evaluation of anti-cancer properties of lichens …...85 shanmugam poornima et al.: evaluation of...

Cancer Research Journal 2016; 4(6): 84-89

http://www.sciencepublishinggroup.com/j/crj

doi: 10.11648/j.crj.20160406.11

ISSN: 2330-8192 (Print); ISSN: 2330-8214 (Online)

Evaluation of Anti-Cancer Properties of Lichens Using Albino wistar Rats as an Animal Model

Shanmugam Poornima1, Ponnusamy Ponmurugan

1, *, Khader Syed Zameer Ahmed

1,

Ganesan Ayyappadasan1, Fahad Khalid Aldhafiri

2, Balakrishnan Santhanaraj

2

1Department of Biotechnology, K. S. Rangasamy College of Technology, Tiruchengode, Namakkal District, Tamil Nadu, India 2College of Applied Medical Sciences, Majmaah University, Al Majmaah, The Kingdom of Saudi Arabia

Email address: [email protected] (P. Ponmurugan) *Corresponding author

To cite this article: Shanmugam Poornima, Ponnusamy Ponmurugan, Khader Syed Zameer Ahmed, Ganesan Ayyappadasan, Fahad Khalid Aldhafiri,

Balakrishnan Santhanaraj. Evaluation of Anti-Cancer Properties of Lichens Using Albino wistar Rats as an Animal Model. Cancer Research

Journal. Vol. 4, No. 6, 2016, pp. 84-89. doi: 10.11648/j.crj.20160406.11

Received: October 20, 2016; Accepted: November 4, 2016; Published: November 29, 2016

Abstract: Studies were undertaken to evaluate the efficacy of purified lichen extracts against cancer induced Albino wistar

rats as an animal model under in vivo condition. Lichen species were collected from different mean sea level of Yercaud hills of

Tamil Nadu, India. Extraction and purification of lichen compounds were done using silica gel column chromatography with

TLC analysis. Different fractions were collected from the crude extract and it was subjected to study the potential of

anticancerous property. Anticancer activity was confirmed that Parmotrema reticulatum exhibited the highest control over the

cervical cancer in cell line model. The compound Benzoic acid, 2, 4 dihydroxy, 6 methyl-methyl ester was identified as a

potent molecule from Rocella montagnei. Anticancer effect of P. reticulatum and P. hababianum and their compounds were

further evaluated to have the effective cancer drug against cervical cancer disease. Cancer cell line induced rats showing a

significant reduction tumor proliferation which were treated with bioactive anti-cancer lichen compounds in comparison with

the standard drug. Histopathology and hematology studies have been done to examine to confirm the level of tumor growth in

the animals and to analyze the blood platelet count in tumors; respectively.

Keywords: Cancer, Lichens, Parmotrema Reticulatum, Rocella Montagnei, Animal Model

1. Introduction

Cancer is of paramount importance today in terms of third

leading cause of cancer-related death worldwide to human

beings [1]. It develops because of the progressive

accumulation of genetic and epigenetic alterations in human

body cells that lead to the transformation of normal colonic

epithelium to colon adeno carcinoma. Indeed, consumption

of high levels of red meat and fat together with low levels of

fruits, vegetables and fibers has been suggested to increase

the risk of cancer incidence [2]. Moreover, scientific

investigations are making the best efforts to combat this

disease, but there is not sure-shot, perfect cure is yet to be

brought into world medicine. So alternative solution to

western medicine has their own severe side effects and the

use of medicinal plant preparations to arrest the insidious

nature of the disease [3]. Many herbal plants have been

evaluated in clinical studies and currently being investigated

phytochemicaly to understand their tumoricidal actions

against various cancers [4]. The rich and diverse plant

sources of India are likely to provide effective anti-cancer

agents. It is of the best approaches in the search for anti-

cancer agents from plant resources is the selection of plants

based on ethnomedical leads [5].

It has been reported that there are over 100 different types

of cancer in the world, and each is classified by the type of

cells that are initially affected. Cancer harms the body when

altered cells divide uncontrollably to form lumps or masses

of tissue called tumors. Normal cells in the body follow an

orderly path of growth, division and death. Programmed cell

85 Shanmugam Poornima et al.: Evaluation of Anti-Cancer Properties of Lichens

Using Albino wistar Rats as an Animal Model

death is called apoptosis and when this process breaks down,

cancer begins to form in human body. Cancer is caused by

various genetic factors, life style factors such as tobacco use,

food diet and physical activity, certain type of infections and

the environmental exposes to different types of chemicals

and radiation. The cancer prevalence is established to be

around 2,500,000 (2.5 million) people with over 800,000

new cases and 5,50,000 deaths occurring each year. More

than 70% of the cases present in advanced stage accounting

for poor survival and high mortality. About 6% of all deaths

in India are due to cancers, which contribute to 8% of global

cancer mortality [6]. The current Indian population is around

1,270,272,105 (1.27 billion) in which the incidence of cancer

in India alone is measured by 70-90% due to disease severity

at matured stage [7].

Some of the most important types of cancer include Adrenal

cancer, Anal cancer, Bladder cancer, Breast cancer, Colon

cancer, Eye cancer, Leukemia cancer, Lung cancer, Liver cancer,

Ovarian cancer, Pancreatic cancer, Oral cancer, Skin cancer and

Thyroid cancer. Some of the sign and symptoms of cancer are

unexplained weight loss, continuous fever, hair loss, skin

changes (skin cancer), change in bowel habits or bladder

function, unusual bleeding or discharge and nagging cough or

hoarseness. There are more than 100 cancer drugs generally

used to control the cancer growth. Presently available therapies

including surgery, radiation and chemical therapeutic drugs, are

still limited for advanced stages of carcinogenesis.

Chemoprevention remains an effective and promising additional

strategy for controlling the incidence of cancer [8]. The some of

the drugs are commonly used such as Abiraterone acetate,

Abitrexate, Bicalutamide, Cabazitaxel, Cytarabine, Degarelix,

Zelboraf and Vorinostat for the control of cancer [9]. The main

objectives of the present study is to collection and identification

of various lichen species obtained from Eastern Ghats (Yercaud

and Kolli hills) of Tamil Nadu, India and subjected to extract,

characterize and purify the bioactive anti-cancerous compounds

using various bioassay techniques. Further, it was evaluated the

efficacy of bioactive anti-cancerous lichen compounds against

cancer induced Albino wistar rats subsequently it was analyzed

the hematological, histopathological and biochemical studies of

both treated and untreated experimental rats.

2. Methodology

2.1. Collection, Characterization and Identification of

Various Lichen Species

Lichen species were collected from different agroclimatic

regions of Yercaud and Kolli hills of Tamil Nadu. The

collected lichen species were brought to laboratory in a

sterile condition. Then it was subjected to identify by using

various colour test (K, C, KC and PD) and morphologically

on chemical basis [10]. The chemistry of the individual

lichen species were done in different solvent system [11].

The identified lichen species were authenticated and

maintained at Herbarium of K. S. Rangasamy College of

Technology, Tiruchengode, Tamil Nadu for further uses.

2.2. Extraction and Characterization of Anticancer

Compounds from Lichens

Based on the available quantity, the lichen species were

subjected to sequential extraction of increasing polarity

(Hexane, Ethyl acetate and Methanol) to extract bioactive

compounds present in it using Soxhalet apparatus. The dried

powder was obtained and which was served as a crude

extract for performing bioassay guided fractionation [12].

2.3. Purification of Lichen Extracts Using Column

Chromatography

The dried powder was mixed with silica gel to purify the

crude extract in column chromatography. The elution profile

was done based on the different ratio of increasing polarity.

Eluted sample was collected based on volume and was stored

in room temperature. Various fractions of lichen sample was

further purified and confirmed using Thin layer

chromatography, GC-MS and HPLC analysis. The bioassay

of lichen fraction was done on the basis of DPPH analysis

which helps to find out the bioactive anticancer compounds

from the lichen species [13].

2.4. Cytotoxicity Assays of Lichen Extracts Against in Vitro

Cancer Cell Line

The four different cancer cell lines were obtained from

National Centre for Cell and Science, Pune, India for the

present study. The cell lines were sub-cultured and

maintained at CO2 Incubator at 37°C. Then it was subjected

to treat with various concentrations of crude extracts as well

as various fractions for doing cell toxicity MTT assay. The

toxicity of cancer cell lines with our extracts was measured

by calculating the inhibitory concentration (IC50). Cell count

and viability was done using Hemocytometer and trypan blue

assay [14].

