated in many human diseases other than diabetes. It is possible that some of those diseases and syndromes could be caused by misregulation of the same (or similar) genetic pathways that are perturbed in type 2 diabetes, which could be one of the explanations for this observation. On the other hand, it raises an exciting possibility that Circulat could be effective for treatment of conditions other than type 2 diabetes. Finally, analysis of genes regulated by the total Circulat and its individual components demonstrated the existence of significant interactions between the active ingredients of Circulat suggesting that the full therapeutic effects can only be achieved by administration of the complete preparation.

Significance of gene expression analyses
The application of full-genome expression analyses to phytopharmacology opens new horizons for carrying out scientific studies of herbal remedies and integration of herbalbased treatments into mainstream medicine. Using this approach, the physiologically active fractions of effective herbal extracts can be isolated and their specific activities can be determined. Such separation of different activities of a particular extract may enable researchers to selectively regulate the expression of specific genes (or gene groups) by varying the composition of the herbal preparation. It is likely therefore, that expressionprofiling-based approaches to studies of herbal medicines will become standard in phytopharmacology in the near future. This section, explained Circulat´s molecular modulating capabilities. Section 4 will examine its clinical effectiveness through a diabetic foot study synthesis.

Circulat´s therapeutic properties in diabetic foot: Synthesis
The World Health Organization estimated that more than 220 million people worldwide have diabetes (WHO, 2011). This number was likely to more than double by 2030. Diabetic foot ulcers are one of the most frequent complications of this disease. The prevalence of diabetic foot ulcers has been estimated, at the time of the study, between 2.2 and 15%. Differences being attributed to risk factor diversity: ethnicity, age, sex, education level, health service quality, and others (Table 4). Diabetic foot ulcers represent a large emotional and economic burden on patients and caregivers, as well (Gulam-Abbas et al, 2002). Foot complications are caused by diabetic neuropathy or peripheral ischemic vessel disease or a combination of both (Ratzmann et al, 1994) and are the most frequent reason for hospitalization in patients with diabetes.   . Approximately 40-60% of all lower extremity amputations are performed in patients with diabetes. More than 85% of these amputations are precipitated by a foot ulcer deteriorating to deep infection or gangrene (Apelqvist and Larsson, 2000).
In people with healed diabetic foot ulcers, the 5 year cumulative rate of ulcer recurrence is 66% and of amputation is 12% (Apelqvist et al., 1993). The high amputation incidence and healing failure after lower extremity amputation for the treatment of diabetic foot ulcer (Malay et al., 2006) is a distinct signal that the efficiency of conventional medical treatments used is less than optimal. This substantiates the need to search for effective therapeutic alternatives and diminish the suffering and high economic and social costs caused by this common diabetic patient complication. Various medicinal plants have been used traditionally for the treatment of circulatory obstructive diseases. In the last couple of decades many of their active principles and action mechanisms have been discovered. Also, traditional healing know-how has been proven to be effective in many cases. This raised the possibility of using herbal therapeutic protocols to complement conventional treatments for complications in diabetes. Specially, since there was mounting evidence which demonstrated that medicinal plants contained synergistic and/or side-effect neutralizing combinations (Thyagarajan et al., 2007;Gilani and Rahman, 2005). In contrast to synthetic pharmaceuticals based upon single chemicals, phytomedicines exert their beneficial effects through the additive or synergistic action of their multitude of constituents acting at single or multiple target sites (Dalby-Brown et al., 2005); because of their primary and secondary metabolite roles (Greenspan et al., 1994) and the adjuvant substances which enhance the activity of components actually responsible for the effect (Gilbert et al., 2003). In order to take the maximum advantage of the therapeutic properties as well as benefits of the synergistic action of the active principles in medicinal plants, it is necessary to use herbal combinations. Herbal formulations have been used for hundreds of years, however, little was known about the methodology to combine plants and obtain effective compositions. The Systemic Theory provided fundamentals which allowed the formulation of an effective herbal composition for treating diabetic foot . The previous section revealed that Circulat's active principles exerted therapeutic effects through a synergistic action, as well as its potential for genetic normalization in diabetic patients (Antoshechkin et al., 2007).

