**Abstract**

Women in India are the major workforce in agriculture and perform almost all the agricultural activities. Women constitute 25.51% of the total work force in the country. The rural women play a vital role in agriculture and other agro based processing activities. They spent more time on post-harvest activities than those pre-harvest activities. Environmental and occupational health issues among agricultural workers expressed high levels of concern about working in hot weather, agricultural injuries, pesticides, awkward posture and drudgery prone activities. User Eco-friendly tools can increase the working efficiency and reduce the working load health hazards on farm activities during agricultural activities.

**Keywords:** Agriculture Activities, musculoskeletal Problem, drudgery, health, work Load, women Participation, postural discomfort

### **1. Introduction**

Women comprise approximately 25.51% of the total work force in India as per [1], of which approximately 18.5% and 24.9% are agricultural labourers and cultivators respectively while 47.5% work in household and other important agricultureaiding fields. Women fulfill many crucial roles in the farming sectors, all the way from being general farmers to farming-entrepreneurs [2]. It was observed that more than 75 per cent women are involved in activities like winnowing, weeding, grading, threshing and cleaning of field farm operations [3]. If women were to be given the same opportunities in utilizing resources as men, the UN Food and Agriculture Organization estimates a rise in yields by up to 30%, which could have a chain effect by which a rise in total agricultural output by 4% is predicted along with reduction of the general hunger in population by 12–17%, which is about 100 million people [4]. The participation of farm women in agricultural activities suffer both by nature wise and extent wise by multiple of things including, local dimension in nature of labor, socio-economic strata of the farm families, ancestral family customs, change in nature of activities thanks to mechanization, introduction of your time and labour saving implements and ever changing nature of agro-climatic conditions [5].

Ergonomics is a multidisciplinary science that activities to make a better fit between the work and the worker to ensure their healthiness and well-being. It emphasizes on designing and arranging things so that workers can use them easily and safely. The International Ergonomics Association [6] defined ergonomics as a science that deals with understanding the interaction between humans and other elements of a socio-technical system. Using ergonomics in agriculture helps in reducing and removing the risks involved in work, machines, vehicles and the work environment which includes tools and materials, method of work, ambient conditions, physical environment and organization of work. Ergonomics and sustainable agriculture should go hand in hand with each other. Women contribute in overall farm production--average contribution--is estimated to be in the range of 60 to 65 percent of the total labour and in certain areas, the same contribution is even higher in percentages. Unfortunately even today, agriculture related activities rank as one of the most hazardous activities as it relies mostly on manual labour and people working in the fields of agriculture are exposed to a multiple varieties of hazards that are extremely damaging to their physical condition and well-being. People from rural communities and background often lack orientation and knowledge on the health related issues that they face. Farmwomen consider pain as a traditional part of work or let us say their *Karma* and seek medical assistance when the condition becomes ruthless or disabling. This same issue carries over to preventive measures designed to reduce the incidence of musculoskeletal injuries or other hazardous work exposures [7]. It was reported by [8] that the share of women labour force in agricultural operations is expected to be 55 percent by 2025 A.D. They participate in several farm operations putting many hours of productive manual labor daily. It is duly reported [9] that women contribute to plentiful responsibilities to undertake extensive range of duties both in the home and outside but their partaking is considered insignificant by the social order. They are at length involved in a variety of farm operations like transplanting, weeding, harvesting, processing, marketing and selling of food grains, fruits and vegetables etc. These activities not only require substantial time and energy but also are the sources of tremendous amount of drudgery. Drudgery, which is largely, envisaged as physical and mental damage, anguish, repetitiveness and hardship that are experienced by farmwomen while performing these farm operations. The drudgery prone situations lead to an assortment of health and mechanical hazards, which creates physical exhaustion fatigue and low productivity.


*<IP_ADDRESS> Aminopeptidase*

Adipocytes are involved in production of a polypeptide hormone named aminopeptidase, which circulates at very high levels in the bloodstream, exerts anti-inflammatory effect. It also expresses an anti-atherosclerotic effect and inhibits intimal thickening and vascular smooth muscle cell proliferation in injured arteries [56]. Angiogenesis, a neo-vessel formation from pre-existing micro-vessels requires sequential steps involving detachment of pre-existing pericytes for vascular destabilisation, extracellular matrix turnover, migration, proliferation, tube formation by endothelial cells, and reattachment of pericytes for vascular stabilisation. Aminopeptidases has been found to regulate the N-terminal modification of proteins and peptides for maturation, activation or degradation, and thereby relate to a variety of biological processes. Three types of aminopeptidases which have been reported are involved in angiogenesis. They include type 2 methionine aminopeptidase, aminopeptidase N, and adipocyte-derived leucine aminopeptidase/puromycin insensitive leucyl-specific aminopeptidase [57]. It has been documented and shown by Costagliola et al. [58] that APN levels in aqueous humour of patients with type 2 diabetes, PDR, and macular edema are higher than in aqueous of control subjects.


**1. Introduction**

The activity of the olive oil industry generates enormous quantities of solid wastes; among them olive leaves represent 10% of the total weight of the olives harvested. They are also generated in high amounts during olive tree pruning (25 kg per olive tree annually) [1]. Disposal of olive leaves help to maintain fields clean and avoid the spread of diseases; they are usually removed by either burning or grinding and scattering them on fields, which involves a very important problem of management and environmental pollution [2]. Nowadays there are no exploitations on an industrial scale for this byproduct, so proposals for evaluation and exploitation can help its better use and valorization.

Olive leaves represent a promising raw source of antioxidants and bioactive substances. Olive leaves contain high amounts of a large variety of phenolic and flavonoid compounds (hydroxytyrosol, verbascoside, apigenin-7-glucoside, rutin, and ligstroside, among others), similar to those present in olives and their derived products [3,4]. Because of that, there is a great interest by the scientific community in the valorization of these residues to achieve extracts enriched in these compounds that can be used in food, cosmetic, and pharmaceutical industries [5,6] to prevent oxidation processes. In addition, olive leaves extracts have other applications in food industry such as to improve the nutritive value of certain foods [7]. Among the bioactive compounds of olive leaves are secoiridoids and flavonoids, oleuropein and the flavone luteolin-7-glucoside being some of the compounds that have attracted more interest and which show well-documented biologic activities including antioxidant, antimicrobial, anti-inflammatory, anticancer, and neuroprotective properties [8]. Oleuropein and luteolin-7-glucoside are the most important phenolic compounds recovered from olive leaves [9]. Oleuropein is the most abundant component of the secoiridoid fraction, whereas glycosylated form luteolin-7-glucoside is the principal flavonoid with concentrations can reach up to 210 and 11 g/kg dry leaf, respectively [10,11].

Literature shows that oleuropein has potent biological and pharmacological activities [12]. Its main pharmacological properties that make it anticancer, anti-diabetes, antiobesity, cardioprotective, gastroprotective, hepato-protective, and radioprotective, among others, are in large part attributed to its putative antioxidant and anti-inflammatory effects [13]. Oleuropein and luteolin-7-glucoside have also been studied for their differentiation-inducing effects on hematopoietic stem cells highlighting their potential use in the ex vivo generation of blood cells components that are very important for the therapy of a wide spectrum of hematological disorders [14]. In a work carried out by Ahmad-Qasem et al. [15] on the olive leaves extracts' bioaccessibility, luteolin-7-glucoside was the most stable polyphenol during the in vitro digestion process simulation which means this compound may be considered as one of the responsible of the bioactivity of olive leaves extracts. Therefore, owing to their numerous health benefits associated, an optimized extraction method is important for their isolation and purification, thus contributing to the advances in research of their biological properties.

