Chung-Hung Chan
email: rykenz87@yahoo.com

Assessment of various pretreatment and extraction methods for the extraction of bioactive compounds from Orthosiphon stamineus leaf via microstructures analysis by

Keywords: bioactive compounds, microstructure, extraction systems, ultrasonic-assisted extraction, microwave-assisted extraction

The impacts of various methods such as mechanical grinding, ultrasonic-assisted extraction (UAE), microwave-assisted extraction (MAE), and also sample pretreatments using acid and alkali on the microstructure of plant sample were studied for the extraction of bioactive compounds from Orthosiphon stamineus leaf. From scanning electron microscopy (SEM) analysis of the extracted sample, UAE and MAE induced significant disruption on glandular trichomes structure, which is the main site for biosynthesis of plant secondary metabolites. This improves the diffusion of bioactive compound and resulted in approximately 86-95% of the total extraction yield quantified by conventional Soxhlet extraction. Chemical pretreatments generally imparted weaker microstructures disruption thus slight improvement on the extraction yields was observed. In this case, acid reagent is more suitable for the pretreatment as the presence of alkali decomposes the bioactive compounds. In a nutshell, the performance of an extraction strongly depends on its degree of disruption on the plant sample microstructure.

INTRODUCTION

Orthosiphon stamineus or Java tea is a traditional herbal plant that can be found in Asia Pacific mainly Malaysia and Indonesia [START_REF] Akowuah | Sinensetin, eupatorin, 3′hydroxy-5, 6, 7, 4′-tetramethoxyflavone and rosmarinic acid contents and antioxidative effect of Orthosiphon stamineus from Malaysia[END_REF]. It contains bioactive compounds such as sinensetin, 3'hydroxy-5,6,7,4'-tetramethoxyflavone, rosmarinic acid and eupatorin and has been traditionally used to treat kidney problems, gout, and diabetes [START_REF] Chin | Toxicity study of Orthosiphon stamineus benth (misai kucing) on Sprague dawley rats[END_REF]. Natural products of this plant are available in the form of herbal tea product and concentrated extract in the market [START_REF] Chin | Toxicity study of Orthosiphon stamineus benth (misai kucing) on Sprague dawley rats[END_REF][START_REF] Yam | Evaluation of the anti-pyretic potential of Orthosiphon stamineus Benth standardized extract[END_REF]. They are conventionally manufactured through series of processes involving drying, grinding and extraction. The fresh leaves from plantation are usually sun-dried to remove the moisture, followed by grinding to break the plant cells for the ease of elution of bioactive compounds in herbal tea processing. Other method such as extraction is relevant in the production of concentrated extract as it extracts the bioactive compounds from the intact cells after grinding process. The conventional extraction process often subjected to a long heating process which causes the degradation of bio-active compounds and energy wastage. Thus, it is important to look into other methods to enhance the extraction yield of natural product effectively.

The methods to be considered are those can impart structural disruption to biomass sample in order to facilitate efficient mass transfer of bioactive anti-oxidant compounds during extraction process.

For instance, grinding of plant sample creates more broken cells, which facilitates the elution of bioactive compounds to extraction solvent. The limitation is that breaking the plant particle to very fine sizes requires huge energy consumption and complicated machinery. Other advanced methods such as ultrasonic-assisted extraction (UAE) and microwave-assisted extraction (MAE) can also improve the extraction efficiency. Ultrasonic treatment creates micro-pores on the structure of plant sample through cavitation [START_REF] Floros | Acoustically assisted diffusion through membranes and biomaterials[END_REF][START_REF] Juárez | New high intensity ultrasonic technology for food dehydration[END_REF]. It has been applied in the extraction of bioactive compounds from tobacco seeds [START_REF] Stanisavljević | Ultrasonic extraction of oil from tobacco (Nicotiana tabacum L.) seeds[END_REF], pomegranate peel [START_REF] Pan | Continuous and pulsed ultrasoundassisted extractions of antioxidants from pomegranate peel[END_REF] and tomatoes [START_REF] Lianfu | Optimization and comparison of ultrasound/microwave assisted extraction (UMAE) and ultrasonic assisted extraction (UAE) of lycopene from tomatoes[END_REF]. On the other hand, microwave treatment provides localised heating which increases the internal pressure of the cells and subsequently ruptures them [START_REF] Zhou | Microwave-assisted extraction of solanesol from tobacco leaves[END_REF]. Its employment can be seen in the extraction of bioactive compounds from cocoa leave [START_REF] Chan | Optimization of microwave-assisted extraction based on absorbed microwave power and energy[END_REF], green tea [START_REF] Pan | Microwave-assisted extraction of tea polyphenols and tea caffeine from green tea leaves[END_REF], and Morinda citrifolia [START_REF] Hemwimon | Microwave-assisted extraction of antioxidative anthraquinones from roots of Morinda citrifolia[END_REF]. Sample pretreatments with acid and alkali are another method of improving the extraction yield of biomass.

