STUDY ON EXTRACTION OF CHLOROPHYLL FROM SIKLWORM WASTE (FEACES BOMBYCUM)

Concurrently, I would like to thank the topics of "Research on chlorophyllextract from silkworm excrement" and "Research on technological process ofproducing chlorophyll food additives I

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VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE

FACULTY OF BIOTECHNOLOGY

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Hanoi, December 2018

Student Tran Thi Oanh

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ACKNOWLEDGEMENTSFirstly, I would like to express my gratitude to my supervisor Dr Bui Thi ThuHuong for providing me an opportunity to do the final work in Vietnam NationalUniversity of Agriculture and giving me all support which made me complete theproject.

I would like to express my deepest gratitute to Dr Hoang Thi Bich and Dr DoTien Lam As my teachers and supervisors, their guidance not only fully supported mystudy but also encouraged, inspired me to complete my thesis

I am grateful to all of those with whom I have had the pleasure to work duringthis thesis semester Each of the members of Department of Protein Biochemistry hasprovided me extensive personal and professional guidance and taught me great lessonsabout both scientific research and life in general I would especially like to thankAssoc Prof Pham Hong Minh, the head of “Clean Technology and MaterialsDevelopment Center”, who gave me permission to perform the necessary works and

utilize the equipment in laboratory, Institute of Institute of Natural Products Chemistry

(INPC) I have furthermore to thank M.S Tran Thi Hoai Van, M.S Vu Thi Thu Le, fortheir support, interest and valuable help under the thesis period Also, I am thankful toand fortunate enough to get constant encouragement, support and guidance from Dr

Do Trung Sy, M.S Tran Huu Quang of Institute of Chemistry.

Concurrently, I would like to thank the topics of "Research on chlorophyllextract from silkworm excrement" and "Research on technological process ofproducing chlorophyll food additives (INS 140), copper chlorophyll (INS 141) andpropylene glycol alginate (INS 405) from natural materials" that provided financialsupport so that I could complete this thesis

It is also give my thankfulness to my family, to all of my friends, for sharing mydifficulties, and giving me various used advices during the process of learning andstudying

Thank you very much!

Hanoi, December 2018 Student

Tran Thi Oanh

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TABLE OF CONTENT

ACKNOWLEDGEMENTS iii

ABBREVIATIONS vii

ABSTRACT viii

INTRODUCTION 1

LITERATURE OVERVIEW 3

MATERIAL AND METHODS 21

RESULTS AND DISCUSSION 26

CONCLUSION AND PROPOSAL 46

REFERENCES 47

ADDENDUM 1 50

ADDENDUM 2 52

LIST OF FIGURE Figure 2.1 Standard absorbance spectra of chlorophyll a and b 4

Figure 2.2 Molecular structure off chlorophyll a 6

Figure 2.3 Molecular structure of chlorophyll b 6

Figure 2.4 Molecular tructure of chlorophyll d 7

Figure 2.5 Molecular structure of 7

Figure 2.6 Molecular structure of 7

Figure 2.7 Spinach (Spinacia oleracea) 9

Figure 2.8 Alfalfa (Medicago sativa) 12

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Figure 2.9 Mulberry leaves (Morus alba) 13

Figure 2.10 Spirulina algae (Spirulina plantensis) 13

Figure 2.11 Silkworms eat mulberry leaves 15

Figure 2.12 Silkworm excrement 15

Figure 2.13 Synthesis of the basic chlorin derivatives having reactive groups 17 Figure 3.14 Fresh silkworm excrement 21

Figure 4.15 Chlorophyll acetone extraction from various materials 26

Figure 4.16 Chlorophyll content and chlorophyll a ratio in some fresh materials .28

Figure 4.17 Chlorophyll content and chlorophyll a ratio in some dried materials .28

Figure 4.18 Experiment to test the level of mulching of silkworm excrement in some organic solvents 30

Figure 4.19 The solubility of the silkworm excrement in some organic solvents .31

Figure 4.20 Chlorophyll content in the silkworm excrement were affected by the extraction solvent 32

Figure 4.21 Effect of different NH4OH concentrations on chlorophyll extraction efficiency from silkworm excrement 34