Several extraction technologies have been used to obtain antioxidant extracts and to isolate bioactive compounds from olive leaves [16]. The optimization of the extraction techniques for these compounds is essential for the development of biorefineries dedicated to the valorization of the by-products generated by olive biomass for obtaining pharmaceuticals, cosmetics, nutraceuticals, and functional foods. The conventional or traditional methods such as Soxhlet solid–liquid extraction have many drawbacks, such as long extraction times and exposure of the extracts to excessive heat. Recently, the demand of novel techniques of extraction with shorter extraction times and less consumption of solvent than conventional methods is increasing. As an alternative of traditional extraction methods, newer and more sophisticated technologies such as pressurized liquid extraction, also known as accelerated solvent extraction, microwave-assisted extraction, plasma-assisted extraction, supercritical fluid extraction, and ultrasound-assisted extraction (UAE) have been developed and used to obtain different types of bioproducts from various biomass [17]. For instance, pressurized liquid extraction has shown to be effective for the recovery of olive leaves biophenols [18]; microwave treatment has been utilized for the extraction of lipids from lyophilized algal biomass [19]; plasma-based processes have been used as pretreatment for the isolation of biopolymers from crustacean wastes [20], and supercritical carbon dioxide extraction is suited for the obtaining of hop extracts mostly constituted by non-polar compounds [21]. Specifically, the UAE process is an effective extraction technology for a broad range of compounds from different samples of biomass [22] and is being specifically used in the extraction of phenolic antioxidant and bioactive compounds from olive leaves [23–26] and other olive oil industry by-products [27,28]. UAE provides many advantages for natural compounds extraction since ultrasound waves produce intense pressure and temperature gradients within the material, which can induce physical structural disruption of cell wall due to the formation of microcavities. This then enhances the extraction rates by increasing the mass transfer and release of intracellular components into the extraction solvent, leading to higher extraction yields with shorter processing time [23]. The application of UAE in food industry is an interesting alternative to the traditional extraction methods. It is considered one of the most economically and feasible large-scale applications [29].

UAE is influenced by several factors such as temperature and amplitude (maximum height of a soundwave). Therefore, the optimization of the extraction procedure is critical to produce extracts with high content of oleuropein and luteolin-7-O-glcuoside. Experimental designs are useful tools for evaluation of statistical significance of the factors implicated and the simultaneous optimization of variables. In comparison to one-variable-at-a-time protocol, the experimental design reduces the experimental trials, analysis time, and the environmental and economic cost and error [30]. Experimental design under response surface methodology (RSM) is a useful mathematical and statistical protocol to assess the influence of multiple factors and their interactions on one or more response variables. RSM has been used by many authors for optimization of bioactive compounds extraction from a number of biomass sources, including olive leaves, using UAE [31].

Although the effect of extraction conditions on the oleuropein content has been investigated, no study reports on the effects of UAE extraction factors on the recovery and simultaneous optimization of the other main polyphenol from the olive leaves extracts, the compound luteolin-7-glucoside. Optimization of UAE has been studied by extraction parameters including solid/solvent ratio, time, and ethanol concentration. However, most of the authors did not include temperature and/or amplitude as design factors [32–34]. Thus, the goal of this study was to optimize the extraction of oleuropein and luteolin-7-glucoside from olive leaves through a study of the influence of two key factors of UAE such as increment of temperature and amplitude using a central composite design (CCD). Additionally, antioxidant capacity of the extracts as well as the total phenolic content (TPC) and total flavonoid content (TFC) were also included. Furthermore, these findings were compared to the traditional Soxhlet extraction method.

### **2. Materials and Methods**

### *2.1. Samples and Reagents*

Olive tree leaves from Picual variety were manually picked from specimens situated on the campus Las Lagunillas of the University of Jaén, Spain. After collection, raw material was dried in an oven for 24 h at 105 ◦C and ground using a cutting mill featuring a 5 mm sieve. The average particle diameter by mass was further evaluated (D50 = 1.21 mm). Finally, they were stored at room temperature in the dark until extraction. For determining the moisture content, a sample of fresh olive leaves was weighed, heated in an oven for 24 h at 105 ◦C, cooled in a desiccator, and then reweighed to constant weight in accordance with NREL analytical procedure (Technical Report NREL/TP-510-42621). Total solids and moisture content in fresh olive leaves expressed on weight basis were 51.44% (w/w) and 48.56% (w/w), respectively.

Acetonitrile, ethanol, and methanol (HPLC grade) were supplied by PanReac AppliChem (Barcelona, Spain). Commercial standards of oleuropein and luteolin-7-glucoside were provided by Sigma-Aldrich (St. Louis, USA).

### *2.2. Extraction Methods*

Two methods were evaluated for the extraction of oleuropein and luteolin-7-glucoside from olive leaves.

Soxhlet extraction was carried out with 10 g of dry olive leaves and 150 mL of 60% (v/v) ethanol-water solution for 4 h. Literature shows that the aqueous ethanol solutions are required to effectively extract phenolic compounds from olive leaves [26,32,35]. Since aqueous ethanol solutions are the most common safe solvents in the food industrial process, they were selected as extraction solvent. The extractions were performed in triplicate. The extracts were filtered at the end of the experiments using grade 1 Whatman® qualitative filter paper. The results were expressed in mean ± standard deviation.

Ultrasound-assisted extraction (UAE) was performed using a sonifier ultrasonic disrupter/ homogenizer 150 (Branson Ultrasonics Corporation, Danbury, USA) operating at a frequency of 40 kHz, equipped with a microtip ultrasound probe, and a temperature probe. Specifically, 2.0 g ground olive leaves were thoroughly mixed with 26 mL of 60% (v/v) ethanol-water solution (solvent/solid ratio of 13:1 v/w) in a glass beaker. The extraction was performed with the ultrasonic probe immersed

1 cm into the mixture. Before applying ultrasonics, all samples were put into a thermostatic bath at 20 ◦C and maintained there until this temperature was reached. When an ultrasound pulse travels through sample, some of the energy is absorbed by the sample and is converted to heat, which in turn produces a temperature rise. The heating rate depends on the amplitude. Amplitude is a parameter used to describe the strength of an ultrasound pulse. Amplitude is a measure of a wave's magnitude of oscillation, that is, the magnitude of the maximum disturbance in the medium. Since sample heating occurs due to absorption of ultrasound energy by the solution and to self-heating of the probe, the extraction procedure was performed in maximum temperature mode and continuous sonics (continuous sonics were run at a set constant amplitude, until a set maximum temperature was measured by the temperature probe, time the assay was terminated). The increment of temperature was used as a design factor and the ultrasonics time was considered as a response to be studied. The effects of these UAE parameters were evaluated on the oleuropein and luteolin-7-glucoside content from olive leaves extracts. An ethanol–water (60%, v/v) solution was used as extraction solvent. This solvent is commonly used for extracting under optimal conditions phenolic compounds from olive leaves [25] because it is a non-harmful, low-toxicity, and easily removable solvent that presents minimum environmental impact and was a good option for the UAE process.

### *2.3. Design of Experiments and Statistical Analysis*

RSM was applied to the investigation of the e ffects of the factors increment of temperature and amplitude on the extraction of phenolic and flavonoid compounds from olive leaves. As the equipment cannot operate at more than 70% the maximum amplitude when using a microtip, in this design a range between 30% and 60% of maximum amplitude was selected. Seven responses were selected: the extraction yield or residual dry matter (R), total flavonoid and phenolic contents (TFC and TPC, respectively), antioxidant capacity (2,2-diphenyl-1-picrylhydroazyl (DPPH) free-radical scavenging capacity), oleuropein and luteolin-7-glucoside contents of the extracts, and applied ultrasonic time to reach the maximum temperature. To determine the relationships between the factors and observed results and to optimize the operating conditions, a design of experiments was performed: a central composite design (CCD) involving two factors, consisting of thirteen randomized trials, and including five replicates of the central point. The coded and actual factors for the experimental design are listed in Table 1, together with the maximum temperature reached by the sample. The factors were coded in accordance with the transformation reported by Vidal et al. [36].