This method is able to break down crystalline cellulose of biomass as observed in the cases of rice husk [START_REF] Ang | Elucidation of the effect of ionic liquid pretreatment on rice husk via structural analyses[END_REF], sugar cane [START_REF] Yoon | Regression analysis on ionic liquid pretreatment of sugarcane bagasse and assessment of structural changes[END_REF], and switch grasses [START_REF] Dien | Chemical composition and response to dilute-acid pretreatment and enzymatic saccharification of alfalfa, reed canarygrass, and switchgrass[END_REF], which may improve solvent penetration into plant sample during the extraction process. Despite the methods above demonstrated promising extraction performance, their performances relative to one another and their mechanisms are not well understood. Therefore, this work assessed the impacts of the pretreatment and the extraction methods on plant microstructure and their resulted effects on the extraction yields. The extraction and pretreatment methods in this work can be incorporated into the existing herbal tea processing to enhance the release of therapeutic bioactive compounds in the product.

In this study, the methods, i.e. grinding for particle size reduction, UAE, MAE and chemical pretreatments with acid and alkali were applied to extract bioactive compounds from Orthosiphon stamineus leaves. To assess the methods, the improvements on the extraction yield contributed by UAE, MAE and chemical pretreatments were determined and compared with conventional Soxhlet extraction, after excluding the contribution due to the particle size of the plant sample. The impacts on plant microstructure by these methods were observed and their acting mechanisms were discussed.

MATERIALS AND METHODS

Materials and reagents

The standards of the investigated bioactive compounds namely eupatorin (EUP), 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (TMF) and sinensetin (SEN) were purchased from Sigma-Aldrich (USA). The standards were dissolved in chromatography grade absolute ethanol (Merck, Germany) and diluted to desired concentration prior to use in high performance liquid chromatography (HPLC) analysis. The pretreatment reagents, 0.1 M hydrochloric acid and sodium hydroxide solutions, were prepared from concentrated hydrochloric acid (Merck, Germany) and sodium hydroxide pellet (Merck, Germany), respectively. The denatured ethanol used in the extraction was obtained from R&M chemicals (Malaysia).

Pretreatment procedure

Fresh leaf of Orthosiphon stamineus was collected from local plantation in Negeri Sembilan, Malaysia. The leaves were washed and dried in a forced convection oven at 45 o C overnight until moisture content achieved below 15 %. After that, the dried sample was grinded and sieved to various particle sizes: size A (>500 μm), size B (250-500 μm), size C (180-250 μm) and size D (<180 μm). The grounded sample was then stored in an air-tight container at 4℃ until further use.

Extraction procedure

To determine the extraction yields due to grinding of plant sample, plant sample with particle size A was rinsed with ethanol at solvent-sample ratio of 100 ml/g. The ethanol extract was then filtered through 0.2 mm RC (Regenerated Cellulose) syringe filter before HPLC analysis. The experiment was repeated with plant sample with particle size B, C and D to investigate the effect of particle size reduction on the extraction yields.

The effects of various methods namely UAE, MAE, sample pretreatments with acid and alkali and

Soxhlet extraction were investigated based on plant sample sized B. These methods are conducted at their typical and suboptimal operating conditions. Note that their treatment time and solventsample ratios have been optimized in the previous work to prevent mass transfer limitation. Their extraction procedures are elucidated as follows: UAE was performed using sonicator (Qsonica Q500, 20 kHz). The sample of 2 g was mixed with 70% (v/v) aqueous ethanol at solvent-sample ratio of 30 ml/g in a container. The mixture was sonicated at 300 W for 30 min under intermittency ratio, α of 4/5 in which α is defined as the fraction of a cycle when sonication was on, i.e. α=τon/(τon+τoff) where τon and τoff are the times in seconds when the sonication was turned on and off, respectively. This intermittency ratio is to prevent overheating of the sonotrode. Upon irradiation, the extract was filtered using fine cloth and RC syringe filter before HPLC analysis. MAE was performed using domestic microwave oven (Samsung MW718). The sample at 2 g was mixed with 70% (v/v) aqueous ethanol at solvent-sample ratio of 50 ml/g in a closed Duran bottle.

The mixture was irradiated at microwave power of 150 W for 20 min. The subsequent analysis procedure was similar to the UAE as previously described.

Sample pretreatments using acid and alkali were performed using reagents, i.e. 0.1M hydrochloric acid (HCl, pH 1) and 0.01M sodium hydroxide (NaOH, pH 12), respectively. The sample was soaked with the selected reagent in a closed Duran bottle for not more than 24 hours. After the pretreatment, the sample was mixed with pure ethanol at solvent-sample ratio of 100 ml/g and the mixture was stirred for about a minute. The mixture was subjected to HPLC analysis using the similar procedure as in UAE.

Soxhlet extraction was conducted using 2 g of sample and 200 ml of ethanol for 6 hours. The subsequent procedure for HPLC analysis is similar as described in UAE.