2.5. Evaluation of Bioactive Anti-cancer Lichen

Compounds Against Cancer Induced Albino Wistar

Rats

Healthy adult Albino Wistar rats weighing around 180-250

gm were maintained at an ambient temperature of 25±20°C

and relative humidity of 55-65% for 12±1 hours of light and

dark schedule in the Animal House of K. S. R. College of

Technology, Tiruchengode, Tamil Nadu. They were fed with

commercially available rat chow (Hindustan Lever Ltd.,

Bangalore) with free access to water [15]. The experimental

protocol is approved by the Institutional Animal Ethics

Committee (CPCSEA Registration No.:

1826/PO/EReBi/S/15/CPC).

After adaptation of one week the rats were cancer induced

with DLA cancer cell lines at Amala cancer research

institute, Tirussur, Kerala. The rats developed swelling in the

lateral side of its body. After 11 days, cancer fluid was

aspirated from the peritoneal cavity of the rats and injected

(1,00,000 cells / ml) into another group of animals after

counting the cells in heamocytometer [16]. Histopathology

Cancer Research Journal 2016; 4(6): 84-89 86

and hematology analysis was done for different organs

against control rats [17].

2.6. Experimental Animal Groups

Group I: Normal control rats

Group II: Cancer treated control rats received no drugs or

lichen compounds

Group III: Cancer treated rats that receive anti-cancer

lichen compounds (100 mg/k.g.b.w)

Group IV: Cancer treated rats that receive anti-cancer

lichen compounds (200mg/k.g.b.w.)

Group V: Cancer Treated rats that receive standard drug

(Vincristine 3mg/k.g.b.w)

3. Results

The lichen species were collected from Yercaud hills and

were subjected to identify by colour tests as well as presence

of isidia, soredia, apothesia, cilia, rhizines and spores. The

results were given in the Table 1 for identification and

authentication purpose. The result revealed that

Parmelliaceae family covering six species were identified

and deposited at herbarium of K. S. Rangasamy College of

Technology, Tiruchengode, Tamil Nadu for research and

voucher specimens. These were further purified for the

bioactive anticancerous molecules present in it. Furthermore

results indicated that there were more abundance of lichens

in the form of foliose lichens than crustose and fruticose. The

high genus level of appearance was observed as parmeliod

lichen species at Yercaud and Kolli hills which was used for

extraction of bioactive anticancer compounds for the present

study.

Table 1. List of various lichen species collected from Yercaud and Kolli hills

of Tamil Nadu, India.

Family Name Genus Name Lichen Name

Parmelliaceae Parmotrema

1. Parmotrema reticulatum (Taylor) M.

Choisy 1952

2. Parmotrema grayanum (Hue) Hale

1974

3. Parmotrema hababianum (Gyeln.)

Hale 1974

4. Parmotrema austrosinense (Zahlbr.)

Hale 1974

5. Parmotrema crinitum (Ach.) M. Choisy

1952

6. Parmotrema tinctorum (Despr. ex Nyl.)

Hale 1974

3.1. Classification and Distribution Pattern of Lichen

Species

Lichen species were distributed less abundant at 350 Mean

Sea Level (MSL) and which increase in increasing with

Mean Sea Level in terms of height of the hills. The results

showed that very high abundant level of lichen distribution at

1500 MSL near to Shevroy temple. Moreover Crustose and

Foliose lichens were equality distributed and contributed to

the entire Yercaud hills whereas Fruticose lichen was less

diversity.

3.2. Extraction of Anticancerous Compounds from Lichen

Species

Lichen compounds were extracted using different solvent

systems like Hexane, Ethyl acetate and Methanol for

separation of anticancerous compounds. The crude methanol

extract was subjected to fractionation using column

chromatography with silica gel of 60-120 mesh as stationary

phase. The two solvent mixtures of hexane (H) with ethyl

acetate (EA) and methanol (M) with ethyl acetate (EA) in

different ratios were used as mobile phase. In both solvent

systems different ration of mixture was used. In the 100%

hexane and 90: 10 (H: EA) no residue was found during the

fractionation process in contrast to this all other solvent ratio

either residue or pigment and in some mixture the

combination of both were found (Fig. 2). All the fractions

were subjected to crystallization by autoevaporation process

in which only six fractions has formed crystals.

3.3. Anticancer Activities of Lichen Extracts Against MG

63 Cell Line and SiHa Cell Line Model

The selected lichen species were subjected to identify the

potential of inhibiting the growth of cancer cell by

performing MTT assay. The observation revealed that more

than 50% of lichen species were showed the ability of killing

the cancer cell population. Thus the screenings of potential as

well as fractionation of lichen compounds were done based

on the anticancer activity under in vitro model. The result

further revealed that IC50 was observed as 100µg/ml of

extract. Anticancer activity of P. reticulatum was studied

against HeLa cell lines. The result was observed that

significant reduction in the confluency with increase in the

concentration of lichen extract. Percentage of inhibition was

calculated to study the potential of lichen compounds. At a

concentration of 50 µg/ml of extract showed the 50% of

inhibition of cell growth. While increasing the concentration

upto 300 µg/ml revealed that there is no viable cell in the

sample.

3.4. Evaluation of Lichen Rocella Montagnei Extracts

Against Cancer Induced Rats Model

The tumor induction had been identified by the bulk

appearance of the rats at the peritoneal cavity. The animal has

been sacrificed at 13th

day and the peritoneal cancer fluid had

been aspired from the animal and injected into another

animal marked as dorsal female. The cancer fluid was also

injected into another animal with no marking, at the

subcutaneous (limbs) for solid tumor induction. The new set

of animals also showed bulk appearance in their body. After

the development of solid tumor, the arts were treated with

lichen extracts on cancer induced rats model with the

concentration of 100mg /k.g.b.w and 200mg/k.g.b.w.

There is a significant variation in haemoglobuline, platelet

counts and red, white blood cells in both treated and

untreated rats (Fig. 5). The results showed that tumor size



and volume has been reduced in the significant level of

treated rats groups. Treatments given for DLS solid tumor

with lichens were most effective compared to standard drug

Vincristine. After the treatments the histopathology analysis

were done for both treated and untreated organs like kidney,

lungs and liver. The necrosis and development of individual

cells were analyzed using H & E stain. The slide was

observed in the light microscope which showed much

significant of reduction of cancer cell populations.

Fig. 1. Collected lichen species at Yercaud hills of Tamil Nadu, India.

Fig. 2. Extraction and purification of anti-cancerous compounds from

lichens.

Fig. 3. Various concentration of lichen extract (Parmotrema hababianum)

against MG63 cell line.

Fig. 4. Study of cancer cell inhibition at different lichen concentrations.

Fig. 5. Maintenance of cancer induced rats for different experimental

groups.

0 1 2 3

0

20

40

60

80

100

Log10 concentration (µg/ml)

% C

ell In

hib

itio

n

Cancer Research Journal 2016; 4(6): 84-89 88

Fig. 6. Hematological analysis of cancer induced and control rats.

3.5. Histopathology Analysis of Control and Treated Rats

Model

Different organs of treated as well as untreated tissue

samples were analyzed for Heamotoxylin and Eosin (HE)

stain to assess the tissue damage. Tissue damage was

measured in the untreated rats model. The result revealed that

tissue was damaged in highest level in the DLS induced rats

model. Whereas treated with lichen showed the significant

level of reduction in tissue damage which helps to formation

of new cells.

4. Discussion

Anticancer activity of selected R. montagnei exhibited in

moderate level of cancer cell population which may be the

presence of lichen bioactive substances present in it. The

present lichen extract showed better a significant growth

inhibition against MG63 cell lines. Similar result was also

obtained for anticancer activity of against various cell lines

such as FemX (melanoma) and human colon carcinoma cell

lines in cytotoxicity assays [18]. The IC50 value of MG63 cell

line in lichen R. montagnei was obtained as 100µg/ml in

contrast to that Hypogymnia physodes showed very less IC50

in the range of 12.72 to 24.63 µg/ml. Anticancer potential of

three Umbilicaria lichen species and revealed that IC 50 in

the range of 28.45 to 97.82 µg/ml. It has stated that lichen

compounds showed better anticancer property against both in

vitro and in vivo methods [19].