Figure 4.22 Chlorophyll content in the silkworm excrement were affected by temperature 35

Figure 4.23 Effect of different material/solvent ratios on chlorophyll extraction efficiency from silkworm excrement 36

Figure 4.24 Effect of sample incubation time on chlorophyll extraction efficiency from silkworm excrement 38

Figure 4.25 Chlorophyll purification process from silkworm excrement 39

Figure 4.26 Chlorophyll purification process from silkworm excrement 40

Figure 4.27 Fragment did not contain chlorophyll 41

Figure 4.28 Fragment did not contain chlorophyll 41

Figure 4.29 Fragment contained chlorophyll (TA1) 41

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Figure 4.30 Technological process of extracting chlorophyll from silkwormexcrement 43

LIST OF TABLE Table 4.1 Chlorophyll content of some materials 27Table 4.2 Solubility and chlorophyll content of silkworm in some organicsolvents 30

Table 4.3 Effect of different concentrations of some solvents on chlorophyllextraction efficiency from silkworm excrement 31

Table 4.4 Effect of different NH4OH concentrations on chlorophyll extractionefficiency from silkworm excrement 33

Table 4.5 Chlorophyll content in the silkworm excrement were affected bytemperature 34

Table 4.6 Effect of different material/solvent ratios on chlorophyll extractionefficiency from silkworm excrement 36

Table 4.7 Effect of sample incubation time on chlorophyll extraction efficiencyfrom silkworm excrement 37

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TA Aceton extract of Bombycis Excrementum

HPLC High Performance Liquid Chromatography

TLC Thin layer chromatography

PTLC Prepared thin-layer chromatography

GPC Gel permeation chromatography

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Silkworm excrement was a potential chlorophyll extraction material with the amount

of chlorophyll obtained from fresh silkworm excrement is 0.568% (moisture content32.71%), corresponding to 0.845% dry biomass The appropriate solvent forchlorophyll extraction was 90% acetone (NH4OH: Acetone = 1: 9), 25oC extractiontemperature, 2% material / solvent ratio, 24h extraction time The highest containedchlorophyll was 0.619%, in which the chla ratio was 63.81% The extraction of 100 g

of fresh silkworm excrement with acetone procedured resulted in 2.94 g of chlorophyllprecipitation After the chlorophyll precipitation was purified by GPC, obtained 0.7 g

of partial purified chlorophyll with 70,58% of chlorophyll content (43,15% of chla and27,43% of chlb) The technological process to extract and purify chlorophyll fromsilkworm excrement was developed that was stable and practical

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INTRODUCTIONPresent day, the deliberate or incidental addition of food colorants that were notallowed or used in excess of the prescribed dosage had been seriously affecting thefood hygiene and safety, affecting to the health and psychological puzzling consumers.Due to its superiority, it is not toxic to humans, ensuring food hygiene and safety thanartificial synthetic pigment (coloring matter) Therefore, in the world today, weconcentrate on studying the extraction of pigment groups from cheap natural materials

or make use of by-products to collect pigment groups used as food colorants

Chlorophyll is a natural pigment that is a component of photosynthesis of plantsand photosynthesizing bacteria It is widely used in food, cosmetics, nanotechnologyand cancer treatment Chlorophyll extract is extracted with turtles, spinat and someleaves used as food colorants, the trade name is Natural green 3 or E140 used in thefood and beverage industry Chlorophyllin is a safe natural, colorless substance thatdoes not detect any toxicity to the human body

Chlorophyll is not only a natural colorant that is safe for human health but alsohad outstanding biological properties for good health Chlorophyll had many differentuses in medicine and industry In medicine, chlorophyll is used as a basic ingredient indiets and medicines Chlorophyll and derivatives were also used as photosensitizers todestroy cancer and anti-viral cells, wound healing and deodorant, blood sugarreduction for elderly patients

Silkworm waste or silkworm excrement (Faeces Bombycum or Exerementum Bombycis) is a popular folk medicine, which had effect on curing glycosuria During

digestion of silkworms, Chlorophyll is not digested and excreted in the digestive tract,

so in the silkworm excrement, the chlorophyll content is higher than that of commonplant materials, algae Therefore, the isolated process is simple, low cost chemicalsolvent and high economic efficiency, and could combine the intake of other preciouscompounds from the silkworm excrement

