Extraction and characterisation of water soluble carotenoids of gardenia jasminoides ellis

Crocin and crocetin glycosyl esters are water soluble Carotenoidswhich are found in nature in stigmas of saffronCrocus sativus Linne and in the fruits of gardenia Gardenia jasminoides El

MINISTRY OF EDUCAnON& TRAININGHOCHIMINH CITY NATIONAL UNIVERSITY.UNIVERSITY OF TECHNOLOGY

THESIS OF DOCTORATE

EXTRACTION AND CHARACTERISATION OF

To my country Vietnam and to my family

for always encouraging me and always believing in me

Thanh Quan Pham

This work was in the scope of the cooperation project between the Food Research and DevelopmentCentre of the Canadian Ministry of Agriculture and Agrofood and the University of Technology ofthe Ho Chi Minh City National University of the Vietnamese Ministry of Education and Training.Most of the work was carried out in the laboratories of the Food Research and Development Centre-Agriculture and Agrofood Canada

*I would like to express the special thanks from my heart to:

Dr Franc;ois Cormier, Head of Bio-Ingredient Section, Food Research& DevelopmentCentre-Agriculture& Agrofood Canada,

Dr Chi Bao Do, Food Research& Development Centre-Agriculture& Agrofood Canada,

Dr Van Hang Tong, Ho Chi Minh City National University, Vietnam

for all of the advices, helps and teaching me during the time I lived, worked and studied in Canada

**I wish to express my thanks more specially to:

Dr Claude B Aube, Director of Food Research & Development Centre- & AgrofoodCanada,

Prof Dr Minh Ve Truong, Rector of University of Technology - Ho Chi Minh CityNational University, Vietnam,

for the financial support and the permission that helped me the conditions of working and studying

***I also wish to express my thanks to:

Dr Marie Rose Vancalsteren,

ttFinally, I would like express my indefinite thanks to:

Prof Dr Quoc Dung Lam, Vice Director of HoChiMinh City National University,

Prof Dr Van Bon Pham, Vice Rector of HoChiMinh City University of Technology,

Prof Dr Minh Tan Phan, Dean of Chemical Engineering Faculty of HoChiMinh CityUniversity of Technology,

Prof Dr Van Thanh Tran, Head of Organic Chemical Department of ChemicalEngineering Faculty of HoChiMinh City University of Technology,

All of my teachers, my professors and my friends in Vietnam,

My parents, my wife and all of my sisters and my brother,

For all of the advices, helps and teaching me for the years I have lived, worked and studied inVietnam

Carotenoids are abundant in many fruits and vegetables and they plays diverse roles in photobiology,photochesrnistry and medicine Crocin and crocetin glycosyl esters are water soluble Carotenoidswhich are found in nature in stigmas of saffron(Crocus sativus Linne) and in the fruits of gardenia (Gardenia jasminoides Ellis).

The best condition for the extraction of gardenia fruits was conducted at 30°C for 48 h in the darkusing 50% EtOH as a extraction solvent for preventing the pigments against the degradation ofenzymatic reaction and oxidation The content of crocin in gardenia fruit from Vietnam was 0.120

±0.016mglg and of geniposide was 5.76± 1.01 % (w/w) of dried fruit of gardenia.Inmost of theextract from gardenia fruits, crocin occupied up to 68 % of the total yellow crocetin glycosyl esters

For further purification of crocin the aqueous solution of 50% acetone is used as a extraction solvent, washing with ether, ionic exchanging, and separation on preparative HPLC The purified crocinwith purity more 99.6 % has been using as the standard and the materials for oxidation monitored

by NMR and LC-MS and for the antioxidant assays

Inboth thermostability assays at 25°C and at 95°C, crocin was stable at pH 6.0 Colour losses ofcrocin over 10 days at 25°C were 39 % in pH 6.0 medium and almost 99 % in pH 3.0 solution.Inthe thermostability trials at 95°C the colour residue of crocin after 60 min was 96 % at pH 6.0 incomparison with the initial colour at time=0 , but it was 73 % at pH 3.0 Attention has to be paidwhen carrying out the oxidation or other reaction of crocin