As temperature is not an independent factor of the amplitude, the increment of temperature from 20 ◦C was fixed as independent variable in experimental design. As mentioned in Section 2.2, prior to the extraction, all samples were tempered at 20 ◦C. The sonifier allowed us to control the way in which ultrasonics were applied to the samples by setting the unit to operate in one of the several di fferent modes. Maximum temperature control mode was determined and specified operating temperatures for each ultrasonic cycle. Several maxima allowable temperatures in the samples or liquids were set for the experimental design (40, 50 and 60 ◦C), so that ultrasonics stopped automatically when the specified temperatures were reached and measured by the temperature probe.

The experimental data were evaluated using the Design-Expert ®v<IP_ADDRESS> software (Stat-Ease, Inc., USA) and response surface methodology. ANOVA test was used to analyze the significance of the data. To explain the behavior of the responses, a quadratic model for each response was used in accordance with Equation (1):

$$\mathbf{Y} = \beta\_0 + \beta\_1 \cdot \Delta \mathbf{T} + \beta\_2 \cdot \mathbf{A} + \beta\_{12} \cdot \Delta \mathbf{T} \cdot \mathbf{A} + \beta\_{11} \cdot \Delta \mathbf{T}^2 + \beta\_{22} \cdot \mathbf{A}^2 + \text{SD} \tag{1}$$

where Y is the response, ΔT is the coded increment of temperature (◦C), A is the coded amplitude, and SD is the standard deviation of the models. The predicted responses were correlated with the following set of coe fficients: the intercept (β0), linear (β1 and β2), interaction (β12), and quadratic (β11 and β22) coe fficients. A significance level of 0.1% was set up for the models (p-value < 0.001) and a 5% for the coe fficients (p-value < 0.05). The lack of fit was statistically significant at the 10% probability level (p-value > 0.1). The terms not significant of the models were not incorporated to the equations in accordance with the p-values indicated above.

**Table 1.** Central composite design (CCD) expressed in terms of actual and coded factors used for optimization of ultrasound-assisted extraction (UAE) conditions and maxima operating temperatures.

> Percentage values with respect to maximum amplitude of the device.

### *2.4. Extraction Yield*

To obtain the extraction yield, the extracts were filtered through a 0.45 μm and an amount of 2 mL was dried using an electronic moisture analyzer model BMO35 (BOECO, Germany) at 105 ◦C to constant weight. The extraction yields are referred to initial dry biomass and expressed as grams of extract per kilogram of dry olive leaf.

### *2.5. TPC and TFC of Olive Leaves Extracts*

1

For the TPC quantification of the olive leaves extracts the Folin–Ciocalteu method was used. Briefly, 1.5 mL of 0.2 M Folin's reagent and 1.2 mL of 0.7 M sodium carbonate solution were added to 300 μL of a suitably diluted aliquot of the extracts. The sample was maintained at 25 ◦C in the dark for 10 min, and then absorbance was measured at 655 nm. The results were expressed as mmol Gallic acid equivalents/kg dry olive leaf (mmol GAE/kg).

The TFC of the olive leaves extracts was evaluated using the method described by Chang et al. [37] with modifications. In brief, 1 mL of extract was reconstituted to 1 mL with methanol-water 20% (v/v). After 5 min of incubation, 0.3 mL of 10% (w/v) AlCl3 and 0.3 mL 5% (w/v) NaNO2 solutions were added. The sample was allowed to stand for 6 min. Then, 2 mL of a 1 M NaOH solution was added, and the final volume of the mixture was brought to 10 mL using double-distilled water. The sample was allowed to stand for 15 min, and its absorbance was measured at 510 nm. The TFC was evaluated from a calibration curve. The results were expressed as mmol Rutin equivalents/kg dry olive leaf (mmol RE/kg).

### *2.6. Antioxidant Capacities of Olive Leaves Extracts*

The DPPH free-radical scavenging method described by Vázquez-Roncero et al. [38] was used for determining the antioxidant capacity of the extracts. The inhibition percentage of the DPPH radicals was measured at 515 nm after 15 min in accordance with the following Equation (2):

$$\% \text{DPPHinability} = \frac{[\text{DPPH}] \text{control} - [\text{DPPH}] \text{extract}}{[\text{DPPH}] \text{control}} 100 \tag{2}$$

The DPPH concentrations ([DPPH]) were calculated by interpolating the calibration curve of absorbance versus DPPH concentration. The inhibition percentage was transformed into antioxidant capacity using Trolox as external standard. For this purpose, another calibration curve graphing the

percentage of inhibition against the Trolox concentration was used. The antioxidant capacity of the extracts was expressed as mmol Trolox equivalents/kg dry olive leaf (mmol TE/kg).

### *2.7. Analysis of Phenolic Compounds by HPLC-DAD*

The determination of oleuropein and luteolin-7-glucoside in olive leaves extracts were carried out in accordance with the COI method (COI/T.20/Doc No 29/Rev.1, 2017) for the determination of biophenols with modifications. HPLC quantification was performed on a Shimadzu Prominence UFLC chromatograph system equipped with an autosampler, a column oven, a degasser, a diode array detector, and a quaternary pump. A C18 reverse-phase column (250 mm × 4.6 mm), type BDS HYPERSIL 5 μm (Thermo Fisher Scientific Inc., USA) was used at 30 ◦C to separate the biophenols. Oleuropein and luteolin-7-glucoside were identified by comparison with their commercial standards through retention times and the UV absorption spectra in the 190–350 nm range. Quantification was carried out by integration of the peaks at 280 nm using five-point regression curves of individual stock solutions of known concentration in triplicate of external standards. The phenolic compounds contents were expressed as g polyphenol/kg dry olive leaves (g polyphenol/kg).

### **3. Results and Discussion**

### *3.1. Influence of UAE Factors on the Extraction of Bioactive Compounds from Olive Leaves*

Extraction procedure is a very important stage for the recovery and isolation of bioactive compounds from olive leaves. Although the e ffect of UAE operating conditions on the extraction yield, total phenolic and flavonoid contents, and antioxidant capacity has been studied, few works have reported the influence of extraction factors on the recovery of specific phenolic compounds from olive leaves [23,25,33] using UAE and RSM, at least from our knowledge of literature, and only one of them tackled the task of how to simultaneously optimize the recovery of some of the main phenolic compounds found in olive leaves extracts (verbascoside and luteolin-4'-O-glucoside) besides oleuropein, but without taking into account amplitude as a factor to study [39]. In order to obtain the maximum extraction yield of oleuropein and luteolin-7-glucoside, two of the key factors involved in the UAE technique such as the increment of temperature, or maximum operating temperature, and amplitude were examined in this study. CCD and RSM were used to perform the tests and interpret the experimental results and optimize the operating conditions of this technique. The experimental design is presented in Table 1. The time duration of the ultrasonic processing needed to reach the maxima temperatures was measured and can be seen in Table 2.

**Table 2.** Responses for the central composite design obtained of the extractions by UAE 1.

1Data were expressed per kilogram of dry leaf.

Table 2 also summarizes the influence of temperature and amplitude on R, TPC, TFC, DPPH, and oleuropein and luteolin-7-glucoside contents of olive leaves extracts by UAE and shows experimental values obtained all of the responses for each the design runs. The oleuropein content ranged from 44.9 g/kg to 71.6 g/kg dry leaf. As for luteolin-7-glucoside content, the quantity changed between 1.08 g/kg and 1.88 g/kg dry leaf. The highest values of oleuropein and luteolin-7-glucoside (runs 5 and 10) was in agreement with the best results of TPC, TFC, and DPPH, showing that these polyphenols can also be the main compounds responsible for the antioxidant capacity of the obtained extracts.

### Increment of Temperature and Amplitude Influence

The mathematical models based on the experimental results for each of the responses are showed in the Table 3. Analysis of variance in accordance with the p-values stated in Section 2.3 showed that the models were reliable for prediction of responses.