In this study, we investigated the extraction of chlorophyll from silkworm, thefirst step of purification and building the technological process to extract chlorophyllfrom silkworm excrement in the pilot scale

“Study on extraction of chlorophyll from silkworm waste”

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Purpose: Extraction and purification of chlorophyll from silkworm waste (silkworm

excrement)

Content:

- Determination of chlorophyll content in some materials

- Examination some factors (solvents, temperature, material/solvent ratio, extracttime) affect to chlorophyll extraction efficiency

- Particity purification of chlorophyll

- Bulding the technology process of chlorophyll extraction from silkwormexcrement

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LITERATURE OVERVIEW

2.1.1 Chlorophyll

Defination: Chlorophyll was a green molecule in plant cells which played

important role in photosynthesis process It absorbed sunlight and used it was energy

to synthesis carbohydrates from CO2 and water (Khaleghi E et al, 2012) Chlorophyllwas one of the valuable bioactive compounds that could be extracted from plants,algal, cyanobacteria It was used as a natural food colouring agent and hadantioxidant as well as antimutagenic properties (Hosikian A., 2010)

Under natural conditions, on average one leaf reflected 10% of the light rays,absorbed 70% and the remaining 20% propagates through the layers of leaf cellsbelow Photosynthesis used only 1% (mainly blue and red light rays) in the 70% oflight absorbed; 49% of the energy used to evaporate and leaves would radiate 20%.Thus, the useful wavelengths for plant photosynthesis were blue light with awavelength (430-460 nm) and red light (630 nm-720 nm) They were two groups ofpigments involved in photosynthesis: chlorophyll and carotenoids Chlorophyll wasthe major pigment that played the most important role in photosynthesis with thefunction of absorbing energy from sunlight, transforming it into an electron-stimulating form of the chlorophyll molecule Chlorophyll had the role of transportingenergy into the reaction center From the first absorbing light chlorophyll molecule tothe center of photosynthesis, it was through a structural system in the thilacoidmembrane, which contained many different chlorophyll molecules Light energymusted pass through the chlorophyll molecules to reach the reaction center (P700) Itparticipated in converting light energy into chemical energy at the P700 reaction center

by phosphorylation to form ATP and NADPH Chlorophyll had the ability toselectively absorb light, some of the light absorbed by the chlorophyll was strong,some areas were less absorbed, and some were less absorbed This had created theabsorption spectrum of chlorophyll In the absorption spectrum of chlorophyll, therewere two areas of light absorbed by the chlorophyll that make up the two peaks ofmaximum absorption.There was the red light area with a maximum of 662 nm and theblue-violet area with a maximum of 430 nm Green light was not absorbed by the

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chlorophyll but is completely reflective, so it was often observed that the leaves weregreen The carotenoid pigment was a yellow, orange pigment They were the satellitepigments of chlorophyll The absorption spectrum of this pigmented group was in theblue light area of 451nm to 481nm.

Figure 2.1 Standard absorbance spectra of chlorophyll a and b

Synthesis of chlorophyll: in plants, chlorophyll could be ombined from

succinyl-CoA and glycine, although pre-precursors to the formation of chlorophyll a and b wereprotochlorophyllide In angiosperms, the last step in the conversion ofprotochlorophyllide to chlorophyll, which is dependent on light and light-coloredplants if grown in the dark Non-vascular plants and green algae, there was acomplementary enzyme that does not depend on light and develops green even in thedark Chlorophyll was attached to the protein and could transfer the energy absorbed inthe required direction (G Mackinney, 1941)

Researching history: Chlorophyll was first isolated by Joseph Bienaim Caventou

and Pierre Joseph Pelletier in 1817 (Delépine, 1951).

Since the discovery of chlorophylls of Pelletier and Caventou in 1818, severalmethods have been used for their extraction and purification Chlorophylls have beenextracted from plant tissue using various organic solvents; however, aliphatic and

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aromatic hydrocarbons were not used because they do not extract chlorophylls(Schwartz and Lorenzo, 1990).