It has been described that around 17 lichen species were

screened in which lichen compounds obtained from

Flavocetraria cucullata showed a remarkable anticancer

activity in apoptosis process [20]. The similar trend was also

observed in R. montagnei lichens in apoptosis by the G2

Phase on cell cycle. Usnic acid showed better anticancer

activity in mouse model. The anticancer activity of R.

montagnei might be the presence of usnic acid and other

lichen bioactive substituents. The result of cell proliferation

and migration was suppressed and represented in MG63 cell

lines in which usnic acid at very less dosage of angiogenic

power in xenografted tumor based mice model which also

described that endothelial cell proliferation, migration and

tubulin formation in cancer cell. The mechanism of cancer

cell reduction might be role of inhibition breast cancer cell

population and suppress of VEGFR2 mediated AKT and

ERK1/2 signaling pathway [21].

Hematological parameters were also revealed that

tremendous variation in blood cells which also in line with

the report of Monica et al. [22] that in vitro and in vivo

evaluations of antineoplastic potential of barbatic acid which

also explained in detail about the mechanism of apoptosis in

cancer cell population. Further, the purified compounds of R.

montagnei will also evaluated in both in vitro and in vivo

conditions against mouse cancer cell line model.

5. Conclusion

The present study revealed that the bioactive compounds

obtained from Parmotrema reticulatum, P. hababianum and

Rocella montagnei exhibited a strong anti-cancerous property

which confirmed through cancer cell-line studies and animal

model experiments. There was a significant reduction in

cancer cell proliferation due to action of lichen bioactive

compounds which in turn reflected in cancer induced Albino

Wistar rats.

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[11] Culberson, C. F. (1972). Improved conditions and new data for the identification of lichen products by a standardized thin-layer chromatographic method. Journal of Chromatography, 72, 113–125.



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S. Karthiga Kannan et al. Cystic oral lesions of salivary gland origin in children. Case series – An Observational study56

Convex Optimization for Energy Efficiency of a Bluetooth Based Wearable Continuous Cardiac Monitoring Node

Jaspal Singh,1 , Santhanaraj Balakrishnan,2 1 Principal Engineer, Centre for Development of Advanced Computing, Mohali – 160071 India.

Email: [email protected] 2 Professor, Department of Medical Equipment Technology, College of Applied Medical Sciences, Majmaah University, Al

Majmaah – 11952, Kingdom of Saudi Arabia. Email: [email protected] on: 20th May, 2016; Accepted on: 25th June,2016

Correspinding Author:Jaspal Singh

email:[email protected]

Abstract

Real time cardiac monitoring is an important component of body sensor networks. Due to its typical bandwidth requirements, Bluetooth is often the protocol of choice for wireless transmission from the cardiac monitoring nodes. In order to make the node small and wearable, improving energy efficiency is desirable as otherwise we end up either making the battery too big or changing it too often. In this paper we focus on optimizing energy consumption in cardiac data transmission while providing low latency. We show that this problem can be posed as a geometric program, which belongs to class of convex optimization problems. We analyse the issue based on data bandwidth requirements and latency constraints. The results are mapped to different data type specifications in Bluetooth and recommendations are drawn accordingly.

Keywords— cardiac monitoring, convex optimisation, Bluetooth, energy consumption

I. IntroductionReal time cardiac monitoring is indicated

in several healthcare scenarios. Single or multiple channel monitoring is performed in most post-operative cases. The Gold standard

امللخصشبكات يف اهلامة العنارص من حلظيا القلب مراقبة تعترب من البلوتوث تقنية حتتويه ملا نظرا و للجسم. االستشعار هي تكون ما غالبا فاهنا نموذجي ترددي نطاق عرض عقد من القلب ملراقبة الالسلكي للنقل األمثل الربوتوكول السهولة ومن صغرية املراقبة عقد جعل أجل من . املراقبة واال هبا الطاقة استخدام كفاءة حتسني من بد ال ارتداؤها، هذه يف . كثريا للتغيري حتتاج أو جدا كبرية البطارية ستكون البيانات نقل يف الطاقة استهالك حتسني عىل ركزنا الورقة هذه أن وضحنا وقد منخفض. كمون توفري مع القلب من املشكلة يمكن طرحها كربنامج هنديس و الذي ينتمي إىل فئة من املشاكل املحدبة النموذجية. نحن نحلل هذه املشكلة بناء عىل متطلبات عرض النطاق الرتددي واشكاالت الكمون. تم تعيني النتائج حسب أنواع البلوتوث املختلفة ، ويتم رسمها

التوصيات وفقا لذلك.

for this monitoring is the use of wired monitors by the patient bedside. But wired monitoring hinders free movements of the patient and thereby, slows down the patient’s recovery process also. So, a large number of researchers

Original article


S. Karthiga Kannan et al. Cystic oral lesions of salivary gland origin in children. Case series – An Observational study 57

have worked on / developed wireless body sensor networks (BSNs) with continuous cardiac monitoring capabilities for various scenarios [1][2][3]. These BSNs enable greater patient convenience and quicker recoveries. Apart from cardiac monitoring, wireless body sensor networks have been used for monitoring various other physiological parameters including heart rate, SpO2 levels etc. for various purposes including post-operative monitoring. Several body sensor networks and their implementations are described in the literature.[2][4][5] The applications of body sensor networks may vary widely, but continuous cardiac monitoring, remains a popular and an integral part of them.

The protocols used for wireless communications in wireless body sensor networks include Bluetooth, Bluetooth low energy, zigbee, ANT, ANT+ etc. [6]. Most of the physiological parameters like HR, PR, SpO2, RR, temperature, motion, etc. require transmission of few bytes in several seconds and thus require very little bandwidth. The parameters like ECG, EEG or EMG whenever monitored require comparatively much larger bandwidth. In such networks, often Bluetooth is the protocol of choice. [7,8,9] Bluetooth is an open standard wireless communication protocol using the 2.4GHz ISM band. It allows up to 8 connections with the master within a range of about 10-30m. The data throughput is good enough to transmit real time multichannel ECG. Recently, Bluetooth version 4.0 (referred to as Bluetooth low energy) was released as an open and license

free standard branded as ultra-low power communication.

While Bluetooth is very convenient and has suitable bandwidth for wireless cardiac monitoring, the major challenge in practical use of Bluetooth for continuous cardiac monitoring remains its energy efficiency.[10] The relatively larger data bandwidth capabilities of Bluetooth make it poorly optimized for continuous cardiac monitoring purposes. This demands use of larger battery packs which decreases the unobtrusiveness of the sensors. On the other hand, battery change / recharging is always a concern. In this paper we present a simple model of energy consumption in a wireless body sensor network and derive a cost function based on energy and latency of the data. The function is shown to belong to convex optimization problem. The simulations are then worked on the function and results are presented. The results are then discussed in the light of practical limits of bluetooth communication protocol.

II. Network modelA wireless body sensor network may

consist of several sensor nodes for measuring physiological parameters like ECG, temperature, SpO2, pulse rate, respiration rate, etc. of a subject. The nodes communicate wirelessly to a personal gateway, which could be a mobile phone, or a dedicated hardware. The hardware is often body worn, but could also be a nearby-placed node in certain scenarios. The data from the sensor nodes is continuously transferred to the gateway.

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The gateway in turn transfers the data to the required destination using wires or wireless media. As most of the sensor nodes, except ECG, are comparatively less straining on the node computing resources as well as wireless resources; we focus our attention to optimisation of ECG sensing nodes. So, for the purpose of this investigation we consider network consisting of one wireless node for continuous single channel ECG monitoring. The node relays the data to a nearby placed gateway which is assumed to be sufficiently resourceful. The overview of the considered node is shown in fig 1. It has an analog front end and a microcontroller that controls sample and hold circuit, ADC and buffering. The sampling circuit continuously samples ECG signal from the amplifiers and reasonably fast ADC digitises it. The digital data is held in the buffer till the controller takes it, packetizes it and transmits to radio for further wireless transmission. Each of these steps as well as steps like generating clocks and other essential activities of controller and that of analog front end consume energy from the battery.

Fig 1. The configuration of ECG Sensor Node

A

Sampling & ADC

Buffer

Microcontroller

Power Source

Radio

We assume the radios on sensor node and gateway node work as per the Bluetooth protocol in a master - slave configuration. For simple pairing the two radios undergo a series of steps which include – device discovery, connection, security establishment and authentication before the data exchange can take place. Several of these steps can be tuned (like using dedicated bonding instead of general bonding) to improve the latency and instantaneous energy consumption [11]. However, for our purpose of continuous monitoring scenario the contribution of initial pairing and bonding exercise to the overall energy consumption is insignificant. We assume the link once established is maintained uninterrupted and the energies involved during continuous transmission are considered. The sensor node radio behaves as a master and communicates with slave in the gateway. It exchanges data packets based on the clock defined by the master. In accordance with Bluetooth classical standard, [12] each 625 microsecond interval time slot is uniquely identified and packet transmissions can begin at the start of such time interval only. The master transmits in even slots and receives in odd interval slots as shown in fig 2. A packet of transmission consists of three entities, access code, header and payload. The access code and the header have a fixed length of 72 bits and 54 bits respectively. Assuming ideal operating conditions, the payload can be anything from 0 to 2744 (or 343 bytes practically 320 bytes only) bits at the maximum.