HPLC-MS analysis

The bioactive compounds were quantified using Agilent 1200 Series HPLC system (USA) equipped with Agilent ZORBAX Eclipse Plus C18 column, 5 μm (4.6 mm x 250 mm). There are several methods available for separating the bioactive compound from Orthosiphon stamineus [START_REF] Yam | A simple isocratic HPLC method for the simultaneous determination of sinensetin, eupatorin, and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone in Orthosiphon stamineus extracts[END_REF][START_REF] Yam | HPLC and anti-inflammatory studies of the flavonoid rich chloroform extract fraction of Orthosiphon stamineus leaves[END_REF][START_REF] Akowuah | HPLC-TOF/MS profile and nitric oxide scavenging activity of Orthosiphon stamineus leaf extracts[END_REF]. However, the following general HPLC method [START_REF] Bonaccorsi | Flavonol glucosides in Allium species: A comparative study by means of HPLC-DAD-ESI-MS-MS[END_REF] was employed as it does not involve the use of buffer solution and it can separate multi flavonoid compounds from a raw extract. The

Scanning electron microscopy (SEM) analysis

The microstructure changes of the plant samples before and after each extraction methods were examined with Quanta™ 200 FESEM scanning electron microscope (FEI, USA) operated at 10 kV accelerating voltage under low vacuum mode. The surface morphology of the extracted plant samples were compared to the raw dry plant sample.

RESULTS AND DISCUSSION

Enhancement of extraction yield by various methods

The effect of particle size reduction on the extraction yields were illustrated in Fig. 1. As expected, more bioactive compounds can be extracted from plant sample with smaller particle sizes due to broken cells. This effect is amplified when the particle size of the plant sample is reduced below the leaf thickness (average value of 240 μm) of the plant sample, as indicated by size D (<180 μm).

At this condition, the extraction yield is comparable to exhaustive Soxhlet extraction yield.

Nevertheless, excessive grinding of plant particles to achieve powder-like sizes is not feasible as only small portion of the plant samples can be converted into the powder form, not to mention the process requires high energy consumption and long duration. The performances of other extraction methods in Fig. 1 were evaluated relative to the intrinsic yield contributed by the particle size (B). UAE and MAE drastically improve the total extraction yields using shorter treatment time. Their extraction results are about 95% and 86% of the total extraction yields by Soxhlet extraction. The yields of the compounds in this study were about the same with the yield quantified by other conventional technique [START_REF] Akowuah | Sinensetin, eupatorin, 3′hydroxy-5, 6, 7, 4′-tetramethoxyflavone and rosmarinic acid contents and antioxidative effect of Orthosiphon stamineus from Malaysia[END_REF], which is in the range of 0.75-4.72 mg/g of dried mass sample. In view of the sample pretreatment methods, acid reagent is suitable to be used in enhancing the extraction yields compared to alkali, as the latter degraded 5% of the intrinsic yield (after soaking for 1 h). This suggests that the bioactive compounds from the plant might be denatured, degraded or undergone other chemical changes under basic environment.

As a whole, chemical pretreatments is less effective compared to UAE and MAE. To further evaluate the methods above, their impacts on microstructure of plant sample is studied and their acting mechanism is discussed in the next section.

Microstructure analysis of plant sample

The SEM micrographs of the raw dry leaf in Fig. 2a and2b show that the leaf has different upper and lower surfaces. The severe corrugation observed on the leaf surfaces was attributed to heat drying during sample preparation. Throughout the upper surface of the leaf, there are numerous interspersed gland-like structures known as glandular trichome. Glandular trichome consists of a stalk that supports a secretory head, and this is the main site of flavonoids biosynthesis in plant [START_REF] Turner | Distribution of peltate glandular trichomes on developing leaves of peppermint[END_REF]. These gland-like structures are also present in many of the herbal plants, such as thyme, peppermint and basil [START_REF] Turner | Distribution of peltate glandular trichomes on developing leaves of peppermint[END_REF][START_REF] De Santana | Rupture of glandular trichomes in Ocimum gratissimum leaves influences the content of essential oil during the drying method[END_REF][START_REF] Marin | Morphology, distribution, and histochemistry of trichomes of Thymus lykae Degen & Jav.(Lamiaceae)[END_REF]. Under the heat drying condition of sample preparation, the glandular trichomes on the raw dry leaf sample was deflated as shown in Fig. 2(a). Unlike the upper surface, the lower surface of the leaf has scarcer number of glandular trichomes. Besides, it also has numerous stomata openings throughout its lower surface. The orderly arrangement of the upper and lower epidermal surfaces demonstrate that the cell integrity of the raw dry sample remained intact. The intact epidermises ensure minimal loss of bioactive compounds from the leaf sample. 2c and2d. Due to the severe corrugation of the leaf surfaces after the extraction, stomata and glandular trichomes were hardly visible at the lower surface. Despite the fact that the leaf samples after Soxhlet extraction remain almost intact or slightly damaged or ruptured [START_REF] Flórez | Microwave assisted water extraction of plant compounds[END_REF], the extraction is able to extract the bioactive compounds from plant sample completely over a duration of time. This is because the Soxhlet apparatus enables repeated event of diffusion of ethanol into leaf cells, which solubilizes the anti-oxidant compounds and transfers of solubilized compounds into the bulk solvent over a duration [START_REF] Bhatt | An investigation of the glandular and non-glandular foliar trichomes of Orthosiphon labiatus NE Br.[Lamiaceae][END_REF][START_REF] Jyothi | Optimization of microwave assisted extraction of withanolides from roots of ashwagandha and its comparison with conventional extraction method[END_REF].