In 1913, Richard Willstatter, who was the German chemist, pointed out that allliving energy was due to the sun plants had a way to keep the sun energy In 1919, heexplained that the substance holded the sun's energy was chlorophyll The higherplants had absorbed radiant energy by itself and converted it into stored energy in thebody

The general structure of chlorophyll was elucidated by Hans Fischer in 1940 In

1960, most of the chlorophyll stereoisomers were known, Robert Burns Woodwardpublished a total synthesis of molecules In 1967, the remaining stereotype was finally

fulfilled by Ian Fleming (Fleming Ian, 1967) Chlorophyll was reported to exist in

cyanobacteria and other aerobic organisms capable of forming stromatolites, 2010

(Chisti Y., 1986) Chlorophyll had the molecular formula C55H70O6N4Mg and the

2-formyl-chlorophyll structure had been analyzed based on NMR, optical and mass

spectrometry (Müller, 2007).

Physical properties: Chlorophyll was soluble in organic solvents such as ether,

acetone, methanol, chloroform and especially pyridine, but it is difficult to dissolve inhydrocarbons

Chlorophyll a and b were black green solid wax, which could crystallize well inether solution Chlorophyll a had melting temperature of 117-120°C, while chlorophyll

b had a lower melting temperature of 86-92°C The chlorophyll ester usually had alower melting point than its free acid

Chlorophyll dissolves in organic solvents for light green and had two absorptionwavelengths of up to 450 nm and 663 nm (chlorophyll a) or 460 nm and 630 nm(chlorophyll b)

2.1.2 Structure and classification

Chlorophyll was a group of diverse compounds consisting of chlorophyll (a, b, c,d), chlorophyll a was the most common in nature, which was in photosynthesis ofhigher plants, algae and micro photosynthesis In higher plants, photosynthesis wascomplex structure and consisted of different types of chlorophyll The separation of

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chlorophyll from each other was very difficult and complex In photosyntheticbacteria, cyanobacteria were simple photosynthetic organisms, so isolation was easier.

Chlorophyll a:

- Chlorophyll a played very important role in the photosynthetic system due to theirability to absorb light and convert photoperiod to high efficiency chemical, thisprocess occurs in the photosynthetic system of higher plants, seaweed andphotosynthetic bacteria

- Origination: in higher plants, algae and Cyanophyta, Prochlorophyta

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Chlorophyll not only made green plants but also protected other colors such asCarotenoid, Flavonoid, Xanthophyll In the composition of the trees, Chlorophylldispersed mainly in the cytoplasm and in the body called mitochondria, chloroplast or

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chlorophyll Chlorophyll content in plants accounts for about 1% of dry matter Inliving organisms, chlorophyll complexed with polypeptide This complex was oftenassociated with Carotene and Tocopherol (vitamin E) Carotenoid and Tocopherol hadthe function of keeping the activation energy of Chlorophyll and Oxi singlet

2.1.3 Application of chlorophyll

Chlorophylls were found in living organisms almost exclusively as proteins complexes Chlorophylls were of interest to agriculture and ecology, wherethey were indicators of the health status of individual plants and communities, andwere often used as a quantitative reference in physiological research They were alsopermitted as food colors (Humphrey, 1980) Last, but not least, chlorophylls haverecently attracted interest as phototherapeutic drugs (Scheer, 1991)

chlorophyll-Chlorophyll was registered as a food additive (colorant) and its E number was E140.Chefs used chlorophyll to create a variety of colors for food and beverages such asgreen pasta and absinthe Chlorophyll was insoluble in water so it was first mixed with

a small amount of vegetable oil to obtain the desired solution Liquid chlorophyllextracts were considered to be unstable and always changed until 1997, When Frank S

& Lisa Sagliano used liquid chlorophyll lyophilization in a Florida university,stabilized it in powder form, preserved

In addition, chlorophyll had many effects in medicine: Helps improve the body'snatural blood purification, anti-anemia, micronutrients during hemoglobin formation,increases red blood cell count, strengthens the cells, enhances the immune response Inthe body, against toxins / Against carcinogens., help the body to dispose of residues,prevent respiratory distress

2.2 Some rich-chlorophyll materials

2.2.1 Some rich-chlorophyll plants

Spinach (Spinacia oleracea): Spinach was a highly nutritious food grown in

Europe and North America; In Vietnam, Spinach had been planted in Dalat-Lam Dong.This was the source of high chlorophyll content, simple Chlorophyll extractionprocess, easy to plan on large scale Spinach grows well in low temperature climatessuch as the Tay Nguyen and some mountainous provinces in the north of the spring

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Chlorophyll content in spinach leaves could be up to 0.6% (Khalyfa, 1992; DeveciM., 2011;).