1 Coefficient of determination; 2 Coefficient of variation.

When setting the amplitude and the temperature increase as operating factors, the operating time is variable and is interpreted as response, so that the model in Table 3 shows the existing interrelation between the three variables. As it is deduced from the model, the time increases with ΔT and decreases with the amplitude and shows a small positive curvature with both factors. In addition, there is a negative interaction between the factors so that at the lower ends of one factor the action of the other increases. Thus, for A = −1 the coefficient of ΔT becomes +5.92 and for A = +1 its value is +1.36, which indicates that for low amplitudes the heating speed is low and for high amplitudes the speed is high (Figure 1). Likewise, for the amplitude, if ΔT is set at its value −1 the coefficient of A is −1.84, the time slowly decreases with the increase of A, and for ΔT = +1 the coefficient is −6.40, the decrease in time is considerably higher than in the previous case. The greater the amplitude, the higher the heating rate and the shorter the time needed to reach a certain temperature.

For the rest of the responses in the Table 3, both increment of temperature and amplitude were statistically significant factors. The effects of interaction between the factors did not show to be statistically significant. As it can be seen, only linear terms and quadratic effect of amplitude resulted as significant. These data are quite consistent with those reported in a previous work [23] in which the quadratic terms of the amplitude were significant for the responses TPC and TFC measured in olive mill leaves extracts. In all models, except for time, ΔT is the most important factor by several orders of magnitude. This indicates that, for these responses, it is much more important to increase temperature than to decrease amplitude, although with low amplitudes the responses are improved.

Since time and temperature are dependant on each other in UAE, optimizing the increment of temperature and/or maximum temperature makes it possible to reduce extraction time. In the case of UAE, in general, it has been reported that an increase in temperature from 20 to 70 ◦C correlates with enhancements in extraction yields of the phenolic compounds from olive leaves as well as occurs in other biomass compared to non-ultrasonicated extractions [22]. This is owing to the induction of matrix bonds ruptures, the increase of the compound solubility, solvent diffusion rate, mass transfer, and reduction in viscosity of the solvent [31]. Effectively, in this work a positive correlation was observed between all responses and the increment of temperature, which was the most influential

factor, indicated that the responses were directly proportional to the temperature reached. A similar positive effect of temperature on total polyphenols recovery and antioxidant capacity from olive leaves has been also described by other authors [3,25].

**Figure 1.** Variation of ultrasonic process duration time with increment of temperature and amplitude during processing of olive leaves by UAE.

In the ultrasonic system used in this work, the system with a coupled probe, amplitude plays an important role in the extraction since the number of compression cycles of ultrasonics depends on it. However, in this work its influence was less significant and showed a negative correlation with all responses slightly decreasing with increasing amplitude. It was observed that high amplitudes resulted in solvent agitation instead of cavitation, and as a consequence in low transmission of the ultrasounds through the liquid media.

The analysis of the simultaneous effects of the independent factors on the responses can also be seen on 3D response surface curves. Response surface plots were constructed in accordance with the equations of the predicted models (Table 3). Figure 2 presents the response surface graphs showing the combined influence of increment of temperature and amplitude on the oleuropein and luteolin-7-glucoside contents. Increment of temperature during extraction process was the most significant factor and the responses linearly increased with raising temperature, separately from amplitude.

### *3.2. Optimization of the UAE Conditions*

The extraction process was optimized by using RSM. A simultaneous numerical optimization of the experimental conditions for all of responses was carried out using the optimization option in Design Expert software to maximize the responses what generated as optimal conditions of increment of temperature and amplitude of 40 ◦C (corresponding to a maximum temperature of 60 ◦C) and 30% (minimum amplitude), respectively (Table 4).

**Figure 2.** Response surface analysis for the oleuropein and luteolin-7-glucoside contents from olive leaves extracts with ultrasound-assisted extraction describing the influence of the factors increment of temperature and amplitude. The maxima of the responses are located at 60 ◦C and 30% amplitude. (**a**) Model graph for oleuropein content; (**b**) Model graph for luteolin-7-glucoside content.

**Table 4.** Comparison of both extraction methods applied in this work.

1 Optimal values resulted from a simultaneous numerical optimization all of the responses, including time; 2 Values reported are averages. The results were expressed as means ± standard deviation of replicate extractions in triplicate.

Other researchers have also proven that the maximum oleuropein content is obtained at an extraction optimal temperature of 60 ◦C [33,39]. Using optimal conditions, the maxima response values R, TPC, TFC, DPPH, oleuropein and luteolin-7-glucoside contents, and time were theoretically calculated (Table 4). The use of temperatures over the optimum should be avoided owing to the fact they lead to solvent loss by evaporation and may also promote degradation of the compounds of interest which has been reported at higher temperatures and therefore for longer periods of time using UAE [40]. As for the other factor considered for optimization, 30% of amplitude was also selected as an optimal value by other authors [35] which indicated significant statistical differences when comparing phenolic extraction yields utilizing amplitudes in a range of 20%–50% [41]. Also, as mentioned before, it is important to indicate that high amplitudes can deteriorate the ultrasonic probe, resulting in liquid agitation instead of agitation.

### *3.3. Comparison against Soxhlet Conventional Extraction Method*

Phenolic profiles from olive leaves extracts obtained by UAE and Soxhlet are shown in Figure 3. As can be seen, the extracts did not exhibit significant differences in composition, showing similar chromatograms as for the types of compounds observed in each one, although the amounts recovered were different. Oleuropein and luteolin-7-glucoside were the most abundant phenolic and flavonoid compounds in olive leaves extracts.

**Figure 3.** Representatives HPLC chromatograms recorded at 280 nm of olive leaves extracts recovered by UAE under the following conditions: 44.1 ◦C of ΔT and amplitude 45% (**a**) and Soxhlet extraction for 4 h; (**b**) the peaks of oleuropein and luteolin-7-glucoside appear marked in the chromatograms.

Table 4 shows a comparison of the responses obtained by conventional Soxhlet extraction under the conditions described in the Section 2.2 and UAE from a simultaneous numerical optimization all of the responses obtained with an operating conditions of 44.1 ◦C of ΔT and 30% of amplitude from CCD stated in Table 1. The best results regarding R, TPC, TFC, and DPPH were reached by Soxhlet method. Based on the differences observed between UAE and Soxhlet extracts in R, TPC, and TFC, it is reasonable to think that Soxhlet extracts may contain other phenolics in higher concentrations than oleuropein and luteolin-7-glucoside but also having solubilized more impurities during the extraction. On the other hand, ultrasounds can ease swelling and hydration, resulting in an expansion in the pores of the cell wall, which enhances the diffusion action, leading to improve mass transfer. However, the second stage which is the limiting step requires additional energy, for example, stirring to quicken the extraction procedure. The ultrasonic breakdown of olive leaf matrix by ultrasonics might not be enough. In addition, in the Soxhlet method each cycle of extraction was done with pure solvent which can be another explanation of the better yield of Soxhlet over UAE [42] in these responses.

The extraction method had also a great effect on the amount of the bioactives quantified in this work. In this case, however, if the influence of extraction method on oleuropein and luteolin-7-glucoside contents in the extracts is analyzed, the data indicate that the same trend does not repeat because UAE provides significantly higher contents of both compounds compared to Soxhlet extraction. In the case of oleuropein, UAE extracts (69.91 g/kg) showed a 6.6% significantly higher content than Soxhlet extracts ones (65.57 g/kg). With regards to the luteolin-7-glucoside content, significant differences were also found between Soxhlet (1.32 g/kg) and UAE extracts (1.82 g/kg). UAE extracts exhibited a 37.9% higher content than Soxhlet ones. Long exposure to temperature for several hours can more likely result in the degradation of these compounds by Soxhlet method. These differences were observed by other researchers; for instance, Cifá et al. [23] optimized the extraction of oleuropein and reported significant increases in extraction yield of oleuropein from 6% to 84% compared to traditional maceration and therefore within the range obtained in this work. Several works show the efficiency of the UAE technique compared to conventional methods. In any case, with regards to the content of the quantified bioactive compounds and from a point of view of the purity of the extracts, this should be seen as an advantage in favor of ultrasound-assisted extraction, since in the end purer extracts in oleuropein and luteolin-7-glucoside were obtained.