UAE is fast and efficient extraction method, where it disrupted the integrity of the leaf surfaces by causing shrinking of epidermis (Fig. 3a and3b). Furthermore, most of the glandular trichomes were visibly deflated and many were ruptured. The ultrasonic waves propagate into the liquid, results in alternating high-pressure (compression) and low-pressure (rarefraction) cycles. In the rarefraction half-cycle of ultrasonic wave, vast amount of small vacuum bubbles were generated by the high intensity ultrasonic waves [START_REF] Awad | High-power ultrasound in surface cleaning and decontamination[END_REF]. This event was followed by cavitation that is the implosion of small bubbles on the surfaces of the sample during the compression half-cycle of the wave. The imploding of bubbles was accompanied by generation of high local temperature, which was evidenced by the increase in liquid temperature during ultrasonication in this study. Cavitation also results in the formation of high pressure shock wave and generation of powerful liquid jet that is expelled at the leaf surface [START_REF] Stephanis | The process (mechanism) of erosion of soluble brittle materials caused by cavitation[END_REF]. The occurrence of these events led to the formation of micropores as observed from the SEM micrograph. Furthermore, micro-streams and micro-turbulent eddies formed during bubble implosion cause micro-stirring in the solvent [START_REF] Sajjadi | Mechanistic analysis of cavitation assisted transesterification on biodiesel characteristics[END_REF] which enhanced the mass transfer of solvent and release of plant cell materials. 3c and3d). It has been reported and reviewed that microwave irradiation of plant samples leads to intense structural destruction, shrinking of plant part and irregularities in the plant surface [START_REF] Flórez | Microwave assisted water extraction of plant compounds[END_REF]. Penetration of microwave heated up the solvent and plant cells by both ionic conduction and dipole rotation mechanisms [START_REF] Eskilsson | Analytical-scale microwave-assisted extraction[END_REF]. The localised heat generated within the cells as well as heat of convection from the surrounding solvent resulted in liquid vaporisation and pressure build up within the plant cells, which in turn ruptured the cells and led to the formation of micropores. The structural disruptions facilitated the permeation of solvent into plant cells and enhanced the extraction of anti-oxidant compounds from cells to bulk solvent [START_REF] Flórez | Microwave assisted water extraction of plant compounds[END_REF]. The better efficiency of MAE is attributed to its more efficient heating than Soxhlet extraction.

Compared to the UAE and MAE, chemical pretreatments did not appear to be the preferred pretreatments prior to extraction. The SEM analysis demonstrated that structural disruption imparted by the acid and alkali was not as intense compared to UAE and MAE, where most of the glandular trichomes were deflated with only a few ruptured (Fig. 3e to 3h). In spite of being classified in the same category, hydrochloric acid and sodium hydroxide act on the leaf sample in a different ways. The acid indiscriminately hydrolysed amorphous hemicellulose/cellulose in the sample to disrupt the surface structure of the sample [START_REF] Ang | Comparative study of various pretreatment reagents on rice husk and structural changes assessment of the optimized pretreated rice husk[END_REF]. Whereas the alkali mainly remove lignin and fractionally remove hemicellulose/cellulose from the sample [START_REF] Weil | Cellulose pretreaments of lignocellulosic substrates[END_REF]. Furthermore, the present of pretreatment reagent alkali in the extraction solvent leads to decomposition of the flavonoids, which was the main cause of the consistently lower extraction yields.

CONCLUSIONS

The performance of extraction method in terms of extraction yields and duration depends on their impacts on the microstructure of plant sample, particularly on the glandular trichome as it is the main site for biosynthesis of plant secondary metabolites. UAE and MAE are able to rupture most of the glandular trichomes of Orthosiphon stamineus leaves thus resulted in higher extraction yields and shorter treatment time. Sample pretreatments using chemicals disrupts the epidermal surface of plant sample severely, but gives poor rupture effect on glandular trichome and it may denature the extracted bioactive compounds. In conclusion, there is a proportional relationship

  mobile phase was linear gradient of acetonitrile in water: 5-20% (0-15 min), 20-30% (15-20 min), 30-50% (20-30 min), 50-100% (30-35 min), 100% (35-40 min), and 100-5% (40-50 min) at flow rate of 1.0 ml/min. The injection volume of sample was 10 μL and the separation is detected by UV-DD at wavelength 340 nm using Agilent 1260 Infinity Diode Array Detector (USA). The retention times of EUP, TMF and SEN were 29.28 and 32.28 and 33.30 minutes, respectively. The extraction yields of the compounds were reported as the mass of extracted bioactive compounds per unit mass of plant sample (mg/g).