Figure 2.7 Spinach (Spinacia oleracea)

Alfalfa (Medicago sativa, Buffalo herb) was a legume, herb with high nutrition

value, which was food for animal, poultry, seafood,… It had been very interested inthe world for about 50 years

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Many areas of alfalfa material had been planted in Vietnam Stems, roots ofalfalfa was used primarily for the pharmaceutical industry, however, alfalfa leaveswere rich in chlorophyll, so they could be utilized for the production of copperchlorophyll (Arnon, 1949; Christian, 2005) Chlorophyll was a byproduct which wasused in the processing of functional foods that would bring high economic efficiency

Mulberry leaves (Morus alba): Mulberry was originated from China, which

was replicated in Sericulture Development places Currently, mulberry trees could be

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found in most Asian countries, some in North America, Europe and Africa Especiallythis tree was very popular in Southeast Asia.

Figure 2.9 Mulberry leaves (Morus alba)

Mulberry leaves contained many compounds such as citral, linalyl acetate,linalool, β-sitsterol, These were compounds which silkworms like There were largeamounts of Protein, Chlorophyll, Xanthophyll, Carotenoids, Calcium and VitaminC….in mulberry leaves

In addition, some plants have high levels of chlorophyll in the leaves, such asroyal rope, bamboo leaves, and soot… (Ta Duy Tien 2008, Ngo Xuan Cuong 2012)

2.2.2 Spirulina algae

Spirulina algae (Spirulina platensis) was a green-twisted microalgae It wasonly possible to observe the filaments formed by multiple monocytes under themicroscope

Figure 2.10 Spirulina algae (Spirulina plantensis)

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Spirulina algae was a high nutrient food It was the highest protein contentfood, 56%-77% dried weight, triple protein content of beef and double protein content

of soybean

It had high vitamin content 1 kg of spirulina algae contained 55mg of vitaminB1, 40 mg of vitamin B2, 3 mg of vitamin B6, 2 mg of vitamin B12, 113 mg ofvitamin PP, 190 mg vitamin E, 4.000 mg of carotenoid (1.700 mg of β-Caroten, tentimes higher than carrots), 0,5 mg of folic acid, 500-1000 mg of inosit Mineral contentmay vary according to culture conditions, commonly, 580-646 mg/kg of Fe (50 timeshiger than spinach), 23-25 mg/kg of Mn, 2.915-3.811 mg/kg of Mg, 0,4 mg/kg of

Se Calcium, potassium, phosphorus were about 1,000-3,000 mg/kg or higher (calciumwas 500% higher than milk)

The majority of fat in spirulina was unsaturated fatty acid, of which linoleicacid was 13.784 mg / kg, γ-linoleic was 11,980 mg / kg This was rare in other naturalfoods (Pham Quoc Long & Chau Van Minh, 2005)

Spirulina could act as a functional food of high economic value or as a rawmaterial for various phytochemical processes Spirulina was cultivated popular inHawaii China with huge annual yields Vietnam was a tropical country with very largesunshine hours during the year, some areas had carbonate-rich waters and importantminerals for the development of spirulina Spirulina cultivation and processing was astrength in agriculture that needs to be invested in large-scale research anddevelopment Spirulina could simultaneously separate two products of chlorophyll(1%) and phycocyanin (15%) Spirulina was available and relatively abundant in manyparts of Vietnam Thanh Mai Co., Ltd was cultivating with the quantity of 10 tons /year, the scale of 2016 was expected to be 20 tons/year

Currently, Spirulina was being used in raw, unrefined form This was the mainsource of food for the functional food industry However, to develop from this materialmust be accompanied by the phycocyanin separation process Spirulina processedproducts would be functional foods, copper chloropyllin as a food coloring wouldbring high economic efficiency, rich-nutrient feed material