Although there are many aspects that can influence the extracts composition, like the cultivar and sampling date, the oleuropein content reached in this work was similar than that reported by other authors utilizing the same extraction technique, i.e., ultrasound application. The maximum oleuropein content was 71.65 g/kg dry olive leaf versus 65 g/kg dry olive leaf determined by Ahmad-Qasem et al. [43].


**1. Introduction**

26 Will-be-set-by-IN-TECH

302 Biodiversity Loss in a Changing Planet

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Zipkin, E., Royle, J. A., Dawson, D. K. & Bates, S. (2010). Multi-species occurrence models to

interacting species, *Ecological Applications* 20: 1467–1475.

143: 479–484.

& Mazzotti, F. J. (2010). A new parameterization for estimating co-occurrence of

anuran assemblage from Louisiana, USA, *Journal of Wildlife Management* 75: in press.

evaluate the effects of conservation and management actions, *Biological Conservation*

The black pod disease due to *Phytophthora* spp is a destructive disease of cocoa. Worldwide, yield losses have been estimated to 30% (Lass, 1985). Côte d'Ivoire, the first cocoa producing country in the world, with 44% of the world market (ICCO, 2000) is also concerned by this disease. Several species of *Phytophthora* are involved in the disease. In Africa, two species, *P. palmivora* and *P. megakarya*, are the most damaging. The first species, which is the most common, causes damage in all the cocoa producing countries in the world, with yield losses between 20 to 30% ; the second, endemic to central and west Africa, is the most aggressive. This pathogen may cause the loss of the whole pod production in some countries (Flood, 2006). In Côte d'Ivoire, since the discovery of *P. megakarya* in the western region in the 90s, the black pod disease problem became more serious (Koné, 1999; Kouamé, 2006). Yield losses increased from an average of 10% to 35-40% (Kébé *et al*., 1996). Thus, the control of the disease became also a priority.

Although chemical control was developed by the research scientists, the dissemination of this method to the farmers was little successful. The low level of adoption of this technology by the farmers could be explained by the high cost of the fungicides as well as the difficulties related to the provision of water and the application of the fungicides. In addition, the requirements of the international market in terms of bean quality, environmental constrains, health issues for the consumers, and the different moratoriums in this area from the market partners (Anonyme, 2006), are numbers of constraints that do not facilitate the development of the chemical control method.

The strategy adopted in Côte d'Ivoire to control the black pod disease is based on integrated management, which is cost effective and environmentally friendly. This approach combines the use of agronomic practices, resistant cocoa varieties and natural antagonistic microorganisms of *Phytophthora*. Research works continue in order to improve agronomic practices and varietal resistance. The use of natural antagonistic microorganisms of

Isolation and Identification of Indigenous Microorganisms of Cocoa Farms in

disinfectant (Arnold, 1999; Evans, et *al*., 2003; Rubini et *al.*, 2005).

because of the gelatinous consistency of the decomposing pod husk.

26 °C for 2 days for the bacteria and 7 days for the fungi.

**2.3 Conservation and identification of the microorganisms**

**2.2 Isolation of the microorganisms**

**2.2.1 Pod endophytes**

**2.2.2 Soil microorganisms**

Côte d'Ivoire and Assessment of Their Antagonistic Effects Vis-À-Vis *Phytophthora palmivora…* 305

(Burbage, 1982), and the NYD medium (nutrient yeast dextrose) (Guizzardi and Pratella, 1996), adapted to a larger spectrum of bacteria were used. For the isolation of fungi, the TME (*Trichoderma* medium E), specific to fungi belonging to the genera *Trichoderma* and *Gliocladium* (Papavizas and Lumsden, 1982) were used. The PDA (potato dextrose agar),

The surface of the pods was beforehand washed with tap water, and then underwent a series of disinfection in ethanol at 95 % for 30 seconds, in sodium hypochlorite at 10 % for 2 minutes, and again in ethanol at 75 % for 2 minutes, in order to eliminate the microorganisms present on the husk. The pods were rinsed three times in sterile distilled water to eliminate any trace of

The sampling zone is chosen and the superficial tissues were removed using a sterile scalpel. Ten cubic shape fragments of 5 to 7 mm were taken per pod in the husk. The samples taken were put in culture on the selective media contained in Petri dishes. The incubation was made in the dark in a steam room, at 26 °C for 2 days for the bacteria and 7 days for the fungi.

The microorganisms were obtained by direct isolation from the soil according to the method described by Davet and Rouxel (1997) and from fragments of pod husk buried in the soil samples. The soil samples were beforehand dried, ground and calibrated by sieving. The fragments of husk were ground in a porcelain mortar to separate each living propagule,

In both cases, 10 g of ground soil were transferred in 90 ml of sterile distilled water contained in an Erlenmeyer. The mixture is then put in agitation for 30 minutes to obtain a good separation of the particles. To obtain a variable concentration of propagules and facilitate the enumeration of the colonies, a series of dilution was performed from the initial solution whose concentration was 10-1 (Rapilly, 1968). To obtain a solution of 10-2, 1 ml of the initial solution was mixed in 9 ml of sterile distilled water. Thus, a series of dilution from 10- 2 to 10-9 was performed in hemolytic tubes. For each dilution, 100µl was pipetted and spread onto the surface of the culture media in Petri dishes. For each dilution and for each medium, 4 Petri dishes were inoculated. The incubation was also made in the dark in a steam room at

The microorganisms isolated were first purified by two or three successive monospore transplantings on specific culture media. Once purified, each isolate was designated by a code number. For strains of bacteria, the code numbers were preceded by the letter B, followed by an order number. The nomenclature of the fungi isolates begins with one or several first letters of the name of the genus, followed by an order number. The conservation of the microorganisms was then made in a freezer at a temperature of - 80°C for the bacteria, and - 10°C for the fungi. In both cases, agar disks taken near the edge of the purified culture were transferred to 1.5 ml sterile Eppendorf microtubes containing glycerol at 50 %. The identification of the isolated microorgamisms was based on the macroscopic, microscopic, biochemical and molecular characters. The molecular characterization was made by the

adapted to a larger spectrum of fungi was used for the isolation of the other fungi.

*Phytophthora* in the control of black pod disease is a new area of investigation explored by research scientists in several cocoa producing countries. Thus, some species in the genera *Trichoderma* and *bacillus* have been described by several scientific teams as potential biological agent for the control of *Phytophthora* spp. on coca (Bong *et al*., 1996; Krauss *et al*., 2003; Mpika, 2002). On other crops, fungi and bacteria belonging to several genera including *Pseudomonas, Burkholderia*, *Streptomyces*, *Serratia*, *Penicillium*, *Geniculosporiun, Gliocladium, Aspergillus*, *Coniothyrium, Ampelomyces, Phytophthora, Botrytis, Colletotrichum, Pythium, Rhizoctonia, Fusarium, Gaeunannomyces* and *verticillium* have been described as antagonists of many fungi, pathogens of plants. Members of these genera are pathogens of plants such tomato, rice, cucumber, maize, cotton and beans (Hebber *et al*., 1998 ; Benhamoun *et al*., 2000 ; Singh *et al*., 1999 ; de Cal *et al*., 1999 ; Paulitz and Linderman, 1991 ; Gerlagh *et al*., 1999 ; Vidhyasekaran and Muthamilan, 1999 ; Bong and Stephen, 1999 ; Tondje *et al.*, 2006a,b).

During this study, the biodiversity was explored in the cocoa ecosystems. Microorganisms, potential antagonists of *Phytophthora* spp., were collected from pods and soils of cocoa farms. A collection of microorganisms was established. The antagonistic effect of these microorganisms on *Phytophthora* was assessed in the laboratory and in the field on the cacao trees.