Fig 2. Indicative scheme of timing slots

I. Optimization framework In this section we address the problem of

designing an optimal packet size and map it to convex optimization problem. We consider the case of continuous transmission of ECG data over Bluetooth link. The constraints are based on Bluetooth specifications for the radio, clinical utility of the ECG signal, buffering requirements based on packet size and the signal latency. Noting the tradeoff between energy consumption in different steps and latency, the problem of interest becomes designing optimal intervals for which the data can be buffered and then transmitted for lowest energy and latency values. A. Energy consumed at the sensor node

Energy consumed at the sensor node consists of energy consumed by the radio for transmission and that consumed by other activities at the node including microcontroller activities.

Based on heath condition or clinical requirements ECG requirements may vary. Let the node sample the ECG at a sampling rate ‘S’ per second. If the ADC is sufficiently fast, in time time, ‘t’, the controller has ‘S.t’ digitised samples. To avoid complications, we assume contents of buffer are transmitted in a single packet. We further assume the

ADC depth of 8, so that each data sample is efficiently placed in the stream and there is no need of eliminating unwanted zeros or such manipulations anywhere in the entire system.

The radio communication is undertaken in slots of 625 µs, in compliance with Bluetooth protocol. Assuming ideal operating conditions for radio, the master node can take anything from one to five time slots of 625 µs each while one 625 µs slot is taken by the slave to acknowledge. So the radio on the node acting as master listens to slave for 625 µs, consuming ‘Ir’ current from the battery. It then transmits using ‘It’ current for one to five slots of 625µs each. It consumes s.625.It charge during the ‘s’ slots of transmission. Let the node go low into energy state for ‘Le’ time slots of 625 µs where the master radio is temporarily put in sleep mode, consuming only a sleep current of ‘Is’. Assuming the complete system works at a voltage v; Energy, E consumed by the system in any time period with n different states each can be given by

E = v*(∑ in * tn ) So, the energy used by radio for one

transmit- receive-sleep cycle can be given byEr = v*(625.Ir + s.625.It + Le. 625.Is) (1)

This corresponds to a time, T

T = (1+s+Le)*625 (2)In this time S.(1+s+Le).625 – samples

would be available and buffered. So, buffer size equal to packet payload size, P is given as

P = S.(1+s+Le)*625 (3)

606060



Total energy consumed by different operations in the controller can estimated as sum of energies spent on analog amplification and digital processes including sampling, digitisation and buffering.

Ec = Eanalog + Esampling + EADC + Ebuffering (4)

As energy for analog amplification for any time is independent of both the sampling rate and transmission packet size, so it is ignored. Energies for sampling and ADC are considered dependent only on sampling frequency, S and do not vary with transmission packet size so, the equation 4 reduces to

Ec = [v*S*(Isa)*T] + Ebuffering (5)where, Isa represents sum of currents consumed for various processes including sampling and digitisation. Using expression for sum of first n natural numbers, current for holding a buffer filling at a constant rate would be,

Ib*((Peak buffered)* (Peak buffered+1))/2where Ib is the current consumed for buffering a sample byte. Thus, Ebuffering comes out to be,

Ebuffering = v* Ib*T((P)*(P+1))/2 (6)

B. Formulation of cost function Overall cost function is based on

energy consumption and latency as the two metrics of the network performance. Energy consumption of the system is sum of energies consume in different processes of the system. using equations 1, 5 and 6 we get the

energy E, consumed at the node as

E = Ec + Er = [v*(625.Ir + s.625.It + Le. 625.Is)] +

v*[S*(Isa)*T] + [v*Ib*T((P)*(P+1))/2] (7)Latency is defined on per sample basis as the

time interval between collection of the sample and its transfer to the gateway node. Ignoring the ADC conversion time, the maximum delay in reaching of a sample is T. so the average per sample latency can be estimated as

L = T/2 = (1+s+Le)*625 /2 (8)

The overall cost function can be given as cost of energy consumption and cost of latency period.

C = E + λ . Lwhere, the E is given by equation (7) above and L is given by equation (8) above. The parameter λ can be interpreted as dimension equalizer. It can also be considered as relative importance given to latency with respect to energy. Combining the constraints that s can be between 1 and 5 only and that the energy given by E above is the energy based on T time periods for packet size P. Thus the optimisation problem can be written as

Minimise [ E/P + λ. T/2] where E is given by equation (7) above and minimization is over time period T. This is a geometric program solvable in polynomial time and thus a convex in optimisation variable T. [13] .



II. Simulation results and discussion In order evaluate the minimisation problem

we substitute the values of different variables and vary the variables to their limits. First we consider the sampling rate. For ECG monitoring, AAMI guidelines suggest a minimum rate of 300 Hz. So, 300≤ S. A higher sampling rate would be preferable, but the simple logic that higher will increase the not only the sampling current but also the data payload and so minimum clinically advisable limit is good enough. So, we set S = 300. From the data sheets of Bluetooth module [14] we substitute Ir = It = 50mA and Is = 20mA (for sniff mode or sleep mode). We vary the s from 1 to 5 for different data formats suggested in bluetooth and summarised in table 1.

We observe ACL data type given by DH5 is most versatile and is used for getting the results. We also note that maximum latency

at the extreme of DH5 comes to be (794 + 6) slots of 625 µsec. This comes out to be average latency of 0.25 secs, which for most practical purposes is tolerable. We set λ = 0 . The energy curves were observed to be monotonously decreasing. The results at extreme values are tabulated in table 2.

We observe that ACL data type of DH5 is best suited for transmission of ECG signal with reasonable latency. Most Bluetooth modules which are configured for serial port profile of EDR (corresponding to payloads of 81 bytes) are actually not really the best alternative. The DH5 although apparently most suitable requires a tolerance of signal latency upto 500 µsecs. It also demands a buffer size of 150 bytes (=S*latency). Thus if the latency of upto 0.5 secs is allowed DH5 mode with 320 bytes of payload, 5 slots of master transmission and 794 slots of sniff mode is the optimal setting for ECg data

Table 1. ACL Data type [12,14]

ACL Data type Range of variables Payload (bytes)

(P)

Slots of 625µsec used by master

(S)

Slots of 625µsec in Sniff or sleep mode

(Ts) DH1 1 - 27 1 only 4 – 70 2-DH1 (EDR) 1-54 1 only 4-142 3-DH1 (EDR) 1-81 1 only 4-216 DH3 1-180 1-3 4-130 DH5 1-320 1-5 4-794

Table 2. Result of simulation at extreme points

Sample payload (bytes)

S Time slots of

625µsec

Le Time slots of

625µsec

Energy metric (mAs)

Average Latency msecs

1 1 4 30.0 1.875 81 1 216 22.1 68.125 320 5 794 20.2 250

626262



transmission. The results can also be interpreted as confirmation of the fact that Bluetooth data packets have a large overhead and it is optimal to have as big payload as possible.

Also it is important to note that we have assumed ideal operating conditions and so have not accounted for ambient noise. Further, it is important to take note of the fact that we have only considered single ECG sensing node. The metrics are going to change drastically if multiple and / or heterogeneous sensors are taken into consideration of wireless body sensor network.

III. Conclusion We have developed a Framework for

optimisation of wireless sensor node for continuous ECG transmission. The model is a geometric program which is a convex optimization problem. It can be solved for optimisation of desired parameters.

The model has been worked for standard Bluetooth data types. From the results, it is clear that making the data available to Bluetooth transmitter from the on-chip ADC is a good idea but the energy metrics are improved further if we buffer the digitised data rather than pass it straight to the radio module. Use of ACL data types DH3 or DH5 marginally improves the energy metrics of ECG sensor nodes provided noise immunity and latency are not critical. Tweaking the data transmission modes with suitable buffering to suit to DH5 can make the sensor node upto about 30% more energy efficient than transmitting signal as it becomes available.

References1. J. Ko, C. Lu, M. B. Srivastava, J. A.

Stankovic, A. Terzis, and M. Welsh, “Wireless Sensor Networks for Healthcare,” Proceedings of IEEE, vol. 98, no. 11, pp. 1947–1960, Nov. 2010.