Objective, research design and methods
The aim was to appraise the clinical efficacy of Circulat in healing diabetic foot, measure the amputation rate and determine patient's tolerance to the treatment. Thus, a retrospective, cohort, study of patients with type 2 diabetes and foot ulcerations from 50 medical centers, from 2004 to 2007. Patients were classified in accordance with The University of Texas Health Science Center Diabetic Wound Classification System . Patients were being administered ten Circulat 800 mg capsules twice per day, on an outpatient basis, during a period of two to four months. Each case was followed-up during a period of six months, after the end of the treatment. A patient was considered to attain clinical improvement if the lesion visibly decreased in size and depth, or closure or scarring of the wound was attained. All patients received conventional treatment for metabolism correction, local topic cures and systemic antibiotics. The Inclusion criteria of the study were: Patients of any age and gender diagnosed with Diabetes mellitus type 2, grades D1, D2 and D3 (University of Texas Diabetic Wound classification).

Results and discussion
The total number of patients which completed the treatment in accordance with the study's inclusion criteria was 174. The mean was 61.3 years of age. The gender classification was: 101 male (58.1%) and 73 female (41.9%). Clinical results are reflected in Table 5. Amputations were prevented in 88.55% (p< 0.00001, 99.9999%) of all 174 patients in the D1-D3 categorization. The treatment was well tolerated; only 4 patients (2.3%) had minor gastrointestinal unrest which did not warrant treatment suspension. Conventional diabetic foot treatments, based on: risk factors control, affected area's functional relaxation, metabolism correction, topical cures, antibiotics, rheology improving agents, prostanoid vasoactive therapy, platelet aggregation inhibitors, thrombolytic agents, tricyclic antidepressants or benzodiacepines for neuropathies and invasive treatments, such as endovascular, endarterectomy, by-pass or sympathectomy surgeries, do not manage to prevent small and large amputations which occur in 1.86 to 5.9 per every 1.000 diabetics per year (Bilenko et al, 2006;Winell et al, 2006;Rayman et al, 2004;Lavery et al, 2003;Trautner et al, 2001). Circulat-in combination with conventional therapy-prevented more amputations than various conventional treatments reported (Figure 8).

Formulation´s potential in chronic ischemic heart disease treatment
Context: The previous clinical study (Olalde et al., 2008) demonstrated a significant improvement in 174 diabetic foot patients. These encouraging outcomes and further evidence that other complex herbal formulations could help reduce atherosclerotic endothelium intima thickness (Tripathi et al., 2005) prompted us to evaluate the formulation by measuring the arterial intima-media thickness, a structural marker of early atherosclerosis that relates to the severity and extent of artery disease (Järvisalo et al., 2001), using high-resolution ultrasound (Celermajer et al., 1992). The study (unpublished) showed that treatment with this herbal combination significantly reduced the arterial intima-media thickness. These results further encouraged the formulation of a new hypothesis: could similar results be obtained in the treatment of IHD? If so, is there a potential for CAM complex herbal formulations' intervention in the treatment of Ischemic Heart Disease? Objective: Assess the hypothesis that myocardial perfusion, in chronic ischemic heart disease patients, might safely improve with Circulat. This was evaluated with Gated Single Photon Emission Computerized Tomography (GSPECT) imaging.