**2. Synthesis of MCC**

The MCC can be synthesized by different processes including extrusion and enzyme-mediated process [25]. Other studies reported that it can also be synthesized by steam explosion and acid hydrolysis process [5, 6]. The acid hydrolysis process is more preferable due to shorter duration than others. It also offered the possibility to be applied as a continuous process rather than a batch-type process. Limited quantity of consumed acid is also the advantage of the process, while, despite the lower unit cost from less chemicals used, this process offered more fine particles of the MCC as the final product [5]. Fibrous plant pulp is hydrolyzed by mineral acid under heat and pressure. In the presence of water and acid, hydrolysis process breaks cellulose polymers into smaller chain polymers or microcrystals. Other celluloses, to which soluble components of cellulose such as beta and gamma celluloses, hemicelluloses, and lignin are dissolved with acid and water, are separated out during washing process by water which continued by filtration. The obtained pure alpha cellulose has then been neutralized and given the slurry final product [3]. This suspension is dried to obtain the insoluble white, odorless, tasteless powder, which has later been characterized as MCC [39]. MCC is hygroscopic in nature, and insoluble in water, but swells when in contact with water.

Another synthesis procedure of the MCC reported by Ohwoavworhua et al. [40] can be concluded as follows: the α-cellulose was hydrolyzed with hydrochloric acid at a boiling temperature of 105° for 15 min. The neutralized slurry obtained from the hydrolysis process was washed, and the fraction passing through 710 μm sieve was stored at room temperature in a desiccator. MCC is commonly dried from the slurry by spray-drying method. By varying spray-drying conditions, the degree of agglomeration and moisture content can be manipulated. In order to obtain smaller particle sizes (below 50 μm), further milling MCC can be performed [1].

**43**

*Microcrystalline Cellulose as Pharmaceutical Excipient DOI: http://dx.doi.org/10.5772/intechopen.88092*

**Type Conc. Hydrolysis condition MCC-Y**

**L/C (vol./ wt)**

*yield of microcrystalline cellulose (MCC-Y) hydrolysis reagent.*

**Particle size (micron)**

Less than 50

*Types of the commercial microcrystalline cellulose [1, 51].*

viscoelastic properties [48].

**Table 1.**

**MCC Type**

PH 101

PH 102

PH 103

PH 105

PH 112

PH 113

PH 200

PH 301

**Table 2.**

Other drying techniques may be used, which may require additional screening steps postdrying in order to control particle size distribution [41, 42]. Higher bulk density grades are also available by using specific cellulose pulps (raw material), and median particle sizes below 50 mm can be obtained by further milling MCC [1]. Several studies have compared microcrystalline cellulose with various sources, including different manufacturers and different sites [4, 43–47]. MCC produced by various manufacturers or in various manufacturing sites may have different properties due to the kinds of pulp used as raw materials and their respective manufacturing conditions [2, 4]. A number of studies have confirmed that the moisture content of MCC influences compaction properties, tensile strength, and

**Temperature (°C)** HCl 2 N 10:1 105 15 n.a [5] HCl 2 N 10:1 45 15 n.a [6] HCl 2.5 N 20:1 85 90 80 [5] HCl 2.5 N 62.5:1 105 15 19 [6] HCl 2 N 10:1 n.a 45–60 n.a [7]

*Hydrolysis reagents (acid type and concentration), liquor to cellulose ratio (L/C), hydrolysis conditions, and*

**(%)**

**Utilization**

spheronization, and in capsule filling processes

used for moisture-sensitive pharmaceutical active ingredients

It is the most compressible of the PH products owing the smallest particle size. Well known as excipient for direct compression for granular or crystalline materials. When mixed with PH-101 or PH-102, specific flow and compression characteristics will be obtained. It has applications in roller compaction

used for high moisture-sensitive pharmaceutical active ingredients

used for high moisture-sensitive pharmaceutical active ingredients

variation and to improve content uniformity in direct compression formulations and in wet granulation formulations

tablet weight uniformity. Useful for making smaller tablets and in capsule filling excipient

50 It is most widely used for direct compression tableting, for wet granulation, for

100 It is used as the PH-101, but its larger particle size improves the flow of fine powders

50 It has the same particle size as PH-101 with lower moisture content (3%), so it is

100 It has the same particle size as PH-102. It has lower moisture content (1.5%). It is

50 It has the same particle size as PH-101. It has lower moisture content (1.5%). It is

180 It has a large particle size with increased flowability. It is used to reduce weight

50 It has the same particle size as PH-101 but is denser providing more flowability and

**Duration (minute)**

**References**

*Microcrystalline Cellulose as Pharmaceutical Excipient DOI: http://dx.doi.org/10.5772/intechopen.88092*

Other drying techniques may be used, which may require additional screening steps postdrying in order to control particle size distribution [41, 42]. Higher bulk density grades are also available by using specific cellulose pulps (raw material), and median particle sizes below 50 mm can be obtained by further milling MCC [1].

Several studies have compared microcrystalline cellulose with various sources, including different manufacturers and different sites [4, 43–47]. MCC produced by various manufacturers or in various manufacturing sites may have different properties due to the kinds of pulp used as raw materials and their respective manufacturing conditions [2, 4]. A number of studies have confirmed that the moisture content of MCC influences compaction properties, tensile strength, and viscoelastic properties [48].

#### **Table 1.**

*Pharmaceutical Formulation Design - Recent Practices*

ing in shorter and more crystalline fragments.

**2. Synthesis of MCC**

to remove impurities including lignin, pectin, and wax [36].

but also in structural organization, i.e., regions which are relatively more crystalline or amorphous. The amorphous regions are more prone to hydrolysis by acid result-

Non-woody lignocellulosic materials have also been developed as source of MCC such as cotton linters [5], cotton stalks [6], cotton rags [5], cotton fabric waste [7], cotton wool [8], soybean husk [9], corn cob [10], water hyacinth [11], coconut shells [12], oil palm biomass residue [13, 14], oil palm fronds [15], rice husk [6, 16], sugar cane bagasse [6, 16–20], jute [21, 22], ramie [23], fibers and straw of flax [24], wheat straw [25], sorghum stalks [26], sisal fibers [27] and mangosteen [28], alfa grass fibers [29, 30], soybean hulls [31], orange mesocarp [32], Indian bamboo [33], roselle fiber [34], and alfa fiber [35]. Seed flosses from milkweed pods (*Calotropis procera*), shrubs, and kapok (*Ceiba pentandra*) trees are also known as cellulosic resources. Due to its high purity of alpha cellulose, most seed flosses must be treated

Wooden sources contain cellulose chains which are packed as layers held by cross-linking hydrogen bonds [37]. Chemically it consists of polymeric matrix of lignin, hemicelluloses, and pectin [38]. Different woods considerably possessed different chemical composition of cellulose (including allocations of cellulose, hemicelluloses, and lignin in cell wall) and structural organization as well. Relatively different crystallinity in particular regions is observed as more amorphous according to softwoods (evergreen conifer) and hardwoods which are termed as deciduous broadleaf [4, 37]. The amorphous regions of cellulose provide a more susceptible property for depolymerization by acid hydrolysis. At optimum acid concentration, the process gave shorter and more crystalline fragments such as the MCC [2, 37].

The MCC can be synthesized by different processes including extrusion and enzyme-mediated process [25]. Other studies reported that it can also be synthesized by steam explosion and acid hydrolysis process [5, 6]. The acid hydrolysis process is more preferable due to shorter duration than others. It also offered the possibility to be applied as a continuous process rather than a batch-type process. Limited quantity of consumed acid is also the advantage of the process, while, despite the lower unit cost from less chemicals used, this process offered more fine particles of the MCC as the final product [5]. Fibrous plant pulp is hydrolyzed by mineral acid under heat and pressure. In the presence of water and acid, hydrolysis process breaks cellulose polymers into smaller chain polymers or microcrystals. Other celluloses, to which soluble components of cellulose such as beta and gamma celluloses, hemicelluloses, and lignin are dissolved with acid and water, are separated out during washing process by water which continued by filtration. The obtained pure alpha cellulose has then been neutralized and given the slurry final product [3]. This suspension is dried to obtain the insoluble white, odorless, tasteless powder, which has later been characterized as MCC [39]. MCC is hygroscopic in

nature, and insoluble in water, but swells when in contact with water.

particle sizes (below 50 μm), further milling MCC can be performed [1].