2. A. K. Triantafyllidis, V. G. Koutkias, I. Chouvarda, and N. Maglaveras, “A pervasive health system integrating patient monitoring, status logging and social sharing.,” IEEE Journal of Biomed. Heal. informatics, vol. 17, no. 1, pp. 30–7, Jan. 2013.

3. M. A. Hanson, H. C. P. Jr, A. T. Barth, K. Ringgenberg, B. H. Calhoun, J. H. Aylor, and J. Lach, “Body Area Sensor Networks :,” vol. I, 2009.

4. P. Rashidi and A. Mihailidis, “A Survey on Ambient-Assisted Living Tools for Older Adults,” IEEE J. Biomed. Heal. Informatics, vol. 17, no. 3, pp. 579–590, May 2013.

5. U. T. Pandya and U. B. Desai, “A novel algorithm for Bluetooth ECG.,” IEEE Trans. Biomed. Eng., vol. 59, no. 11, pp. 3148–54, Nov. 2012.

6. M. Seyedi, B. Kibret, D. T. H. Lai, and M. Faulkner, “A survey on intrabody communications for body area network applications.,” IEEE Trans. Biomed. Engineering , vol. 60, no. 8, pp. 2067–79, Aug. 2013.

7. R. Balani, “Energy consumption analysis for bluetooth, wifi and cellular networks,” [Online. http//nesl. ee. ucla. edu/fw/documents/reports/balani.htm, assessed



2nd December 2013.]8. U. R. Shoaib, O. Rehman, Z. Akbar, and

J. Iqbal, “Performance Evaluation of Bluetooth and Zigbee Using Monte Carlo Simulation,” International journal of computer science issues, vol. 9, no. 1, pp. 235–237, 2012.

9. H. Li and J. Tan, “Body sensor network based ECG segmentation and analysis.,” Conf. Proc. IEEE Eng. Med. Biol. Soc., vol. 2007, pp. 5166–9, Jan. 2007.

10. J. Linsky, “Bluetooth and power consumption : issues and answers,” rfdesign.com, November 2001, pp. 74–80, .

11. T. Bourk, P. Hauser, and D. Roberts, “Simple pairing - Whitepaper” Bluetooth SIG, 2006, revision- 10r00.

12. “Bluetooth specifications : Logical link control and adaption protocol” SIG, July 1999, pp. 247-314.

13. Aharon Ben-Taly and Arkadi Nemirovski, ”LECTURES ON MODERN CONVEX OPTIMIZATION”, Georgia Institute of Technology, Fall 2013. Accessed online, http://www.isye.gatech.edu/faculty-staff/profile.php?entry=an63 , June 2014.

14. “Advanced user manual”, Rowing networks, version 4.77, 2009.

Int.J.Curr.Microbiol.App.Sci (2016) 5(11): 472-477

472

Original Research Article http://dx.doi.org/10.20546/ijcmas.2016.511.054

Antibacterial Activity of Green Tea Leaves

Ponnusamy Ponmurugan1*, Fahad Khalid Aldhafiri

2 and Santhanaraj Balakrishnan

2

1Department of Biotechnology, K.S.Rangasamy College of Technology,

Tiruchengode - 637 215, Namakkal District, Tamil Nadu, India 2College of Applied Medical Sciences, Majmaah University, Al Majmaah,

The Kingdom of Saudi Arabia *Corresponding author

A B S T R A C T

Introduction

In recent years it has been turned the

attention from chemotherapeutic agents that

to focus on green pharmacy which may be

due to immediate action and metabolism.

According to World Health Organization

(WHO) report, traditional medicine systems

serve the health need of about 80% of the

world’s population (WHO, 2013).

Traditional system of medicine is being

followed in preparing novel drug products

from medicinally important plants. The

increasing concentration of drugs towards

green pharmacy may be due to emergence of

antibiotic resistance organisms, side effects

and economic concern too. The alarming

situation on the steady increase of antibiotic

resistance microorganisms throughout the

world, which resulted increased illness

followed by deaths (Levy, 2002) and

highlighting the search for a novel

antimicrobial agents (Stepanovic et al.,

2003).

It is reported that green tea leaves are known

for antimicrobial activity against numerous

human pathogenic microorganisms. In

recent years, green and black teas are being

tested against both The major difference

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 5 Number 11 (2016) pp. 472-477

Journal homepage: http://www.ijcmas.com

The aim of the present investigation is to study the antibacterial activity of different

green tea leaves subjected for solvent extraction against seven clinically isolated

antibiotic resistant bacteria. Different types of tea leaves such as mother, scale

(cataphyll), first, second and third and buds of tea plant were extracted successfully

with petroleum ether, ethyl acetate and chloroform and tested for their antibacterial

activity by well diffusion method. The results showed that a remarkable

antibacterial activity of green tea samples of first and second leaves followed by

leaf bud against the tested organisms was recorded. However zone of inhibition is

moderate in third leaf and mother leaf which may be due to several factors

including origin of tea leaves, vegetation habits, age of the leaves and extract

preparation. Ethyl acetate extract of tea leaves was found to be the most effective

against human pathogenic organisms in terms of growth inhibition.

K e y w o r d s

Tea leaves, Camellia

sinensis,

Antibacterial

activity, Antibiotic

resistant bacteria.

Accepted:

23 October 2016

Available Online:

10 November 2016

Article Info

http://dx.doi.org/10.20546/ijcmas.2016.511.054


473

between green and black teas is in the

fermentation practice takes place during

manufacturing process which is required to

produce black tea powder. In case of black

tea powder production, three leaves and a

bud are fermented and/or oxidized after they

have been dried in manufacturing process.

The phytochemicals present in tea leaves

and buds are highly sensitive to oxidation

process. According to Toda et al. (1989)

daily consumption of green tea lead to kill

pathogenic microorganisms like

Staphylococcus aureus, Vibrio

parahemolyticus, Clostridium perfringens,

Bacillus cereus and Pleisomonas

shigelloides. Green tea contains around 30-

40% polyphenols and related compounds

such as Epigallocatechin gallate (EGCG),

Epicatechin gallate (ECG), Epigallo

catechin (EGC) and Epicatechin (EC) but

black tea contains only 3-7% polyphenols

(Diane et al., 2007). Among these EGCG is

the most luxuriant component in tea extract

and the most potent chemical tested for

biological activity in terms of inhibiting the

growth of pathogenic microorganisms

(Archana and Abraham, 2011).

Tea leaves are polymorphic in nature and

known for its antibacterial activity against

many pathogenic microorganisms. It is

beeing shown to have a wide range of

antioxidant, anti-inflammatory, anti-

carcinogenic and antimutagenic properties

against pathogens. Antimicrobial activity of

green tea and black tea extracts were studied

using clinically important pathogenic

microorganisms and compared by several

Investigators (Diane et al., 2007; Archana

and Abraham, 2011). But no literatures are

available on antibacterial activity of

different green tea leaves against a wide

variety of pathogenic microorganisms.

Hence the present investigation made an

attempt to study the antibacterial activity of

different green tea leaves against selected

bacterial pathogens isolated from clinical

samples.

Materials and Methods

Tea (Camellia sinensis (L.) O.Kuntz) leaves

and buds were collected from Parry Agro tea

plantations, Valparai, Tamil Nadu, India

during August 2016 for the present study.

Three leaves and a bud are generally used

for black tea manufacturing which is used to

stimulate central and autonomous nerves

system of human beings to get freshness. In

addition, other leaves such as mother and

scale (cataphyll) leaves were also collected

for the present investigation. The solvents

such as petroleum ether (10.5%), ethyl

acetate (15.5%) and chloroform (15.5%)

were used as solvent extracts (Sawaya et al.,

2004). Phytochemical screening of tea

leaves revealed the presence of antioxidant

compounds like polyphenols and catechin in

petroleum ether extract, caffeine, theaflavins

and thearubigins in ethyl acetate and

saponins and tannins in chloroform extracts

as per the report of Diane et al. (2007).

A total of 7 clinical isolates of bacteria such

as Salmonella typhi, Escherichia coli,

Bacillus subtilis, Klebsiella pneumonia,

Pseudomonas fluorescence, Aeromonas

hydrophylla and Staphylococcus aureus

were used. The selected organisms are

resistant to antibiotics commonly used for

treatment of various diseases caused by

these bacteria and the MAR index value also

calculated. Mueller Hinton agar (Himedia)

plates were prepared and seeded with 16

hour old cultures of the said pathogenic

microorganisms. Before seeding the

inoculum, the turbidity was adjusted to the

turbidity of 0.5% of McFarland solution and

challenged with different concentration of

tea leaf extracts by well diffusion method.