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2.2.3 Silkworm waste/excrement (Faeces Bombycum)

Silkworm excrement, its scientific name was Faeces Bombycum or Excrementum Bombycis, was harvested in the spring summer (June 2-6) from the

mature silkworms Fresh silkworm excrement were soft seeds, which were 4mm indiameter After a few days, the silkworm excrement would be dried and solidified intogray grains, about 2mm in diameter

Figure 2.11 Silkworms eat mulberry leaves Figure 2.12 Silkworm excrement

Chemical composition of the silkworm excrement: 87% of organic compounds,13% of ash content, and 2,8% of total Nitro Organic compounds included proteins,chlorophyll, plant stimulants

- Development of mulberry production

Presentday, with the development of science and technology, Sericulture wasnot strenuous and difficult as before Residents changed from traditional silkwormraising to raising on sliding trays, silkworm making cocoons on the wood trays, usingmachine to pick cocoons to increase productivity, quality and income

Only in Thai Binh province, during the period of 10 years from 2006 to 2015,despite the decrease in the area of mulberry, the number of silkworm householdsdecreased due to the change in the structure of the plants, but in terms of depth, theproduction of mulberry had made significant progress Strawberry yield increased

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13.1%, cocoon productivity increased 159%, there were 2.5088kg / ha strawberry.Cocoons production value of 1 hectare of mulberry cultivation increased (Nguyen ThiDam 2008, Le Hong Van 2017)

Apart from making silk, silkworm pupae used in food, the amount of gain washuge silkworm excrement were used extensively in herbal or organic fertilizer forplants valuable as orchids, roses, needles

Effects of silkworm

According to Oriental medicine, dried silkworm excrement was a cure for manydiseases that many people do not realize Dried silkworm excrement was also used as aremedy to cure many diseases such as inactive bloating, haemorrhage (in postpartumwomen), diabetes, urticaria,

Silkworm excrement extract had many biological active ingredients that havebeen shown to have anti-inflammatory, antipyretic and anti-oxidant effects Inaddition, it had amyloidogenic resistance and nerve protection associated withAlzemer's disease (Moon M., 2014)

Chlorophyll from silkworm excrement

In addition to the precious biological active ingredients, the silkworm containsvery high chlorophyll content due to during digestion of silkworms, Chlorophyll wasnot digested and excreted in the digestive tract, so in the silkworm excrement, thechlorophyll content was higher than that of common plant materials, algae Onekilogram of chlorophyll was obtained per 20 kg of silkworm excrement

According to Urakova, (1987) chlorophyll, carotenoids in the silkworm werethe source of attention for finding cheap sources because chlorophyll and carotenoidlevels were four times higher in strawberry leaves

Instate of used Spirulina, Yoon Il et al (2013) used Chlorophyll paste extractfrom silkworm excrement which had mainly composed of Chlorophyll a andChlorophyll b to produce methyl pheophorbide a (MPa), a major derivative for theconversion into chlorinated species such as methyl pyropheophoride (MPPa, 2), PP-18methyl ester 4 and chlorin e6 by simple steps

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Figure 2.13 Synthesis of the basic chlorin derivatives having reactive groups

These derivatives were the strongest light-sensitive reagent for cancer therapy,which was being tested in clinical trials Optical therapy had been used to treatmalignant tumors of the skin, glands, mucous membranes of the oral cavity, tongue,lower lip, larynx, stomach, lungs, small intestine, bladder and rectum

2.3.1 In the world

Since ancient times, it had been known to use natural plant colors to color food

In addition to colorants, natural colorants also contain other bioactive componentssuch as vitamins, organic acids, glycosides, aromatics, trace elements Therefore,natural colorants not only improved the appearance but also increased the nutritionalvalue of food

For more than a century, scientists were interested in extracting pigments fromthe green leaves of higher plants for used in medicine and the food industry Over the

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past 100 years, chemists had extracted green matter from leaves and called themchlorophyll

Lichtenthaler (1983) used methanol extracted chlorophyll fromNannochloropsis gaditana algae and supplemented with cell wall breaking techniques

to increase extraction efficiency Technique of freezing with liquid N2 had been found

to be more effective than using ultrasound for 15 minutes to extract more stablepigments, such as carotenoids Water-based solvents such as methanol played a majorrole in the extraction, without compromising the use of physical or mechanicalinterruptions

Lee WY (1990) tested the chlorophyll derivatives in silkworm excrementtantrums that might be toxic to cancer cells (MOLT-4 human blood cancer cell lines

and Sp-2 / 0) in vitro.