Fig. 1

 1 Fig. 1 Enhancement of the total extraction yield of eupatorin, 3'-hydroxy-5,6,7,4'tetramethoxyflavone and sinensetin from Orthosiphon stamineus plant by various methods. Unshaded region indicates the degree of enhancement. Particle size reduction: rinsing with ethanol, 100 ml/g, (A) >500 μm sample, (B) 250 -500 μm sample, (C) 180 -250 μm sample, (D) < 180 μm sample; Ultrasonic: 250 -500 μm sample, 70% EtOH, 30 ml/g, 300 W; Microwave: 250 -500 μm sample, 70% EtOH, 50 ml/g, 150 W, 20 min; Acid: soaking with 0.1M HCl, 250 -500 μm sample, 24 h, 25 °C; alkali: soaking with 0.01M NaOH, 250 -500 μm sample, 1 h, 25 °C; Soxhlet: 250 -500 μm sample, EtOH, 100 ml/g, 6 h

Fig. 2

 2 Fig. 2 SEM micrographs of Orthosiphon stamineus leaf showing (a) upper and (b) lower surfaces of freshly dried leaf sample, and (c) upper and (d) lower surfaces of leaf sample subjected to Soxhlet extraction. GT: glandular trichome; S: stomata; RGT: ruptured glandular trichome

Fig. 3

 3 Fig. 3 SEM micrographs of extracted Orthosiphon stamineus leaf. (a) Upper and (b) lower surfaces of UAE extracted leaf; (c) upper and (d) lower surfaces of MAE extracted leaf; (e) upper and (f) lower surfaces of acid pretreated leaf; (g) upper and (h) lower surfaces of alkaline pretreated leaf

ACKNOWLEDGEMENTS

This work was performed under the Centre for Separation Science and Technology (CSST), University of Malaya and financially supported through Fundamental Research Grant Scheme (FRGS: FP076-2015A) and University of Malaya Research Grant (UMRG: RP002A-13AET).

between the extraction performances and the degree of disruption on microstructure of plant sample.


Eric Lao
email: eric.lao@lip6.fr

Marie-Minerve Louërat
email: marie-minerve.louerat@lip6.fr

Jean-Paul Chaput

Semi-Automated Analog Placement based on Margin Tolerances

Digital circuit design is extensively assisted by modern automation tool unlike analog design which is still a manual task because of the complexity of the interactions between devices. This paper presents a semi-automated analog placement based on margin tolerances controlled by the designer by creating analog circuits organized in row similar to digital circuits structure. The results show the ability of our tool at generating multiple layouts respecting designer's constraints.

I. Introduction

Modern system-on-chip contains both digital and analog circuit. Even though most functions in an integrated circuit are implemented with digital circuitry, some functions rely on analog circuit and are the link with the external world. Despite the fact that the digital design flow has been well-defined over the last years, the analog design flow lacks a common methodology making it difficult to capture a design procedure.

An automatic placement tool should produce analog device-level layouts similar in density and performance to the high-quality manual layouts. To achieve this task, the capability to deal with layout constraints, in order to eliminate unwanted parasitics due to the process variations, is mandatory. [START_REF] -H. Lin | Recent Research and Development and New and Challenges and in Analog and Layout Synthesis[END_REF] enumerates the most common placement constraints that are respected by modern analog placer. Among them, symmetries and symmetry-island [START_REF] Lin | Analog Placement Based on Symmetry-Island Formulation[END_REF] are the most used constraints. Other constraints like common-centroid [START_REF] Ma | Analog placement with common centroid constraints[END_REF], proximity and range are also often considered [START_REF] Strasser | Deterministic analog circuit placement using hierarchically bounded enumeration and enhanced shape functions[END_REF]. Taking into account current and signal path improves performance accuracy [START_REF] Long | Signal-Path Driven Partition and Placement for Analog Circuit[END_REF]. Devices can be placed depending on their thermal impact on the chip [START_REF] Lin | Thermal-driven analog placement considering device matching[END_REF]. Regularity [START_REF] Nakatake | Structured placement with topological regularity evaluation[END_REF] and boundary constraints [START_REF] Lin | Performancedriven analog placement considering boundary constraint[END_REF] enhance routability and suppress parasitics induced by extra bends of wires and via cost.

Recent research focused on using simulated annealing algorithm (SA) in combination with topological representations to respect these placement constraints. Topological representations encode the positioning relations between devices and SA optimizer alters their relative positions. The most popular representations used in analog placement over the last decades are Sequence-Pair [START_REF] Ma | Simultaneous Handling of Symmetry, Common Centroid, and General Placement Constraints[END_REF], B*-Tree [START_REF] Lourenc ¸o | Floorplan-aware analog IC sizing and optimization based on topological constraints[END_REF], Transitive Closure Graph [START_REF] He | Symmetry-aware TCG-based placement design under complex multi-group constraints for analog circuit layouts[END_REF], Ordered-Tree [START_REF] Xiao | Analog placement with common centroid and 1-D symmetry constraints[END_REF] and slicing floorplans [START_REF] Unutulmaz | On the convex formulation of area for slicing floorplans[END_REF] and they were coupled with some constraints to respect at the same time. Although most of the recent works focus on simulated annealing algorithm [START_REF] -H. Lin | Recent Research and Development and New and Challenges and in Analog and Layout Synthesis[END_REF], we believe that giving more control to designers and using their interventions to set some constraints yield good analog placement results. In this paper, we introduce a semi-automatic analog placement approach guided by designers' preferences. This semi-automatic approach also helps designers to debug more efficiently and makes adjustments easier since they will control the overall relative placement of the circuit but at the same time, some tiresome and error-prone tasks are automated.

The remainder of this paper is organized as follows: Section II describes the analog/mixed-signal placement approach. Section III presents our methodology. Section IV explains the experimental results. Conclusion and future challenges are given in Section V.