The plates were incubated at 37C under

controlled condition and the zone of


474

inhibition of pathogenic microorganisms due

to solvent extracts containing tea

phytochemical compounds was recorded

subsequently.

Results and Discussion

All the extracts of tea samples (leaves and

buds) showed good activity against the

tested pathogenic microorganisms. Among

the tea leaves, first and second leaves

showed greater activity than other leaves

such as mother and scale followed by tea

leaf bud extracts (Table 1-3). Ethyl acetate

extracts of both tea leaves and buds showed

a higher growth inhibition zone followed by

chloroform extracts and comparatively

leaves extract was found to be more active

than tea bud extracts. This tends to show

that the active ingredients in the tea leaves

were better extracted with ethyl acetate than

chloroform and petroleum ether. The least

performance in terms of growth inhibition in

mother leaf may be due to minimum active

participation in physiological maturity and

proximity. Being an autotropic nature of tea

leaves, the fully expanded mother leaf

supplies adequate amount of sugars to sinks

which is physiologically and biochemically

active (Ponmurugan et al., 2007). This gives

credence to its ethnopharmacological use as

a remedy to treat infections and diseases

caused by above microorganisms.

Table.1 Antibacterial activity of tea mother and scale (cataphyll) leaf extracts a

---------------------------------------------------------------------------------------------------------------------

Bacteria Zone of inhibition in mm

---------------------------------------------------------------------------------------------------

Streptomycin Ciprofloxacin Petroleum ether Ethyl acetate Chloroform

1% 2% 1% 2% 1% 2%

---------------------------------------------------------------------------------------------------

Tea mother leaf extracts

Salmonella typhi 11.3 13.3 6.5 10.0 11.0 15.5 9.3 16.7

Escherichia coli 13.5 14.5 2.3 04.5 6.3 14.5 5.7 12.5

Bacillus subtilis 10.3 13.5 6.7 14.5 8.5 18.7 8.5 16.5

Klebsiella pneumonia 10.7 11.0 2.3 04.3 6.7 13.0 6.0 10.0

Pseudomonas

fluorescence 15.5 14.7 2.0 05.5 12.3 18.7 10.0 15.5

Aeromonas

hydrophylla 16.0 16.5 4.0 06.5 10.5 14.3 8.5 16.5

Staphylococcus aureus 16.3 15.3 4.3 06.7 10.5 14.3 9.7 17.3

Tea scale (cataphyll) leaf extracts Salmonella typhi 11.3 13.3 2.3 4.0 4.5 6.5 2.7 4.5




Pseudomonas

fluorescence 15.0 14.5 1.3 2.5 2.3 5.5 1.7 3.0

Aeromonas

hydrophylla 16.3 16.7 1.5 2.7 2.3 4.5 2.7 4.5


--------------------------------------------------------------------------------------------------------------------- a

Values are the means of five replicates


475

Table.2 Antibacterial activity of tea first, second and third leaf extracts a

---------------------------------------------------------------------------------------------------------------------


---------------------------------------------------------------------------------------------------


1% 2% 1% 2% 1% 2%

---------------------------------------------------------------------------------------------------

Tea first leaf extracts





Pseudomonas

fluorescence 15.3 14.7 2.3 4.5 8.0 15.3 8.3 15.5

Aeromonas

hydrophylla 16.5 16.0 3.3 7.3 8.5 14.5 6.3 12.5


Tea second leaf extracts





Pseudomonas

fluorescence 15.3 14.0 4.5 8.5 9.5 17.3 8.5 15.0

Aeromonas

hydrophylla 16.0 16.3 6.3 10.7 10.0 17.5 9.3 17.0


Tea third leaf extracts





Pseudomonas

fluorescence 15.5 14.3 4.5 08.5 08.7 18.7 8.7 15.5

Aeromonas

hydrophylla 16.3 16.3 5.5 10.5 10.3 19.3 10.0 17.5


--------------------------------------------------------------------------------------------------------------------- a



476

Table.3 Antibacterial activity of tea bud extracts a

---------------------------------------------------------------------------------------------------------------------


---------------------------------------------------------------------------------------------------


1% 2% 1% 2% 1% 2%

---------------------------------------------------------------------------------------------------





Pseudomonas

fluorescence 15.5 14.5 1.0 3.0 3.0 4.5 1.5 2.5

Aeromonas

hydrophylla 16.3 16.3 0.5 1.5 2.3 4.5 1.0 2.5


--------------------------------------------------------------------------------------------------------------------- a


The composition of tea leaves is highly

complex and its biological activity fully

depend on tea plantation hills height where

it was collected and abiotic factors such as

temperature, relative humidity, rainfall and

sunshine. The factors may influence the

antibacterial activity of green tea leaves

which include origin of tea plants, genetics

breeding, extract preparation and nature of

tea clones and seedlings (Grange and Davey,

1990) and geographic differences

(Boyanova et al., 2005). Therefore the effect

of biological activity of tea leaves varies

according to the origin is inevitable. In view

of supporting this, Brumfitt et al., (1990)

found no inhibition of Streptococci by tea

extracts was noted but it is controversial

with other reports (Nieva Moreno et al.,

1999). However in the current investigation,

the results of six samples of tea leaf extracts

revealed only minor variation on its

antibacterial activity, it may be due to mixed

type vegetation and the study area locality is

near with one another. Further it was

exhibited bacteriostatic activity in general

and bactericidal activity at high

concentration in particular (Drago et al.,

2000) against the microbial pathogen.

The vegetation system in the study area is a

mixed type, hence the minor variations were

observed in the antibacterial activity which

collected from different localities. Drago et

al., (2000) mentioned the reasons for the

difficulties in comparison the results of

antimicrobial properties of medicinal plants.

In the present investigation, raw tea leaf

materials were tested against the clinically

isolated organism hence the inhibition

activity may be minimal even though the

high volume of extract was used (Hegazi

and El Hady, 2001). Further the vast work

was done on tea which is used as

commercial product and this work was a

new attempt in terms of using different leaf

materials such as mother, scale (cataphyll),

first, second, third leaves and leaf bud,

hence the comparison with the earlier result

was difficult. The important finding of this

study is displayed the ability of antibacterial

activity of Indian tea leaf against antibiotic

resistant bacteria, which give a new


477

dimension in green pharmacy. The present

investigation may be concluded that three

leaves and bud of green tea were found to be

effective in inhibiting the pathogenic

microorganisms through antibacterial

studies.

References

Archana, S. and Abraham, J. 2011.

Comparative analysis of antimicrobial

activity of leaf extracts from fresh green

tea, commercial green tea and black tea

on pathogens. J. Appl. Pharmaceut. Sci.,

01(08): 149-152.

Boyanova, L., Gergova, G., Nikolov, R.,

Derejian, S., Lazarova, E., Katsarov, N.,

Mitov, I. and Krastev, Z. 2005. Activity

of Bulgarian propolis against

Helicobacter pylori strains in vitro by

agar-well diffusion, agar dilution and disc

diffusion methods. J. Med. Microbiol.,

54: 481-483.

Brumfitt, W., Hamilton-Miller, J.M. and

Franklin, I. 1990. Antibiotic activity of

natural products. Microbios., 62: 19-22.

Diane, L., McKay, R. and Jeffrey Blumberg,

B. 2007. Roles for Epigallocatechin

gallate in cardiovascular disease and

obesity: An introduction. J. American

Col. Nutrit., 26 (4): 362-365.

Drago, L., Mombelli, B., De Vecchi, E.,

Fassina, M.C., Tocalli, L. and Gismondo,

M.R. 2000. In vitro antimicrobial activity

of Propolis dry extract. J. Chemother. 12:

390–395.

Grange, J. M. and Davey, R.W. 1990.

Antibacterial properties of Propolis (bee

glue). J. R. Soc. Med., 83: 159–160.

Hegazi, A.G. and El Hady, F.K. 2001.

Egyptian Propolis: 1-antimicrobial

activity and chemical composition of

Upper Egypt Propolis. Z. Naturforsch.,

56: 82–88.

Levy, S.B. 2002. Factors impacting on the

problem of antibiotic resistance. J.

Antimicrob. Chemoth., 49: 25-30.

Nieva Moreno, M.I., Isla, M.I., Cudmani,

N.G., Vattuone, M.A. and Sampietro,

A.R. 1999. Screening of antibacterial

activity of Amaicha del Valle (Tucuman,

Argentina) Propolis. J. Ethnopharmacol.

68: 97-102.

Ponmurugan, P., Baby, U.I. and Rajkumar, R.