Introduction
Ischemic heart disease (IHD) is the leading cause of death -in both genders-worldwide and a major public health problem in the world. WHO (2009) has estimated this to be 7.2 million deaths per year. IHD is the generic designation for a group of pathophysiologically related syndromes resulting from myocardial ischemia (an imbalance between supply -perfusion-and demand for oxygenated blood by the heart). IHD is caused by the atherosclerotic narrowing of one or more coronary arteries and endothelial dysfunction (Thadani, 2004(Thadani, , 2003(Thadani, , 1999. IHD brings oxygen insufficiency and reduces the availability of nutrients as well as the removal of metabolites. For this reason, IHD is generally less well tolerated by the heart than pure hypoxia, such as may be seen in severe anemia, cyanotic heart disease or advanced lung disease. Today much attention is being paid to Chronic Ischemic Heart Disease. This last is increasingly recognized as a dynamic condition. In addition to over acute myocardial infarction, which can precipitate at any time in patients with stable angina pectoris, clinical and sub-clinical ischemic www.intechopen.com events may accumulate and in the long term generate diverse states of chronic cardiac dysfunction. Repetitive episodes of ischemia, whether stress induced or spontaneous, symptomatic or silent, may progressively impair myocardial contractile performance through myocardial stunning or hibernation, and eventually lead to left ventricular remodeling and heart failure. Evidence is accumulating that genetic variability and altered gene and protein expressions contribute to clinical outcomes in ischemic heart disease. Severe ischemic heart disease remains a clinical challenge; many patients have undergone myocardial revascularization procedures due to the extension and diffuseness of the disease. Also, viable options are becoming available for the 'no option' patients with chronic IHD. Instead of revascularization of the highly diseased epicardial coronary arteries, scientists and clinicians are looking -among other-at providing symptomatic relief in these patients via a biological bypass such as a 'master' cardiac stem cell for intracoronary and intra-myocardial injections (Bu et al., 2009). Current treatments include pharmacological agents such as nitrates, aspirin, beta-adrenoceptor antagonists and calcium channel blockers as well as invasive therapies aimed at restoring blood flow, e.g. coronary artery bypass graft (CABG) and improved percutaneous coronary intervention -PCI (Tin-Hay et al., 2010). On the other hand, various medicinal plants are being usedand researched-for the treatment of coronary heart disease and angina pectoris in China and East Asia, and are referenced in Chinese Materia Medica (Tam et al., 2009;Ling et al., 2008;Duan et al., 2008;Zhao et al., 2007). This last does not preclude quite the contrary it demands, additional pharmacotherapy analysis and herbal medicines drug interaction research (Izzo et al., 2005). Nevertheless, the study and development for synthetic (Yamaguchi et al., 2009;Hirata et al., 2009) and medicinal plant in cardiac treatments continues (Chen et al., 2010;Luo et al., 2009).

Study materials and methods
Inclusion Criteria: Patients diagnosed with Chronic Ischemic Cardiopathy. Exclusion Criteria: Acute Coronary Syndrome, severe aortic valve stenosis, arrhythmia with hemodynamic repercussion, acute pericarditis, acute myocarditis, decompensated cardiac insufficiency, acute aortic syndrome, severe anemia, pulmonary embolism, severe arterial hypertension, severe pulmonary arterial hypertension, chronic debilitating diseases, second or third degree atrioventricular block, hypertrophic obstructive cardiomyopathy and valvular cardiomyopathy with hemodynamic compromise. Patients: 20 patients diagnosed with Chronic Ischemic Heart Disease were evaluated. Prior to treatment, tests had determined that 4 of these patients had cardiac ischemia, assessed with cardiac angiography and non invasive methods, and the other 9 patients tested positive for cardiac ischemia with the exercise treadmill testing protocol (Bruce). Initial cardiac ischemia diagnosis was also corroborated by GSPECT control imaging prior to treatment. Patients were receiving treatment with aspirin, ß-blockers, statins, nitrates, clopidogrel and anti-hypertensive medication (diuretics, ACE inhibitors, angiotensin II receptor antagonists, calcium channel blockers). Patients were given a complete physical examination at screening, each month and at the end of the treatment (sixth month). The patients' baseline factors, relevant cardiac conditions and ongoing treatments were established in Table 6. The most pervasive baseline condition risk, evidenced from patients' clinical history (Table 6) was Hypercholesterolemia (14/20; 70%). On the other hand, the most common treatment was aspirin (17/20; 85%).    Figure 9.

Assessment of regional myocardial perfusion defect
Patient's -before and after Circulat treatment-post-effort -left ventricle-myocardial perfusion defect percentages were assessed by GSPECT imaging with technetium 99mtetrofosmin injection. This technique uses imaging procedures for detection with the help of gamma rays. In the procedure, the radioisotope is used on the patient and the gamma ray emitted by the technetium 99 that is in the radioisotope is captured on a special gamma camera. The total scanning time for the heart of a patient takes around twenty minutes which is enough time given the half life span of technetium 99. This isotope has a short half life -about six hours-and this small longevity is very useful for medical purposes. Recollection of data [imaging] is quick and the amount of radiation which a patient undergoes through is very low in intensity. GSPECT myocardial perfusion imaging is a widely used nuclear imaging procedure for diagnosis and management of coronary artery disease -which is the most common cause of heart failure. It is extensively available with superb standardization and reproducibility . For this study, the Phillips Forte (2007 model) AZ Spect Dual Headed Camera with an AUTOQUANT -quantitative algorithm-program for image processing was used. A 20 segment analysis was performed. Results of Perfusion Defect evolution are reflected in Figure 10.