Another synthesis procedure of the MCC reported by Ohwoavworhua et al. [40] can be concluded as follows: the α-cellulose was hydrolyzed with hydrochloric acid at a boiling temperature of 105° for 15 min. The neutralized slurry obtained from the hydrolysis process was washed, and the fraction passing through 710 μm sieve was stored at room temperature in a desiccator. MCC is commonly dried from the slurry by spray-drying method. By varying spray-drying conditions, the degree of agglomeration and moisture content can be manipulated. In order to obtain smaller

**42**

*Hydrolysis reagents (acid type and concentration), liquor to cellulose ratio (L/C), hydrolysis conditions, and yield of microcrystalline cellulose (MCC-Y) hydrolysis reagent.*

**Table 2.** *Types of the commercial microcrystalline cellulose [1, 51].*

It was generally recognized that batch-to-batch variability from a sole manufacturing site was less important than differences observed between multiple sources. Only a few studies have tried to correlate the manufacturing conditions of microcrystalline cellulose with its physicochemical properties and its performance in tableting applications [2, 49, 50]. The effect of some parameters on hydrolysis process on yield value of production is shown in **Tables 1** and **2**.


**2. Normal physiology**

Normal sinus rhythm is when the heart beats in an orderly predetermined sequence. The atria contract initially in response to the firing of an impulse by the Sino-Atrial (SA) node located at the junction of the superior vena cava and the right atrium. The SA node contains specialized tissue with 'pacemaker cells', which can initiate repetitive rhythmic action potentials. These potentials then travel via internodal atrial pathways to the AtrioVentricular (AV) node located at the right posterior portion of the interatrial septum. The AV node slows conduction into the bundle of HIS which then leads to its right and left branches. The left bundle branch further divides into anterior and posterior fascicles. The final pathway of conduction is the Purkinje system, which consists of a network of fibers that transmit the electrical impulse to the myocardium near the apex of the heart. (1)

The Electrocardiogram (ECG) is a reliable and practical way to document the underlying cardiac rhythm. It essentially consists of a recording obtained by 12 surface leads which trace the electrical activity of the heart from different directions. The 12 leads include 6 limb and 6 precordial leads. The limb leads include 3 bipolar leads (I, II, III) meaning they have 2 electrodes of opposite polarity. The other limb leads are aVR, aVL, aVF which are the unipolar leads meaning they have only one electrode connecting to a central terminal. The precordial leads are all unipolar and include V1-V6.

The limb leads are the frontal plane leads representing electrical current along the coronal plane of the heart, i.e. right/left and superior/inferior. The precordial leads represent the horizontal plane of the heart measuring transverse currents, i.e. right/left and anterior/posterior. Lead I traces currents from right shoulder to left shoulder, lead II from

Post Operative Arrhythmias 243

Step 4. Identify the P waves – upright in lead II and III and negative in aVR usually

The above steps should help one to identify the salient features of any rhythm and place it in one of the following mentioned categories. (Figure 1) We would like to point out that this scheme is only one of many and sometimes more than one arrhythmia can be present in a patient. This scheme also, at times over simplifies natural heart rhythms. For example, a heart rate of 40 beats per minute (bpm) during sleep or in an athletic patient can be normal, while patients with an abnormal conduction system can have supraventricular arrhythmias

identifies sinus rhythm, P waves are absent in atrial fibrillation, saw tooth

Step 1. Determine the ventricular rate – tachycardia is >100 / Bradycardia is < 60 Step 2. Measure the QRS complex – Narrow is < 0.12ms / Broad is > 0.12 ms Step 3. Determine the regularity of the QRS complex – Regular/irregular

appearance at an atrial rate of 300 bpm may indicate atrial flutter

 │¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯│ │¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯│ Regular Irregular Regular Irregular ↓ ↓ ↓ ↓ Sinus tach AF VT VF A Flutter\*\* MAT SVT \* AF \*

(AF – Atrial Fibrillation, AT – Atrial tachycardia, MAT – Multifocal atria tachycardia, SVT – Supra

ventricular tachycardia, VF – Ventricular Fibrillation, VT – Ventricular tachycardia)

The Brugada criteria can be used to identify any broad complex tachycardia (4)

Step 4. Is there any typical bundle branch morphology in leads V1 or V6?

\*\* Flutter can be irregular occasionally when the AV block is variable Fig. 1. Various cardiac rhythm disturbances noted in clinical practice

Step 1. Are there RS complexes in any of the chest leads? Step 2. Is the onset of the R wave to the nadir of S > 100 ms?

Step 5: Measure the PR interval – helps identify AV delay

with heart rates less than 100 bpm.

 ↓ ↓ Sinus Brady Heart blocks Sinus pause Atrial Fibrillation

 AVRT AVNRT AT

Step 3. Is there any AV dissociation?

\*With Aberrancy

Rate

 <60 │ >100 │¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯│ Bradycardia Tachycardia │ <0.12ms │ >0.12ms │¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯ │ │¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯│ Regular Irregular Narrow Broad

right shoulder to left leg, and lead III from left arm to left leg. Lead aVF traces from central terminal, which corresponds to zero potential to the left leg, aVL from centre to left arm and aVR from centre to right arm. The precordial leads work in a similar fashion in that leads V1 – V6 trace their axis center out from right to left respectively, so that V1 represents right of the interventricular septum, V2 and V3 the interventricular septum (anterior wall), V4 the apex (anterolateral), V5 and V6 the lateral ventricular wall. Any current flowing towards the lead causes a positive deflection and current flowing away from the lead causes a negative deflection and vice versa. The strength of the deflection depends on the amount of potential recorded and is affected by cardiac and extracardiac structures. To understand the electrophysiological basis of the 12-lead tracings on an ECG is important, because it gives clues about the origin of an arrhythmia and sometimes guides their therapies.

The first deflection on an ECG is the P wave which represents atrial depolarization. In sinus rhythm without any discernable atrial pathology, P wave is an upright, smooth, rounded wave with relatively low voltage. The PR interval consists of the P wave and the normally isoelectric segment up to the initial deflection of the QRS complex. The PR interval represents the conduction through atria, AV node, bundle branches and Purkinje system. The QRS complex follows the PR segment. The initial negative deflection is the Q wave, a positive deflection which can occur either initially or after the Q is the R wave while any negative deflection which follows the R is the S wave. QRS complex represents intraventricular conduction and depolarization. The J point represents the junction between QRS and the ST segment. The ST segment corresponds with the end of ventricular depolarization and start of the ventricular repolarization. The T wave, which follows the ST segment, represents ventricular repolarization. As such the QT interval represents the complete ventricular depolarization and repolarization period. Occasionally a small hump-like U wave follows the T wave, and is felt to be due to repolarization of the purkinje system. (2,3)

The bedside monitors which are routinely used for continuous cardiac monitoring are typically wireless, i.e. telemetry systems. These can be either 5 lead wire or 3 lead wire systems. The 5 lead wire system allows for monitoring all of the limb leads or the precordial leads while the 3 lead wire system allows monitoring one lead at a time, usually lead II, because the P wave is best visible in this lead. Depending on the monitoring system available it is essential for health care providers to be able to recognize the cardiac rhythm changes based only on a few select leads seen on the monitor as there may be no time to record a 12 lead ECG.

### **3. Recognition of arrhythmias**

Postoperative arrhythmias though transient are usually sudden in onset. It is essential to recognize a rhythm disturbance and institute treatment as quickly as possible in most cases. A 12 lead ECG is recommended but may be impractical if the rhythm disturbance is an immediate threat to the patient's life. The wave forms visible on the telemonitor or a rhythm strip in one lead tracing may be the only available clue.

It is worthwhile to note some salient points early. Is the patient stable as assessed by the blood pressure, oxygen saturation or mental status? If deemed unstable then more aggressive steps are warranted.