Crocin has an antioxidative activity at concentrations of 10-40 ppm The antioxidative property ofcrocin as evaluated by thiocyanate method was better than with the thiobarbituric acid (TBA)method When crocin reacted with oxygen using FeS04 the intermediates such asmonohydroperoxides, polyhydroperoxides, polyhydroxy-polyhydroperoxides of crocin were formedand detected by MS or LC-MS The main monohydroperoxides were mainly 15-monohydroperoxideand 14-monohydroperoxide based on the fragments at the C13-C14 and CI5-CI5' double bondsobtained by MS The adduct between linoleic acid radical and crocin was detected by LC-MS

The semiempirical PM3 geometry optimization calculation and the semiempirical ZINDO/S spectralcalculations have been carried out for crocetin , crocetin glycosyl esters - the water solublecarotenoids and the hydroperoxides of crocetin The 12-mono hydroperoxide of crocin is the most

stable and the 14-monohydroperoxide of crocin is the most unstable The maximum wavelengths ofthe crocetin glycosyl esters calculated by the semiempirical ZINDO/S spectral calculations agreedwith those obtained by spectroscopy in Calculations have also been performed for several torsionangles of the monohydroperoxides of crocetin All of themonohyd~operoxides have the minimumbinding energy when the torsion angle is around 160-180° Calculations have also performed for

all-trans and several cis crocetin The B-cis crocetin is the most stable cis-form with an enthalpy

ofcis-trans transformation from all-trans to B-cis crocetin of 0.15 kcal/mol The enthalpy of

transformation from the singlet state to triplet state of crocetin is 11.59 kcal/mol corresponding tothe energy of radiation with the wavelength of 2470 nm

The process of extraction and production of yellow pigment from gardenia fruits was investigated Iridoids were eliminated from the extract of gardenia fruits

modelling, HPLC,NMR, MS

Chapter I: Extraction, Purification and Identification of the main compounds from

2 Crocetin derivatives and iridoids fromGardenia jasminoides Ellis 6

2 Objective and Direction of Study 44

ChapterIII: Antioxidative activity of crocin from Gardeniajasminoides Ellis 49

2 Objective and Direction of Study 123

GENERAL INTRODUCTION

There is considerable interest worldwide in the development of food colorants from natural sources

A variety of fruits, vegetables and flowers have been studied as potential sources of food colorants.The technological success of the pigments in anatto(Bixa orellana L.)and saffron(Crocus salivus

Linne) combined with their relatively high price led to search for other plant sources with the samepigments The same pigments could be obtained in the greater quantities and at a lower price fromthe fruit ofGardenia lasminoides Ellis [1]

The fruits ofGardenia jasminoides Ellis contain three major groups of pigments: water soluble

carotenoids (crocetin derivatives), iridoids, and flavonoids [2-5] The water solubility of carotenoidcrocin and their derivatives leads to more application in foods and other products The production

of food colorants from gardenia fruits is apparently being investigated at the present time [66] Thecolorants from gardenia appear to have good potential because of their wide range of available colorsand their apparent excellent stability [6]

Being a carotenoid, crocetin derivatives have been shown to possess antioxidant properties [2,3] Theuse of natural antioxidants has received special attention because of the worldwide trend to avoid

or reduce the use of synthetic food additives The application of natural antioxidants in foods andbiological systems has received considerable interest because of their presumed safety andpotentially nutritional and therapeutic effects [7]

Another aspect of their application of the water-soluble carotenoid crocin and crocetin deri vati ves

is that they can be used as an anticancer agent against different kinds of tumors and cancers [8]Among the most significant activities of crocin and crocetin derivatives are their free radicalscavenger properties [9-12] The free radicals are known to be very harmful to cellar components

as a precursor of more reactive species, contributing to the various diseases such as inflammation,multagenesis, carcinogenesis, tissue injury by circulatory disturbance and aging [13,14]

apply further the natural compounds in many fields of industries Pigments from fruits ofGardenia jasminoidesare more and more replaced to pigments from saffron Because they contain the smallamount of iridoids the use of this pigment is restricted in North America and Europe but they areaccepted in Asian countries Works need to be done for the researching the process to eliminate theirridoid compounds in the extract of gardenia fruits in order to upgrade the food natural colors from

Gardeniajasminoides.The project on this research between Food Research&Development Centre,Agriculture and Agri-food Canada and Ho Chi Minh City University of Technology, Vietnam isestablished to wishtosolve this problem

The objective of the the thesis is aimed at to the research on:

The extraction and characterization of the properties of crocin from the fruits ofGardenia jasminoides.