II. Analog/Mixed-signal Design Approach

A. Design Flow

The Cairo Hurricane AMS (CHAMS) project [START_REF]Laboratory LIP6[END_REF], developed by the LIP6 Laboratory, proposes a complete flow which would be able to create a library of reconfigurable analog Intellectual Properties, to automatically generate and optimize the layout with little intervention from designers. The flow provides a reliable and efficient solution taking into account parasitic effect-aware layout generation with enough flexibility to adapt to different designers needs and concerns. Our layout generation tool supports any technology with the new nanometric layout dependent parasitic parameters.

The proposed CHAMS analog design (Fig. 1) is a two Fig. 2. Example of placement (a) and its slicing tree representation (b) where "H" stands for horizontal cut and "V" for vertical cut way communication between the sizing and layout generation. The idea is that the sizing tool provides the electrical parameters of the transistor such as the width, length, number of fingers, etc... to the layout generation tool. The tool automatically generates the layout from a library of parameterized cells and sends back to the sizing engine the layout-dependent parasitic parameters such as the drain and source areas and perimeters, the stress effect parameters, etc... to re-evaluate the performance. This internal loop is repeated several times, with minimal designers intervention, until the target specifications are met. This paper presents the placement approach of our layout generation tool. As mentioned in the previous paragraph, we emphasize that it is part of an internal loop which will be repeated several times. Therefore our placement algorithm does not provide an optimized analog layout in one run. It results from multiple adjustments (sizing and shape) performed by the loop shown in (Fig. 1). Having these considerations in mind, we propose this placement approach because we believe it can provide a better control over the circuit placement and especially easier to adjust compared to simulated annealing approaches.

B. Placement in row

Digital and analog circuits have a dedicated area on a system-on-chip circuit so they can be independently designed within a specific space. Digital circuits are well-known for their regular row structure where standard cells are placed and routed accordingly to their netlist. In a similar way, we plan to organize the analog circuit layout in rows of devices and the analog circuit area should be placed and routed within its dedicated area.

It is common to design analog circuit in rows of devices where the height of each row of devices should be adjustable so it can match its dedicated area. Therefore, we choose the slicing tree representation for several reasons:

• Slicing trees are a natural choice since they are adequate to the row structure. Rows are represented by horizon-tal slices which will be divided into vertical slices determined by the area of each device.

• Unlike most of modern analog placement methods, our placement strategy is semi-automatic and will be guided by designers' preferences. Slicing trees are easy to handle and let designers choose the overall topology. That shows some advantages that will be explained in the following section.

III. Methodology

Modern analog automation tools are able to generate analog layout respecting various placement constraints and they mainly use simulated annealing approach. Nevertheless, such approach might not produce predictable and easy-to-adjust results. We believe that giving more control to designers will generate analog layout, easier to predict compared to optimization-based approach, but also easier to adjust in case modifications are required. Our analog/mixed-signal placer is semi-automatic: the device generation is automated and generated correct-by-construction and guided by designers' constraints over the circuit. The following subsections will describe the designers interventions.

A. Slicing Tree Construction

In order to organize devices in row, we use the slicing tree structure (Fig. 2) which will be described the designers and not automatically generated by the tool. A slicing tree is a slicing floorplan that represents an area that has been divided multiple times either vertically or horizontally, forming a set of rectangular regions representing the place filled by each device. These slices are organized hierarchically so they form a graph where the hierarchical node are either horizontal or vertical cuts. Fig. 2 shows an example of a slicing tree representing a circuit of 9 devices organized in three rows. We insist on the fact that the slicing tree is specified by designers who will also precise the analog constraints in the slices of the slicing tree. Unlike previous topology extraction approaches, our analog layout generation tool considers the designers' slicing tree as an input of the flow.

B. Devices Variations

Our placement approach consists in organizing devices in row, that is to say to have rows of devices with similar height, and the analog part's height would be a multiple of standard cell's heights from the digital circuit part. To obtain a row of devices, the height of each device needs to be similar and this is the reason why, we need to consider several possible aspect ratios for each device by varying the number of fingers like in [START_REF] Long | Signal-Path Driven Partition and Placement for Analog Circuit[END_REF] so we can find heights of devices that match a given height.

As [START_REF] Wu | Exploring feasibilities of symmetry islands and monotonic current paths in slicing trees for analog placement[END_REF] mentioned, changing the number of fingers of a device can considerably change the device property, especially the source/drain bulk capacitance. Nevertheless, our placement phase is part of an internal loop (Fig. 1), which is repeated until a result meets the required performances (Fig. 1). Also, it is the task of designers to decide the number variations for every device which would limit the number of finger variations.

C. Margin Tolerances

We aim at creating rows and it is important to define what we consider as a row of devices since the devices will rarely have exactly the same height. We introduce a tolerance margin, which represents the difference between the smallest and the tallest device's height in a vertical node. Fig. 3 (a) shows an example of devices organized in one row where the area A, B, C and D represents devices. Devices A and B show respectively the smallest and highest height inside the row. This difference of height is compared to the tolerance parameter to establish if this is considered as a row. We apply the same concept to the horizontal node but instead of using the height as a comparison, we use the width. It is up to designers to adjust this tolerance margin and it will impact on the number of accepted possibilities. Different margin tolerances can be considered at each hierarchical nodes. At the same time, having a small tolerance will reduce the waste of space induced by the slicing tree representation.