Dere S (1998), Gitelson A (2000) Determination of chlorophyll content toassess nutrient levels of marine areas Chlorophyll was often used as a nutritionalindicator, due to the relationship between the pigment content and the amount of algaebiomass was quite direct

Pepe et al (2001) had introduced two different extraction methods for thequantification of chlorophyll a there were using ethanol or methanol as extractionsolvents The difference observed in the extraction efficiency was explained by thephytoplankton composition, according to the dominant microalgae strain Ethanolextraction methods have better eficiency results for the dominant strainsChlorophyceae and Dinophyceae, while methanol extraction had been shown to bemore favorable for other strains such as Bacillariophyceae This meaned that theextraction capacity of the solvent depends on the hydration and permeability of the cellwall

Louda and Monghkonsri compared the chlorophyll spectrophotometry withresults obtained by high performance liquid chromatography (HPLC) They concludedthat the evaluation of chlorophyll spectrum by using UNESCO and the Jeffrey [26]and Humphrey equations gave excellent results The collaborators in these two studiesshowed no significant difference between the two methods being introduced

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Karsten et al (2005) used 90% acetone to extract chlorophyll on algae Thesewriters used micro-bead granules during assimilation to increase their ability to breakdown the cell wall by three times the other methods The efficacy of chlorophyllrecovery in this study was 39-85%.

In another study Ronen, R., & Galun, M, (1984), the authors used dimethyl

sulfoxide (DMSO) to extract chlorophyll from lichen (Ramalina duriaei) Ronen and

his colleagues also used 90% acetone with MgCO3 to extract chlorophyll at coldtemperatures and light

In addition, Japanese researchers Irijama, K & cs (2011) also conductedresearch on chlorophyll extraction from spinach with acetone, methanol in cold anddark conditions

Yoon Il et al (2013) used Chlorophyll paste extracted from silkwormexcrement with major components of Chlorophyll a and Chlorophyll b to form methylpheophorbide a (MPa) - a major derivative for chlorine metabolism as methylpyropheophorbide a (MPPa, 2), PP-18 methyl ester 4 and chlorin e6 Thesederivatives were the strongest light-sensitive reagent for cancer therapy, which wasbeing tested in clinical trials Optical therapy had been used to treat malignant tumors

of the skin, glands, mucous membranes of the oral cavity, tongue, lower lip, larynx,stomach, lungs, small intestine, bladder and rectum

2.3.2 In Vietnam

Vu Van Tac (2013) researched chlorophyll content in water applied in themonitoring of water quality as well as assessment of primary biological productivity incoastal marine ecosystems

Nguyen Thanh Binh et al (1999) extracted ecdysteroid from silkwormexcrement This active ingredient contained hormone activity that related in themolting and metamorphic processes and had been used extensively in agriculturalbiology

Ngo Xuan Cuong (2012) had studied the technology of producing pigment fromtea, creating products with beautiful color, high biological value, safe and healthy forconsumers The research had developed a technological process to produce somegreen, yellow and brown tea from the pilot scale and cost 15-20% cheaper thanimported products of the same type The subject also tested the color compounds on

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some food products such as crackers, banana candy, strawberry candy, lemon wine,cherry wine All trial products reached VSATTP standards and were stored at roomtemperature for a period of nine months without discoloration.