The antioxidative activity of crocin and the determination of the mechanism of

antioxidative activity of crocin

The development of the extraction process of the pigments from gardenia fruits

Chapter I:

Extraction, Purification and Identification

1928, both Paul Karrer and Richard Kuhn began working on carotenoids and their researchesproduced very important results which are still relevant to modern carotenoid chemistry Karrerrecognized the symmetrical nature of lycopene, beta-carotene, zeaxanthin and crocetin.In1947, Isler

et al. synthesized crystalline vitamin A: this opened up the field of carotene and carotenoidsyntheses.In1971 the number of known carotenoids increased to about 300 and at present time thenumber of known naturally occurring carotenoids has increased to about 600 Every three years thefUPAC-Symposia on Carotenoids is held to show how much the field has broadened [15]

The natural occurrences of carotenoids are provided by the yellow-orange colors of flowers (e.g.,sunflower, marigold) the orange-red color of fruits (e.g., tomato, orange) and the orange roots ofcarrots, from the Latin name of which(Dacuss carola)the name "carotene" and "carotenoids" arederived The greatest production of carotenoids occurs in the photosynthetic tissue of plants andalgae with the estimation of 100 million tons! year (especially fucoxanthin, lutein, violaxanthine,neoxanthin) Here, thecarotenoids are found universally in the photosynthetic apparatus, though theirpresence is generally masked by the green of the chlorophyll and reveal only when the chlorophyll

is degraded

Carotenoids also occur widely in microorganisms and animals Carotenoids are often responsible foryellow, orange or red colors in non-phototrophic bacteria, moulds and yeast for protection againstdamage by light and oxygen Carotenoids are also found in animals such as the feathers of birds (e.g.,ketocarotenoids in pink-red feathers offlamigos), the skin or flesh of some fish (e.g., astaxanthin ingold fish, salmon), the shrimps and the lobsters (astaxanthin in the carotenoprotein form) [16]

Inplants and animals they occur in several forms:

a) in solution in fat droplets (as in carrots)

b) in colloidal dispersio:l in lipid media (as in the grana of the chloroplast)

c) combined with protein in the aqueous (as in the xanthophyll or fruit carotenoids)

d) esterification with fatty acids (as in ripe fruits)

The industrial production of p, P'-carotene began in 1954 Carotenoids are mainly producedbecause of their coloring properties They are used for the direct coloring of foodstuffs as well asfor pigmentation animal products.

b) Structuresandproperties: [16)

The carotenoids are aliphatic or alicylic unsaturated members of a terpene group They are composed

of eight isoprene units, joined in a tail to tail manner at the Centre of the molecule, which give thestructure symmetry (Fig 1.1)

The carotenoids own their color to the absorption of light by a conjugated system of carbon-carbondouble bonds that is known as the "chromophore " It is possible to have up to 15 conjugated doublebonds in the chromophore of a C4Q carotenoid, though structures with 7 to II such double bonds are

(vitamin A) orthe diapocarotenoids such as crocetin Most natural carotenoids occur predominantly

in the all-E form (trans form) which are more stable than the corresponding Z form (cis form).However, some carotenoids with a Zconfigur~.tionabout one or more double bonds such as naturalbixin have been isolated [17]

Most of the carotenoids are lipophilic substances and are generally insoluble in water unless somestrongly polar groups are present such as the dicarboxylic acid norbixin, the carotenoid sulphatesand crocin which are esterified with sugars: gentiobiose and glucose

Gardenia jasminoides Ellis (= G Grandiflora Lour.), a native of China and Indochina, is anornamental and medicinal woody plant This plant, belongingto the Rubiaceace is an evergreensmall shrub with white, solitary and fragrant flowers The double-flowered form is usually used forornamental purposes, while the single-flowered form is used as a medicinal plant, since the formerdoes not bear fruits, a medicinally used organ