2007. Growth, photosynthetic and

biochemical responses of tea cultivars

infected with various diseases.

Photosynthetica 45(1): 143-146.

Sawaya, A., Souza, K.S., Marcucci, M.C.,

Cunha, I. and Shimizu, M. 2004. Analysis

of the composition of Brazilian Propolis

extracts by chromatography and

evaluation of their in vitro activity against

Gram-positive bacteria. Brazilian J.

Microbiol., 35: 104-109.

Stepanovic, S., Antic, N., Dakic, I. and

Milena, S. 2003. In vitro antimicrobial

activity of Propolis and synergism

between Propolis and antimicrobial drugs.

Microbiol. Res. 158: 353-357.

Toda, M., Okubo, S., Hiyoshi, R. and

Shimamura, T. 1989. Antibacterial and

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How to cite this article:

Ponnusamy Ponmurugan, Fahad Khalid Aldhafiri and Santhanaraj Balakrishnan. 2016.

Antibacterial Activity of Green Tea Leaves. Int.J.Curr.Microbiol.App.Sci. 5(11): 472-477.

doi: http://dx.doi.org/10.20546/ijcmas.2016.511.054

http://dx.doi.org/10.20546/ijcmas.2016.511.054

Majmaah Jouurnal of Health Sciences ,Vol.5, issue 1, May 2017 - Shaban 1438

1

ORIGINAL ARTICLE

Towards a Simple System for Indicating Temporal Variations in Blood Pressure

Jaspal Singh1, Dr. Santhanaraj Balakrishnan2 1. Principal Engineer, Centre for Development of Advanced Computing, Mohali – 160071 India.

[email protected] 2. Professor, Department of Medical Equipment Technology, College of Applied Medical Sciences, Majmaah University, Al Majmaah –

11952, Kingdom of Saudi Arabia. Email: [email protected] on:16th November 2016; Accepted on: 4th January 2017

Abstract

Blood pressure is an important physiological

parameter to be monitored. For several medical

conditions, it is important to quickly know the

rise or fall in blood pressure. While researchers

are working to evolve a reliable continuous non-

invasive arterial pressure (CNAP) monitoring

devices based on sophisticated signal processing

with various technologies including Peñáz

principle & pulse wave velocity (PWV); we

present a very simple system to continuously

indicate the variations in blood pressure. Using

the typical shapes of the pulse wave obtained

by infra-red photoplethysmography (PPG) from

brachial and radial artery, we estimate the changes

in pulse transit time (PTT). This change is effected

onto a change in tone of an audio beep. The simple

circuit was assembled and tested. The details are

presented in the paper.

Keywords— pulse wave velocity, pulse transit

time, blood pressure, differential amplifier,

photoplethysmogaphy

امللخص

التي يتعني رصدها ضغط الدم هو املعلومة الفسيولوجية املهمة

أو ارتفاع معرفة رسعة املهم فمن ، مرض اي تشخيص عند

انخفاض ضغط الدم عند العديد من احلاالت الطبية.

يف حني أن الباحثني يعملون عىل تطوير أجهزة رصد موثوق هبا

عىل أساس إشارات مستمرة و متطورة

هذا يف املطور الرصد جهاز حيتوي الرشياين. الضغط لقياس

البحث عيل خمتلف التقنيات بام يف ذلك مبدأ بيناز ورسعة موجة

النبض

االختالفات رصد ملواصلة جدا بسيط نظام البحث هذا يقدم

النبضية للموجة النموذجية األشكال طريق عن الدم ضغط يف

احلمراء حتت األشعة طريق عن عليها احلصول تم التي

)فوتوبثيزموغرايف( من العضد والرشيان الكعربي،

ومن ثم نقوم بتقدير التغيريات يف وقت عبور النبض . يتم تنفيذ

هذا التغيري عىل اختالف يف نغمة صوت صافرة اجلهاز . تم جتميع

الدائرة البسيطة واختبارها. التفاصيل هي املقدمة يف هذه الورقة.

Jaspal Singh. Towards a Simple System for Indicating Temporal Variations in Blood Pressure


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I. INTRODUCTION

Blood pressure is the pressure exerted by the blood on the walls of vessels it flows through. It can be measured in different parts of the body using invasive or non-invasive techniques depending on the availability of access to vessel or underlying medical requirements. Routinely, it refers to the arterial peak (systolic) and lowest (diastolic) pressures exerted in arteries as the heart pumps the blood. As per WHO guidelines, the blood pressure is measured in the brachial artery held at heart level [1]. The gold standard instrument for non-invasive measurement of blood pressure is sphygmomanometer used in ausculatory method. The ausculatory and the other method, called oscillometric method, both involve inflating a cuff tied around the arm to occlude the brachial artery of the subject. Both the methods are widely used for cardiac screening as well as monitoring for various cardiovascular and other health issues. Intermittent obtaining of blood pressure with either of these methods is, in general, sufficient and good indicator for most cardiovascular diseases. The commonest cardiovascular issue, Hypertension or high arterial blood pressure, is routinely screened and monitored by either of these methods.

The blood pressure of an individual keeps on varying in response to various physiological stimuli. This is an adaptive and necessary response of body. However, there are certain medical conditions [2] like critical pregnancies, certain surgeries, cerebrovascular diseases [3], etc.in which blood pressure is required to be monitored rather continuously. Under certain unstable hydration conditions (due to dialysis or pharmaceutical effects) also, regular and continuous non-invasive blood pressure can avoid acute conditions [4]. Even gross indication of continuous variations in blood pressure can also be useful in most cases. The standard non-invasive method of occluding the artery and measuring blood pressure does not lend itself well to repeated or continuous measurements. Although, some situations may warrant invasive monitoring but, wherever possible, non-invasive (without using the cannula or pricking the skin) monitoring would

be preferable. It not only drastically reduces the chances of infection but also is convenient for both the patient and the caregiver. Researchers have tried several methods towards continuous non-invasive measurement of blood pressure. Finapres equipment [5] works on Peñáz principle based on exerting an opposing force on the finger cuff to prevent physical disruption of blood pulse. The emerging methods based on Pulse wave velocity (PWV) or pulse transit time (PTT) also promise to become more convenient alternatives to the occlusion methods [6].

The blood pumped by heart rushes into the arteries as a pulse. This mechanical wave of pulse spreads into complete vasculature every cardiac cycle. Stiffening of arteries and reduction of radius of arteries leads to increased blood pressure. Higher blood pressures have been related to increased pulse wave velocity [7]. Standard method of pulse wave velocity measurement consists of measuring the pulse wave recorded by one of the several available methods at carotid artery and at femoral artery (ca-PWV) [8].The PWV is given as the physical distance between the two sites of measurement per unit time elapsed (pulse transit time) between pulse arrival times at two sites. This PWV method of estimating blood pressure and underlying thickening of arteries requires availability of trained technician and a relatively complex hardware. For measuring blood pressures it requires elaborate calibration and does not easily lend itself to automated measurements [7].

For meeting the situations where the objective is only to grossly estimate the temporal variations in blood pressure, we propose a simple technique using conveniently obtained pulse waveforms from the arm of the subject. It avoids precise identification of pulse arrival times and calibration. A simple circuit based on the technique was designed, assembled and tested. The developed system indicates the temporal variations of blood pressure as audio signals. The following paragraphs elaborate this. It can further be developed into an unobtrusive ubiquitous non-invasive blood pressure measurement device.



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II. THEORY

As indicated above, increased blood pressure shows as heightened pulse wave velocity. Also, PWV is

PWV = ɭ / PTT (1)

Where ɭ = physical length of the arterial segment and PTT is the pulse transit time. Formally, PTT is given as difference in pulse arrival times at proximal and distal ends of artery. So, the main goal for indicating blood pressure continuously is to continuously monitor the pulse arrival time at two points of an artery. This is either done manually or requires sophisticated processing. To circumvent this, we can exploit the typical shape of pulse waveforms. Using the basic idea of pulse shapes at different body parts from the literature and based on convenience of wearing and maneuverability of device to heart levels, the brachial artery and the radial artery were chosen as two suitable points. Palm and fingers, although equally convenient, were not selected as the pulse waves at these points show significant reflected waveforms and were considered not suitable.

Typical pulse pressure waveforms obtained at brachial and radial artery are shown in fig 1 below. The times corresponding to point A and B indicate the pulse arrival times at respective locations. The time gap between these two time points indicate the pulse transit time. For measuring this PTT, we need to identify and locate the points on the two waveforms. This would evidently require complex signal processing.