D. Placement Constraints

As said in section III-A, it is the task of designers to describe the slicing tree with the help of our tool. This will directly impact on the topology of the circuit. This means designers will have the total control over the relative relation between the blocks. Having designers building their own slicing tree also means that they will have control over placement constraints such as proximity, boundary, current/signal path and regularity constraints based on their knowledges and preferences.

Among the most common constraints, symmetries can be respected with the appropriate slicing tree organization. We also consider alignment constraints inside a slicing tree in horizontal or vertical node. In Fig. 3 (b), we have an example of possible alignments: devices A and B are aligned to the bottom of the row, device C is centered and device D is aligned to the top of the row. In horizontal slices, devices can be

E. Placement Choice

Similar to [START_REF] Chen | Challenges and Solutions in Modern Analog Placement[END_REF], once all the possible variations are set for each device, we evaluate the accepted variations at hierarchical nodes based on the margin tolerances. These accepted variations are propagated from the bottom of the slicing tree to the root. After this bottom-up propagation, designers process the different placements based on height, width or global ratio criteria. They can choose the most optimized placements according to the Pareto front curve like in [START_REF] Saif | Pareto front analog layout placement using Satisfiability Modulo Theories[END_REF], but they are free to choose any other possible placement that would eventually have more white space if the circuit affords more space.

IV. Results

Our tool was implemented in C++ programming language on a Intel(R) Core(TM) i5-4590S CPU @ 3.00 GHz workstation with 6 GB RAM. To illustrate the capability of our tool, we experiment it on a fully differential transconductor [START_REF] Iskander | Hierarchical sizing and biasing of analog firm intellectual properties[END_REF], I. These figures all have the same scale.

designed under a technology CMOS 130 nm.

The fully differential transconductor is composed of a total of 32 devices and we consider 2 possible variations for each device. The slicing tree takes into account 11 symmetries for this circuit and tolerance margins are set in a way to have reasonnable amount of accepted possibilities. Our algorithm found 384 possible placements in less than 1 second, some of them are listed on Table I and can be seen on Fig. 5.

Table I shows the characteristics of those layouts with their total area, their width, their height and the pourcentage occupation of the circuit in the total area. Fig. 4 illustrates a zoomed area of the first and the sixth placement from Table I in order to show the variation of fingers. Fig. 5 illustrates the layouts described in Table I and show the evolution of the rows for different global aspect ratios. Designers can choose their final placement based on their experiences and preferences. Our tool presents the placement results plotted on a graph with heights and widths as axis and can be selected interactively to be placed in a few seconds.

V. Conclusion

We presented our semi-automatic analog placement approach using the slicing tree floorplan representation. The idea is to create an analog circuit organized in rows of devices where the acceptance of a row depends on a margin tolerance defined by the designer. By creating the slicing tree, the designer has more control over the placement phase than with an optimization-based approach. A placement solution is then selected according to height, width or global ratio criteria.

Slicing tree structure is a structure that has been studied over the last decades and its drawbacks are well-known. It is in a way restrictive since there are some topologies that cannot be represented with a slicing tree which can be a problem. Moreover, it does not extend to non-rectangular structures and has a bad white space distribution. Nevertheless, we are quite satisfied with the slicing tree structure which is able to handle the organization in row. For future considerations, even though the white space distribution can be a disadvantage, we plan to exploit it during the routing step that needs to be performed. Our floorplan representation and algorithm has been thought according to the analog routing step that we plan to develop in the future.

Fig. 1 .

 1 Fig. 1. Cairo Hurricane AMS Design Flow

Fig. 3 .

 3 Fig. 3. Tolerance (a) and alignment (b) in slicing tree representation

Fig. 4 .

 4 Fig. 4. Evolution of 2 rows from layout 1 and 6 of Table I for different global aspect ratios. These figures have the same scale.

Fig. 5 .

 5 Fig. 5. Layouts of the fully differential transconductor [19] from TableI. These figures all have the same scale.


Alexis Duque
email: alexis.duque@insa-lyon.fr

Razvan Stanica

Herve Rivano

Adrien Desportes

Demo: Off-the-shelf bi-directional visible light communication module for IoT devices and smartphones

In this demonstration, we propose a line of sight bidirectional communication system between an ordinary LED and an off-the-shelf smartphone. We designed a cheap multi sensors device as a proof of concept of a near communication module for the Internet of Things.

To illustrate a typical use case, we have programmed the micro-controller to broadcast the sensors values and the battery level using the LED. The same LED, reverse-biased, is also used as receiver to get some data, without the need of additional hardware modifications.

Also, thanks to the the serial bus, the board can be connected to an embedded device, such as a Raspberry Pi, to serve as a VLC module, offering numerous IoT applications.

On the other side, a user with his smartphone and our Android application can then retrieve the transmitted information, or send a configuration command thanks to the flashlight, letting him change the color of the RGB LED.