G.jasminoides, as well as its variety, G.jasminoidesvar ovalifoliaNakai, is called gardenia andused as a garden tree in Europe and North America, and also as a pot plant in Greece This plant wasonce called Cape jasmine in North America because of its fragrant flowers In China it is called Zhi-

zi, and the dried fruits have been medicinally used for curing various inflammatory diseasesincluding hepatitis and cystitis (Yen 1982) The dried fruits of G.jasminoides and of its form, G

jasminoides F grandiflora Makino, are called Shan-shi-shi in japan and have been used as adyestuff and antiphlogistic, diuretic and haemostatic drug in Chinese traditional medicine (NaITU1a1980) A demand for the fruits as food coloring has been rapidly increasing and currently more than

150 tons of the dried fruit s have been imported to Japan every year (Japanese Association forIndustrial crops 1985) [18]

G.jasminoides plants which are called"danh danh" or"chi tu" or"son chi tu "in Vietnam havebeen cultivated widely in Vietnam for many years Far from being time the extract form the gardeniafruit has been applied traditionally in daily food products such as stick rice, cake, food colors and

in traditional medicals The fruits of gardenia is a traditional Chinese medicine used as a diuretic,

an antipyretic, an anti hepatitis and anti-inflammatory agent [19]

Danh danh trees grow freely and are planted in many regions of the north of Vietnam (Ha Bac,Quang Ninh) and south of Vietnam (Ninh Thuan, Long An) In the mountain reaions they!ITOW

freely in the banks of the water streams.Inthe farms the gardenia plants are planted as a source offlowers for decoration and their fruits are used as medicals and food colorant The harvest season

of the mature fruits ofGardeniajasminoides is on AuguststoNovember The fruits are exposed tosunlight or dried and stored at the dry place Up to now there is no exact estimation about theproduction capacity of gardenia fruit and their products

The fruits ofGardenia Jasminoides Ellis contain three major groups of pigments: water soluble

carotenoids (crocin and crocetin derivatives), iridoids, and flavonoids (Fig.I.2 to Fig.IA) Thecarotenoid crocin and the related compounds develop in the fruit ofGardenia Jasmilloides during

8-23 weeks of growth, but the irridoid pigments develop 1-6 weeks after flowering [1)

Crocin - the main pigment responsible for the coloring power of gardenia(Gardenia jasminoides)

and saffron(Crocus sativus) was first studied in 1818 Aschoff who called it crocin.In1915, Dekkerdemonstrated it glycosidic nature and later the schools of Karrer and Kuhn established its structureand molecular formula (C44H6402.).It is also known as gardenin since it was found in the fruit ofseveral varieties of gardenia The same pigments also occur inC.albifloris C luteus Cedrela toona, Nyctanthes arbor-tristis, Verbascum pholollloides (20)

Crocin is the bis ester (6-0-P-D glucopyranosyl-p-D-glucopyranosyl) of the carotenoid crocetin(C2oH2.O.), which is 8,8'-diapo-ljJ, ljJ'- carotenedioic acid Mw.·976.97 Very soluble in water andpoorly soluble in organic solvents Crystallized in methanol it presents brilliant red needles whichmelt at 180°C With sulfuric acid it progressively changes colors and is finally blue (20)

On acid hydrolysis in the absence of air, crocin yields crocetin and glucose, while hydrolysis withalcoholic ammonia results in crocetin and gentibiose Crocin is extremely sensitive to dilute aqueouspotassium hydroxide giving a quantitative yield of crocetin (potassium salt) Treatment with methylalcoholic potash yields mono and trimethyl ester of crocetin Crocin like carotene, dissolves inconcentrated sulphuric acid, forming a deep blue solution on standing change to violet, red and

The composition of the yellow pigments from gardenia fruit is as in Table 1.1 [2]

Table 1.1: The composition of the yellow pigments from gardenia fruit.