We propose to simply take the difference of the two pulse waveforms with a differential amplifier and subject the output to a dynamic threshold, such that the output is

Vo= V1-V2 if (V1- V2) > 0.3* max.(V1- V2)

0 otherwise.

(2)

where, V1 and V2 are the pulse waveforms.

Fig1. Pulse Waveforms

This effectively gives us a clean workable signal which is positive for a time equal to two third of sum of PTT and upstroke time of distal pulse waveform. If tVo’ and tVo’’ indicate the zero crossing times of the signal Vo, then the time for which the Vo remains positive, TVO is given as,

TVO = tVo’ – tVo’’ = 2/3 ((PTT) + (upstroke time)) (3)

Now let the waveforms V1 and V2 shift relatively due to change in blood pressure. The zero crossing Times of Vo shall vary accordingly. For noting the change in blood pressure we notice the change in TVO. The change, Δ TVO from equation (3) above would be

Δ TVO = 2/3 ((Δ PTT) + (Δ.Upstroke time)) (4)

As, the upstroke time of pulse waveform relates to the ventricular compression time, it can be safely approximated to be constant. So, Δ .Upstroke time = 0. Thus, we get Δ TVO proportional to Δ PTT. This fact is exploited for indicating changes in blood pressure. In order to keep the system simple, analog processing was used. The indication is given in terms of an audio tone, the frequency of which varies with the change in TVO times. This scheme is shown in the block diagram (Fig 2) below.

Conditioning

Conditioning

Differential Amplifier

Ramp Generator

Audio Signal (freq ~ Vpeak)

Radial Pulse Signal

Brachial Pulse Signal

Clipping

Fig 2. Schematic Diagram of the Technique



4

III.IMPLEMENTATION AND TESTING A. Sensor Design

Researchers have used different methods to detect the pulse wave and to analyze the pulse wave velocity [9]. Researchers have also analyzed the contour of pulse wave for different clinical estimates [Predicting Arterial Stiffness From the Digital Volume Pulse Waveform, A knowledge-based approach to arterial stiffness estimation using the digital volume pulse.]. In our design, being primarilyinterested in arterial pulse waveform, Infra- red PPG was selected for its convenience and suitability based on [6]. Of the two, reflection and transmission modes, reflective sensors are much more unobtrusive and amenable to different body locations. So, two reflective PPG sensors were decided.

We used IR LED (Kingbright KP-1608 with peak λ 940nm and viewing angle 120°) and phototransistor (Vishay VEMT-2020X01, 790 to 970nm with half sensitivity angle = 15degrees) mounted on small circuit board. The LED was placed in the middle and photodetector was mounted at a distance from it. This test-sensor was applied to wrist and arm using a band and the signal obtained. This was repeated for different source –detector distances. Based on test data, two identical sensors were then made with a source – detector distance of 8mm.

B. System Design

The system was implemented on a specially designed circuit board and assembled using hand soldering. The PPGsignals obtained from sensors are band- passed between 0.5Hz to 20 Hz. These were then amplified using Operational Amplifier (LM348 with variable gain resistor). The amplified signals were fed to the two ends of differential amplifier (THS 4121, Texas Instruments). The output of the differential

amplifier is fed to a peak holding circuit and three resistances are used to get one third ofthe peak signal. Simple comparator

circuit (using LM348) applies this threshold to the signal. Output of the comparator powers (gates) a RC ramp generator. So, longer the PTT the longer the gating and so, greater the ramp output. The peak of the ramp output, with peak holding, decaying circuit and Voltage controlled oscillator (VCO, MAX 038 of Maxim Integrated), is translated into an audio beep whose frequency changes with PTT. The tone of this audio beep was thus indicative of changes in blood pressure of theindividual.

C. Test Set-up

The two sensors were placed at brachial artery and radial artery at wrist respectively and secured with bands. The sensor outputs were given to the designed circuit assembled on a printed circuit board. The output audio beeps were listened to carefully for testing the system. The amplified sensor outputs and the clipped output were tapped out to a custom built acquisition system consisting of three ADCs sampling at 300 Hz in round robin fashion and of microcontroller (AVR evaluation kit) interfaced to PC via serial port. A software program was made to display the waveforms in real time. The Fig 3 below shows the test set up and fig 4 shows a random screenshot of output.

Fig 3. Test Set-up



5

Fig 4 Random Screenshot

The system was applied to a normal healthy volunteer and audio tone listened. His systolic, diastolic and mean blood pressure was also recorded using automatic blood pressure monitor (Omron HEM-7120). The volunteer was then shown a random video clip from the collection of movies in library and the above process repeated. After sufficient rest, he was asked to undertake strenuous exercise and all measurements were repeated. The process was repeated randomly for the same individual without disturbing the sensors attached. This was done for each of ten normal healthy volunteers recruited for the purpose. The results are tabulated in table 1 below.

IV.RESULTS AND DISCUSSIONSThe experiment was run for seventy instances of change in blood pressure. Observations by listening to the audio tone have correlated well with the change in systolic blood pressure as measured using standard NIBP instrument. The Fisher’s

TABLE I. Summary of observations

No of instances of change

in Systolic BP (mm of Hg) Total

≤ 20

No Change ≤ 20 >20

After watching

movie

Actual 1 6 3 0 10

Observed by tone 0 5 5 0 10

Afterexercise

Actual 0 1 3 6 10

Observedby tone 0 0 3 7 10

exact test on the results show a p=0.0064 (and total ‘unusual tables’ probability = 0.952). thus, the results are significantly related. This experiment is an initial result of feasibility and suitability of proposed novel system for indicating changes in blood pressure of the subject. The system has well indicated the changes in systolic blood pressure of an individual. Various possibilities of error and bias of the observer have not been really addressed. So, validation of the system with extensive experiments and on a larger population is imperative. The simplicity of the system comes from the selection of pulse waveforms and the method to estimate changes in PTT. Its suitability in patients with various cardiovascular issues remains to be explored. Nevertheless, it can be further developed and customized to precise measurements of blood pressure for most cases. Further, the application of sensors is prone to lot of errors requires extensive experimentation for standardization. In this experiment we have applied sensors by looking at the real time waveforms and reapplying (or adjusting the pressure) if optimal waveform matched with previously stored waveform is not obtained. In practice, robust method of locating the pulse and applying the sensors need to be worked out.

V. CONCLUSION We have designed a simple circuit that

outputs an audio beep indicating changes in blood pressure. It uses the PPG signals obtained from brachial and radial artery of the single arm. By virtue of the typical signal shapes, passing the signals through a differential amplifier gives a waveform indicating current pulse transit time and therefore, the blood pressure. The circuit was assembled and tested. While exact measurements would require rigorous processing and calibration, the presented system was found suitable for gross indication of changes in blood pressure.



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measurement in humans,” in AHA Circulation, vol. 111, pp. 697-716, 2005.

[2] Fortin, J., Marte, W., Grüllenberger, R., Hacker, A., Habenbacher, W., Heller, A., Wagner, C., et al. (2006)., “Continuous non-invasive blood pressure monitoring using concentrically interlocking control loops”. Computers in biology and medicine, vol 36(9), pp 941–57.

[3] Brickman AM, et al., “Long-term Blood Pressure Fluctuation and Cerebrovascular Disease in an Elderly Cohort”, in Archives of Neurology 67 (5): pp- 564–569, 2010.

[4] http://en.wikipedia.org/wiki/continous_non-invasive_arterial_pressure accessed 6th June, 2014.

[5] Wesseling, K. H. “A century of noninvasive arterial pressure measurement- from MAREY TO PENAZ & FINAPRES,” in Homeostasis om health and disease, vol 36, no.2-3 (1995): pp 50-65.

[6] J. Blacher, R. Asmar, S. Djane, and Safar ME, “Aortic pulse wave velocity as a marker of cardiovascular risk in hypertensive patients”. In Hypertension 33 (5), (May 1999, pp -1111–7

[7] JM Padilla, et al. , “Assessment of Relationships between Blood Pressure, Pulse Wave Velocity and Digital Volume Pulse,” in Computers in Cardiology 2006, 33, pp- 893−896.

[8] Josep Sola, S F Rimoldi and Y. Allemann, “ ambulatory monitoirng of cardiovascular system : role of Pulse wave velocity,” in New developments in biomedial engineering, Intech Open, 2012.

[9] J. Bhattacharya, P. P. Kanjila, and V. Muralidhar, “Analysis and characterisation of photo-plethysmographic signal,” IEEE Trans. Biomed. Eng., vol. 48, no. 1, pp. 5–11, Jan. 2001

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