Introduction

With the rise of the Internet of Things (IoT), consumer electronics products, which yesterday were single function, tend to be smarter and connected to the user, through his smartphone. However, providing wireless connectivity with Bluetooth Low Energy (BLE) or WiFi means adding an extra radio chip, increasing the object size and price.

This kind of hardware modification is not without impact for the manufacturers: even if the radio chip cost is negligible for a single unit, it may become huge when millions of products are sold. Also, the health impact of radio waves is not yet fully understood.

However, many of these products already have a micro-controller unit (MCU) and light-emitting diodes (LED), which are the only requirement to enable visible light communication [START_REF] Schmid | An LED-to-LED Visible Light Communication system with software-based synchronization[END_REF]. While photodiodes are commonly used as a light receiver, the transmitting LED reverse-biased can be used as receiver [START_REF] Giustiniano | Low-complexity Visible Light Networking with LED-to-LED communication[END_REF], enabling half-duplex communication.

To further reduce the cost of the system, an off-the-shelf smartphone can receive information through visible light thanks to its camera [START_REF] Danakis | Using a CMOS camera sensor for visible light communication[END_REF], while its flashlight can be controlled to send data [START_REF] Hesselmann | FlashLight -Optical Communication Between Mobile Phones and Interactive Tabletops[END_REF], as shown in figure 1. This demonstration aims to illustrate such a scenario, which targets short-range (tens of centimeters) and low throughput (tens to hundreds of bits per second) communications for the IoT.

Use cases

Even if the throughput and the range of this kind of communication are very limited [START_REF] Duque | Unleashing the power of LED-to-camera communications for IoT devices[END_REF], potential applications exist. VLC can then be a cheap alternative to traditional radio communication, when the application reads only small payloads coming from a sensor, such as a battery level or a temperature value, or occasionally configure a device. In fact, any electronic product for which the wireless function is secondary and rarely used might be a candidate for our solution.

In this regards, we propose a comprehensive VLC transmission system using just a low-cost, low-power colored LED that is most probably already part of the design of candidate devices. On the user side, our application runs on an unmodified smartphone. The additional costs to make a traditional device part of the IoT are therefore minimal. We designed a 40x20mm printed circuit board (PCB) shown in Figure 2 that supports a red 5mm LED, a micro controller unit (MCU), a 6-axis and temperature sensor. Also, we place a surface mount device (SMD) RGB LED whose color can be easily changed.

The MCU is a low cost and low power Cortex M0+ STM32L051. We configured the clock speed at just 8MHz using the high speed external crystal oscillator. The board is powered by a 3.7V polymer lithium battery, rechargeable through USB.

The firmware we developed works in the following way. After enabling all needed peripherals, the MCU is placed in low-power mode, waiting for the beginning of a transmission, coming from the smartphone flashlight.This is achieved by periodically waking-up the MCU, sensing the reversebiased LED, and taking the decision to go back to the lowpower mode or stay active to handle subsequent transmissions.

User smartphone

On the user side, we use a Huawei Nexus 6P smartphone running Android Nougat version number 7. Using the Cam-era2 API, we configure its CMOS sensor to take advantage of the rolling shutter effect.

For this demonstration, we have developed an application that can transmit a short message to the LED using the flashlight and also receive information from the same LED as depicted in Figure 1.

A background thread processes the signal from the camera and decodes the payload. To not display on screen the dark and striped pictures produced by the rolling shutter effect, captures are made in burst mode: the first frame, taken with regular parameters, is displayed in the app, while the other one, taken with a short exposure time, is sent to the background thread.

In this way, by moving his smartphone, the user can see the ambient temperature, the board orientation or the battery level in a popup that follows the LED on the screen, in an augmented reality fashion.

On the other hand, to illustrate the smartphone to LED link, using our application, the user can change the onboard RGB LED or select the sensors values to be broadcast.

Demonstration scenario

This demonstration will illustrate different use cases and potential applications of our work. We set up three scenar-ios using the board as a standalone device, or as a module connected to a Raspberry Pi (RPI) or a laptop.

RGB LED and board configuration: using the application, the user can wake up the board, change the color of the RGB LED and select which sensor values are broadcast. Also, it can place the board in low power mode.

Sensors values broadcast: according to its configuration, the board broadcasts through VLC the selected sensor values: orientation, acceleration, temperature, battery level. The user, by aiming at the LED, can see them in real time on the screen.

VLC Raspberry Pi module: the board is connected to a RPI through its serial bus, working as an external VLC interface. On the smartphone side, the user can type a text and send it to be displayed on the RPI LCD screen.

Conclusions and further works

This demonstration proposes a comprehensive VLC application for the IoT by using a low-cost, low-power colored LED and an off-the-shelf smartphone. This work spotlights the VLC technology as a great opportunity for smart and connected consumer electronic products, providing deviceto-device half-duplex communication.

However, some issues have to be solved. Access control and network layer protocols are still missing and have to be designed to make the transmission efficient and reliable. Also, packet addressing and larger message size should be taken into account and will be addressed in further works.

Figure 1 .

 1 Figure 1. Aiming the VLC board with his smartphone, the user is getting the board battery level. It can change the small RGB LED color by pressing the corresponding button in the app.

Figure 2 .

 2 Figure 2. On the left, the demonstration block diagram. On the right, our demo board