Crocetin-monogentiobiosyl-monoglucosyl ester(trans)

Crocetin-monogentiobiosyl ester(trans)

Crocetin-monogentiobiosyl ester(cis)

Crocetin-diglucosyl ester(trans)

Crocetin-monoglucosyl ester(trans)

Crocetin-monoglucosyl ester(cis)

Composition (%)

6834.52.55.315.32.50.9

Crocin which occurs naturally in the alltransform is the main pigment of the yellow pigments fromgardenia The alltransform crocetin glycosyl ester have the maximum in the regions of 435-440 nmand of 453- 465 nm [4,5J Thecis13- form of crocetin glycosyl esters are present in small quantity

of the color matters in fruit of gardenia and have another maximum wavelength at 324-326 nm

(cis-peak) in visible spectra [4,5J The polarities and water solubilities of crocetin glycosyl estersdecrease from compound (I) to compound (7) [5]

Among the most significant activities of crocin andcrocetin glycosyl derivatives are their free radicalscavenger properties [9-11JThese carotenoids are used in sperm cryoconservation for theirsuperoxide scavenger capacity [8,12]

The flavanoid pigments are yellow, but their contribution to the overall color of gardeniapreparation is unclear [1,6]

The most abundant iridoid in gardenia is geniposide (Fig, 1.3), with the composition of 4-6 % driedweight of fruit [22] Genipin, an aglucone formed geniposide by hydrolysis, has been shown toexhibit laxative (Yamauchiet at 1974) and choleretic (Aburada et ai, 1978) activities as well as

inhibitory effect on gastric acid secretion based on its anti-choligenergic ac:ion, The a1ucone ofgardenoside was shown to have antimicrobial activity againstStaphylococcus aureusandKlebsiella pneumoniae(lshiguroet a11983)

The production of food colorants from gardenia is being investigated at the present time, A stableyellow colorant from gardenia is stabilized by mixing with sugars (e,g" lactose and dextrin) andspray drying the mixture, This is then refluxed with acetone, filtered and the residue dried to gi vethe stabilized yellow pigment Most patent procedures involve extraction of the fruit with water,treatment with enzymes having P-glucosidic activity or proteolytic activity (bromelain), and reactionwith primary amines from either aminoacid or protein such as those in soy, Depending on reactionconditions such as pH, time, temperature, oxygen content, degree of polymerization, conjugation,etc, a series of colorants are produced with the colors from yellow to green, red, violet and blue,Several of the patents involve culturing preparation of Gardenia with microorganism such as B, subtilis, Aspergilusjaponicus,oraRhizopu,Seven such preparations, invo! ving 4 green, 2 blues, andone red, have been commercialized in Japan, The colorants from gardenia appear to have goodpotential because of their wide range of available colors and their apparent excellent stability, [6]

1.1.3 Objective and Direction of Study:

The objective of this part is aimed at:

- Extraction of the fruits of gardenia to find the best conditions,

Working Scheme

Gardenia fruits

~

Extraction (H20, MeOH, Acetone, EtOH)

Separation, Isolation and Purification (Preparative HPLC,

Superoxide anion assay

!

Identification

Determination of mechanism of antioxidant activity of crocin:Oxidation reaction of crocin monitored by RMN and LC-MSMolecular modeling for the intermediates of oxidation

!

Determining method for the elimination of geniposide

and the development of extraction process

1.2 Materials and Methods:

1.2.1 Materials.

Dried fruits ofgardeniajasminoideswere collected from Vietnam HPLC grade solvents: ethanol,methanol, ether, acetone were purchased from Fisher Co The HPLC grade standards geniposide,geniposidic acid, shanzhiside, gardenoside were obtained from Wako Crocin purified from thefruits ofGardenia jasminoidesis used as the standards

1.2.2 Extraction

Kamirura and Nakazato (1985)[84] compared the effect of different solvents on crocin extractionfrom saffron and gardenia fruits using acetone, ethanol and ethanol-water solutions, concluding thatthe smallest yield was obtained with acetone and the greatest yields with ethanol-water solutions

containing higher proportion of water Iborra et al (1992) [70] also compared crocin extraction

from saffron using water and 50% solution of ethanol-water observing that the former led to justlittle greater yields [21]

The pigments from gardenia fruit are extracted by water or polar solvents: ethanol, methanol,acetone The solvents chosen are water and ethanol with different concentration (v/v) Theextractions are carried out at temperature below 40°C and in dark to prevent the oxidation Agitation

or stirring do not affect so much in the extraction of gardenia fruits (see Annex 3) Scheme ofextraction experiments is described as follows:

Scheme of extraction experiments:

Dried fruits of gardenia

Triplicate, dark, stoppered

Find Amax-Amax, A.wo - mg pigment, and find optimal time and optimal solvent ofextraction

1.2.3 Isolattion and purification of crocin:

One hundred grams of gardenia fruits were extracted in the cold by triturating in 50% acetone,centrifugating, and filtering under vacuum The residues were re-extracted in the same manner Thetotal filtrates were pooled, evaporated under vacuum at20-25°C using a rotary evaporator to 100 ml.The crude extract of gardenia was dried under a nitrogen stream and lyophilized at - 40°C undervacuum This dried crude extract was used in the assays

For purification of crocin the crude extract of gardenia fruit was washed with ether (lOOml x 3) andpassed through a Dowex X2-200 cationic exchange column (I Omm i.d x 250mm length) by elutingwith MeOH, then immediately through a Dowex lX2-200 anionic exchange column (I0mm i.d x250mm length) by eluting with MeOH The extract was further purified by preparative HPLCcolumn to separate the crocetin derivatives The conditions of the preparative HPLC were as follows:column: ODS-2 (C,s) 10~m(25 mm i.d x 100 mm length); injected volume: 5 ml; detector: 440

nm The mobile phase delivered at a flow rate of 8 ml/min was water for the first 15 min, thenchanged linearly to 62% (v/v) MeOH over 20 min, maintained at 62% MeOH for 15 min., thenincreased linearly to 100% MeOH over 10 min., and finally maintained at 100 % MeOH forIS min.The crocin fraction having a retention time of 31.4 to 32.2 min was collected to evaporate undervacuum and under a nitrogen stream to dryness, and recrystallized with methanol, evaporated under

a nitrogen stream and lyophilized at- 40°C under vacuum Crocin having a purity of more than 99.5

% analyzer by analytical HPLC was used in the assays (See scheme in the next page)

Washing with acetone 5~

(lOO ml x 3 times) Centrifugation and Filtration

Ether

(lOO ml x 3)

Evaporation (under vacuum at 20-25 ·C, down to 100 ml) - - - - Acetone, H20

~

MeOH

(100 ml x 21

Vacuum evaporation - - MeOH, H

2 0, acid acetic (down to 25 ml )

The results presented are means of three replicates.

Preparative HPLC for crocin [5, 67](see also Annex 2) (Method SAFFRAN)

The cOOlditions of the preparative HPLC were as follows: column: ODS-2 (CIS) 10 mm (25 mm i.d

x 100 mm length); injected volume: 5ml;detector: 440 nm The mobile phase delivered at a flowrate of 8 mlImin was water for the first 15 min., then changed linearly to 62% (v/v) MeOH over 20min., maintained at 62% MeOH for 15 min., then increased linearly to 100% MeOH over 10 min.,and finally maintained at 100 % MeOH for 15 min The crocin fraction having a retention time of31.4 min to 32.2 min was collected to evaporate under vacuum and under a nitrogen stream todryness, and recrystallized with methanol, evaporated under nitrogen stream and lyophilized at - 40°C

1.2.5 Determination of iridoid content:

Dried fruit powder (1.000 g) was placed in 30 ml of 50%MeOH(v/v),then heated under reflux on

a water bath at8SOC. After cooling it was centrifuged at 2500 g'and decanted The residue wasrefluxed with 25 ml x 2 of 50%MeOH(v/v).The total extract was placed in a 100 ml of volume andadjusted to 100 ml by 50%MeOH(v/v).A 20 J.lI of this solution was injected to HPLC after filtrationwith 0.45J.lmmilipore [22]

HPLC conditions are the same in method SCAN Standard samples were supplemented with variousconcentrations of iridoids (0.03125mglmlto 1.000mglml).The standard calibrating curves of thegeniposide, geniposidic acid, shanzhiside methyl ester, and gardenoside were estaJlishec The peakhigh was calculated in order to construct a calibration graph Every sample was analyzed in triplicateand the results were average