Discussion on ischemic heart treatment
The results answered the formulated [raised] hypothesis. Indeed, CAM -and Circulat-can have a role in the treatment of Ischemic Chronic Heart Disease. This is supported by the number of patients (15 out of 20) which quantitatively improved their left ventricular perfusion capacity. Effects of this treatment are thought to be caused, among others, by vascular endothelium structure and function improvement, angiogenesis and tissue perfusion enhancement. Also to be noticed is patients' disease evolution according to initial perfusion defect -reflected in Figure 10. There seems to be a correlation suggesting that initial stages of ischemia development -corresponding to lower Pre-Treatment perfusion defect-are more precociously responsive to a short (6 month) treatment, thus advocating use of the formulation as a preventive remedy against chronic ischemic heart disease. However, to measure the treatment's potential effectiveness, in patients with acute perfusion defect, a longer lasting treatment -of at least one year is necessary. Just as pathologies -with critical conditions-warrant longer management periods.

Conclusions on chapter
A theory is as good as the praxis that backs it up. On one hand the gene expression study (Antoscheckin et al, 2007) established that a complex herbal formulation, designed in accordance with Systemic Theory precepts, had synergistic properties as well as the capacity to modulate genes implicated in diabetes development, energy metabolism, protein synthesis, glucose metabolism and signaling pathways. It also demonstarted that four genes (IL6, HMGA1, SLC19A2 and C4A) involved in the development and progression of diabetes are strongly regulated by Circulat. These bio-molecular results were confirmed with the further evaluation of diabetic foot management (Olalde et al., 2008) which provided clinical substantiation that a complex herbal formulation could be successful in providing therapeutic response to such a circulatory chronic disease. However, the result obtained in the reduction of perfusion defect in Chronic Ischemic patients was unexpected. It also www.intechopen.com requires further validation. The authors are prolonging the length of the treatment -and the study-to one year. From this distance a better image of its potential therapeutic use can be assessed. However, there is room for cautious optimism. Evidence is accumulating that genetic variability and altered gene and protein expressions contribute to clinical outcomes in ischemic heart disease. It is likely therefore, that expression-profiling-based approaches to chronic degenerative diseases such as diabetes and ischemic heart disease with herbal medicines will become standard in phytopharmacology in the very near future. Resuming: a)The confirmation of Circulat's synergistic synergistic gene modulating capabilities (in 32 genes, among which are four significant genes related to diabetes; b) The clinical outcomes of diabetic foot management which prevented the amputation of 88.5% of the study's total population ; and c) The good response to therapy in 75% of the patients (p ≤ 6.104e-05; % = 99.9999) by the composition in a GSPECT evaluated limited Phase II Chronic Ischemic Heart Disease suggest that treatment of circulatory pathologies with substances which act only on function and structure might be an incomplete approach. Whether the findings in circulatory chronic disease studies -and their results-can be generalized in confirming the systemic approach (E, I and O) is something that should be continued in medicinal science. Since this may pave the way for a new integral vision of therapeutics in general having started to proove its validity in circulatory chronic diseases in particular.

Acknowledgement
The authors wish to thank Dr. Edwin L. Cooper for his trust and invaluable guidance. Few can vouch such a supportive professor. Also, our appreciation to En Jose Olalde whose vision prompted a new therapeutic approach to chronic degenerative diseases recognizing that '…the essence of science…' is to have the valour to '…ask an impertinent question…' and get us on the road '…to a pertinent answer…' (Jacob Bronowski). Last but not least to Daniel Tucci, who although does not appear as an author provided figures and graphs, the art, that rounds up the science we all are so passionate about. Obesity and type 2 diabetes are increasing worldwide problems. In this book we reviewed insulin secretion in both healthy individuals and in patients with type 2 diabetes. Because of the risk associated with progression from insulin resistance to diabetes and cardiovascular complications increases along a continuum, we included several chapters on the damage of endothelial cells in type 2 diabetes and genetic influences on endothelial cell dysfunction. Cardiovascular complications occur at a much lower glucose levels, thus a review on the oral glucose tolerance test compared to other methods was included. The medical conditions associated with type 2 diabetes such as pancreatic cancer, sarcopenia and sleep disordered breathing with diabetes were also discussed. The book concludes with several chapters on the treatments for this disease offering us hope in prevention and successful alleviation of the co-morbidities associated with obesity and type 2 diabetes.

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