A rapid and accurate interpretation of the ECG can be tricky and readers are advised to develop a personal strategy to identify any given cardiac tracing so that a quick diagnosis can be made. One approach is to identify and describe 5 basic features of the electrocardiogram (2):

right shoulder to left leg, and lead III from left arm to left leg. Lead aVF traces from central terminal, which corresponds to zero potential to the left leg, aVL from centre to left arm and aVR from centre to right arm. The precordial leads work in a similar fashion in that leads V1 – V6 trace their axis center out from right to left respectively, so that V1 represents right of the interventricular septum, V2 and V3 the interventricular septum (anterior wall), V4 the apex (anterolateral), V5 and V6 the lateral ventricular wall. Any current flowing towards the lead causes a positive deflection and current flowing away from the lead causes a negative deflection and vice versa. The strength of the deflection depends on the amount of potential recorded and is affected by cardiac and extracardiac structures. To understand the electrophysiological basis of the 12-lead tracings on an ECG is important, because it gives

The first deflection on an ECG is the P wave which represents atrial depolarization. In sinus rhythm without any discernable atrial pathology, P wave is an upright, smooth, rounded wave with relatively low voltage. The PR interval consists of the P wave and the normally isoelectric segment up to the initial deflection of the QRS complex. The PR interval represents the conduction through atria, AV node, bundle branches and Purkinje system. The QRS complex follows the PR segment. The initial negative deflection is the Q wave, a positive deflection which can occur either initially or after the Q is the R wave while any negative deflection which follows the R is the S wave. QRS complex represents intraventricular conduction and depolarization. The J point represents the junction between QRS and the ST segment. The ST segment corresponds with the end of ventricular depolarization and start of the ventricular repolarization. The T wave, which follows the ST segment, represents ventricular repolarization. As such the QT interval represents the complete ventricular depolarization and repolarization period. Occasionally a small hump-like U wave follows the

The bedside monitors which are routinely used for continuous cardiac monitoring are typically wireless, i.e. telemetry systems. These can be either 5 lead wire or 3 lead wire systems. The 5 lead wire system allows for monitoring all of the limb leads or the precordial leads while the 3 lead wire system allows monitoring one lead at a time, usually lead II, because the P wave is best visible in this lead. Depending on the monitoring system available it is essential for health care providers to be able to recognize the cardiac rhythm changes based only on a few select leads seen on the monitor as there may be no time to

Postoperative arrhythmias though transient are usually sudden in onset. It is essential to recognize a rhythm disturbance and institute treatment as quickly as possible in most cases. A 12 lead ECG is recommended but may be impractical if the rhythm disturbance is an immediate threat to the patient's life. The wave forms visible on the telemonitor or a rhythm

It is worthwhile to note some salient points early. Is the patient stable as assessed by the blood pressure, oxygen saturation or mental status? If deemed unstable then more

A rapid and accurate interpretation of the ECG can be tricky and readers are advised to develop a personal strategy to identify any given cardiac tracing so that a quick diagnosis can be made. One approach is to identify and describe 5 basic features of the

clues about the origin of an arrhythmia and sometimes guides their therapies.

T wave, and is felt to be due to repolarization of the purkinje system. (2,3)

record a 12 lead ECG.

**3. Recognition of arrhythmias**

aggressive steps are warranted.

electrocardiogram (2):

strip in one lead tracing may be the only available clue.

The above steps should help one to identify the salient features of any rhythm and place it in one of the following mentioned categories. (Figure 1) We would like to point out that this scheme is only one of many and sometimes more than one arrhythmia can be present in a patient. This scheme also, at times over simplifies natural heart rhythms. For example, a heart rate of 40 beats per minute (bpm) during sleep or in an athletic patient can be normal, while patients with an abnormal conduction system can have supraventricular arrhythmias with heart rates less than 100 bpm.

(AF – Atrial Fibrillation, AT – Atrial tachycardia, MAT – Multifocal atria tachycardia, SVT – Supra ventricular tachycardia, VF – Ventricular Fibrillation, VT – Ventricular tachycardia)

\*With Aberrancy

\*\* Flutter can be irregular occasionally when the AV block is variable

Fig. 1. Various cardiac rhythm disturbances noted in clinical practice

The Brugada criteria can be used to identify any broad complex tachycardia (4)

Post Operative Arrhythmias 245

definition for postoperative AF includes the need of medical treatment or electrical cardioversion (14) or confirmation on a 12 lead ECG (13) altering the incidence rates further. The peak incidence of postoperative AF (POAF) has been consistently described on day 2 and 3 after surgery. (10) It usually is transient with 80% of the patients converting to sinus rhythm within 24 hours. The recurrence rate has been quoted to be as much as 50% but still only 10% of patients are still in AF at 6 weeks post operation. (13) Late Postoperative AF is less frequent but was seen in nearly 5% of the postoperative patients after discharge from the hospital. This was found when patients were being followed for cardiac rehabilitation and documented in the ISYDE and ICAROS registries in Italy. (15) Despite better coordinated postoperative care and advances in cardiothoracic surgical and anesthetic practices, the incidence of AF seems stable with no reduction over the last 2

AF is generally due to reentry of multiple wavelets circling the atria. It is likely that a preexisting substrate is needed to allow peri-operative triggers to initiate AF. It is thus a specific

Age is the most consistent risk factor seen in past studies. Advancing age increases the risk with each decade. The incidence of POAF is around 6% when less than 40y of age, 18% in less than 60y olds and increasing to as much as 50% in patients older than 80 years. (13,14,16) Other risk factors include male gender, history of prior AF, heart valve disease (especially if the mitral valve is affected), prior cardiac surgery, prior cardiac structural changes like increased left atrial size and left ventricular hypertrophy. Preexisting medical conditions like obesity, chronic lung disease, peripheral vascular disease, hypertension, prior stroke are associated with increased incidence of POAF. However certain other morbid factors like preexisting diabetes, chronic kidney disease, hyperlipidemia, smoking have not been shown to be individual risk factors for POAF in some studies. (17) Pericarditis which is usually a consequence of the cardiac surgery itself is mechanistically involved. Other unique factors such as preoperative use of Digoxin or Dopamine, raised Brain natriuretic peptide (BNP) and right-sided coronary artery disease have been associated as well. ECG features like increased P wave duration of more than 140 ms, which is suggestive of atrial conduction delay can increase the susceptibility to AF. (18) Withdrawal of preoperative Angiotensin converting enzyme inhibitor (ACE I) or Beta blocker therapy is also contributory if not

Certain operative features like aortic cross clamping, pulmonary venting, bicaval venous cannulation, increased length of cardiopulmonary bypass time and mitral valve surgery can increase the propensity for POAF. It has also been noted that at times cardioplegia via coronary sinus does not stun the atria completely and may be associated with occurrence of POAF(19) Direct cardiac injury due to operative techniques causing inflammation is

The postoperative period is a critical stage as the body is yet to recover from the operative stress completely. Many proarrhythmic features such as pericardial inflammation, acute blood pressure or volume changes, acute cardiac ischemia, electrolyte imbalances,

interaction of preexisting and perioperative risk factors which can lead to AF.

decades.

**Preexisting factors:**

**Pathophysiology of Postoperative AF:**

immediately initiated after the surgery. (13)

perceived as plausible cause as well.

**Intraoperative factors:**

**Postoperative features:**

If the answer to any of questions 1-3 is yes or to question 4 is no, the rhythm is probably ventricular tachycardia. There are several additional ways to distinguish between supra ventricular and ventricular arrhythmias, which exceed the objectives of this review.


*Edited by Rıza Hakan Erbay*

Knowledge of intensive care medicine is growing logarithmically. In addition, intensive care units and their number of beds are increasing in hospitals. Nowadays, the average human life is prolonged due to the growth of medical knowledge and skills. Intensive care units, both as a cause and as a consequence of long-life expectancy, are increasingly important. This book contains current topics on intensive care such as critical care for neonatal, neurological, and cardiological patients; fluid management in these patients; and intensive care infections. We wish the readers find this book to be helpful.

ISBN 978-1-78923-708-5

Current Topics in Intensive Care Medicine

Published in London, UK © 2018 IntechOpen © mkToy / iStock