Vu Ngoc Boi et al., (2015) studied the optimization of polyphenol and

chlorophyll extraction from dried asparagus (Asparagus officinalis Linn) Results

obtained polyphenol reached 43.7821 mg gallic acid / g DW, chlorophyll contentreached 309.622 μg / g DW, total antioxidant activity equal to 3.2164 mg ascorbic acid/ g

Doan Duy Tien et al., 2012 extracted chlorophyll a from blue algae and studiedthe conversion of chlorophyll into chlorin-6-trimethyl ester as a cancer treatment

World studies showed that silkworm excrement was a potential source ofchlorophyll, making chlorophyll derivatives used in phototherapy, chronic diseases,and cancer In Vietnam, there was little research on chlorophyll on silkwormexcrement This was the new research direction in order to make use of the rawmaterial for production of products for industrial, medical and pharmaceuticalapplications

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MATERIAL AND METHODS

Figure 3.14 Fresh silkworm excrement

3.2 Chemistries and equipments

3.2.1 Chemistries

Dinitro salicylic acid reagent - DNS (Sigma); Silicagel (PA) silicagel , NaOH,

NH3, Al2O3, HNO3, H2SO4,, KOH, Potassium citrate, Sodium citrate SolventsEthanol (C2H5OH) 94%, Isopropyl Alcohol (CH3)2CHOH) 99%, Methanol (CH3OH)94%, Dimethylsulfoxide (DMSO) 97%, Acetone (C3H6O) 99.9%, Acetonitrile (C2H3N)99.99 %, Methanol, H2SO4 (China)

3.2.2 Equipments

UV-VIS U2900 Spectrophotometer (Hitachi), EYELA Vacuum Turbine (Japan),Kenjdan Fertilizer, heated magnetic stirrer, thermostatic tank (Memmer-Germany),drying cabinet (China), Refrigerator (Sanyo), Mixer,

3.3 Methods

3.3.1 Determination of moisture content (TCVN 1867:2001)

Drying at 100 – 105oC to constant volume, then the amount of free water in thesample would evaporate

Calculate results:

W = ((M1 - M2)/M) x 100%

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M1: Total weight before drying of the sample and porcelain cups (g)

M2:Total weight of porcelain cups and samples after drying (g)

2g of tissue was weighed and ground to a fine pulp with the addition of 100 ml

of aqueous aceton solution (0,1N NH4OH: aceton= 1:9) After that, the milling solutionwas poured into a glass bottle (silver foil wrap protects against light), to stay in thedark over night This solution was centrifuged at 5000 rpm for 5 minutes and thesupernatant was transferred to a 100-ml volumetric flask The procedure was repeateduntil the residue becomes colorless The mortar and pestle were washed thoroughlywith 80% acetone and clear washings were collected in the volumetric flask This wasmade to 100 ml with 80% acetone This absorbance was recorded at 663 nm and 645

nm against the solvent (80% acetone) blank The amount of chlorophyll present in theextract mg chlorophyll per g tissue was calculated using the following equations:

3.3.3 Research on some factors that affect the chlorophyll extraction process

Experiment 1: Investigation of the effect of different solvents on chlorophyll

extraction efficiency

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2g of Faeces Bombycum were milled in a mortar with 100ml of solvent

(acetone, ethanol, methanol ), put into a dark colored glass bottle of 250ml, in thedark for 16 to 24 hours Then determine the amount of chlorophyll a, b and totalchlorophyll

Experiment 2: Investigation of the effect of solvent concentration on

chlorophyll extraction efficiency

2g of Faeces Bombycum were milled in a mortar with 100ml of different types

and concentrations of solvent, put into a dark colored glass bottle of 250ml, in the darkfor 16 to 24 hours Then determine the amount of chlorophyll a, b and totalchlorophyll

2g of Faeces Bombycum were milled in a mortar with 100ml of the most

effective extract solvent in previous experiment with different NH4OH concentration,put into a dark colored glass bottle of 250ml, in the dark for 16 to 24 hours Thendetermine the amount of chlorophyll a, b and total chlorophyll

Surveyed HN4OH concentration: 0.025; 0.05; 0.1; 0.125; 0.15; 0.175; 0.2N

Experiment 4: Investigation of the effect of different extract temperatures on

chlorophyll extraction efficiency

2g of Faeces Bombycum were milled in a mortar with 100ml of the most

effective extract solvent with the most NH4OH concentration in previous experiments,put into a dark colored glass bottle of 250ml, in the dark for 16 to 24 hours at differenttemperature Then determine the amount of chlorophyll a, b and total chlorophyll (Usethermostatic tank to reach the necessary temperature: 10oC, 15oC, …, 50oC.)

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