1.2.6 UV-Visible Spectroscopy

were scanned from 200 nm to 600 nm at 600 nmlmin The Iml-quartz cuvette is used

1.2.7 NMR analysis:

The analyses were run on a Chemagnetics Infinity 300 NMR spectrometer operating at 300.07MHz

for'H,using a Nalorac inverse 5-mm broad band probe Samples were run inD20 at 25°C, and werereferenced to the residualHDOat 4.7 ppm For each sample, 128 or256 scans were acquired to havesufficient signal-to-noise, depending on the concentration ofcrocetin in each sample The scans wererun with a 45 ° pulse angle and a pulse delay of 5 T ,'s as estimated from the null point of an inversionrecovery sequence The identification of peaks was based on the relative integrations and chemicalshiftsascompared to the values published by Van Calsterenet ai.(1997) (5)

1.2.8 MS analysis:

MS

The mass spectrometry analysis of the reaction mixture was performed using the electron spraytechnique on a Finigan LCQ mass spectrometer The potential applied to the electrospray probevaried from4.0 to 4.5 kV, and the acquisition took place in the negative ion mode Each sample wasdissolved in distilled water All mass obtained corresponded to [M - H]

LC·MS

Analysis of the sample of reaction mixture was performed using a Finigan LCQ desktop-type liquidchromatograph mass spectrometer (LC-MS) The HPLC conditions were as for the HPLC analysisabove, and detection in the negative mode with the electron spray ionization method

1.3 ResultsandDiscussions

1.3.1 Extraction:

The results are presented on Fig.1.5 to Fig 1.8 (see also Annex 3) The spectrum of crude extraction

of gardenia fruits after 16 h of extraction at 25°C with 50% ethanol as presented on Fig 1.5 showsthree peak regions: 230-250 nm, 320-330 nm and 430 - 460 nm corresponding to the three kind ofcompounds in gardenia fruit: iridoids, flavonoids and crocetin derivatives The UV region of 230-

250 nm is partly due to iridoids with some contribution from the yellow fraction and waste materials.The region of320 - 330 nm is partly due to flavonoids with the yellow fraction (cis-peak) The region

of 430-460 nm is mainly due to crocetin derivatives [66]

2.50 ~ -,

550 500

450 400

350 300

0.00+ _ !?-: : :: : : +_ _ + _-+-_ +_ _+ _ -1

o

Ture(day)Fig I 6: O1anges in absorbance at 440 run of the extract of

Gardenia jasminoides fiuits during the extraction with water and

ethanolic solutions at'l5'c.gardenia/solvent ratio=1.0/3.0(wIv)

- - Water

-+-50% ROO_.~. ROO

_IO%Roo

-+-7(JJ1oROO

.- 30% ROO

·x ·9J%ROH

0.00.J -~-+-~ _+-~+_~_+~-i ~_+_~ + 60

01:02 01:03 01:04 01:05 01:06 01:08 01:10

Gademia/50%ErOH ratio(w/v)

Fig L 7: Qocin contents and % crocin in total yellow pigments of the extract

of Gardeniajasminoides fruits at 25°C with different gadenia/solvent ratio (

analysed by HPLC) _ _ Content ofcrocin (~/g) +-Crocin purity (%)

When using the gardenia/50% EtOH ratio of 1: 1 and 1:2, the solvent is not enough to wet thematerials The HPLC analytical results show that the crocin amounts obtained varied in the range

of 0.08 to 0.11 mg/g of crocin and crocin purity varied in the range of 68 to 72 % as the same results

of rchiet al.(1995) [2] when the gardenia/50%EtOH ratio(w/v)changed from 01 :02 to 01: 10 (Fig.1.7) This means that the gardenia/solvent ratio did not affect so much to the crocin amounts in theextraction of gardenia fruit with the aqueous 50% (v/v) EtOH The results show that using thegardenia/50% EtOH(w/v) =1:3 to 1:4 is suitable for the extraction and the expense for theevaporation of solvent is not high The 50% ethanol is used in the next experiments, the extractiontime is 72 hours and the gardenia/solvent ratio is 1:3 to 1:4 (w/v).

fruits at different temperatures gardenia/50%EtOH ratio =I :3 (w/v).

The high temperature accelerates the degradation ofcrocetin glycosyl ester [2,24,25) At temperaturehigher than 35°C the color of the extract is destroyed faster (Fig.I.8) The temperature suitable forthe extraction is between 18°C to 25°C At the temperature below 18°C the extraction time is longer.The shorter extraction time should be chosen because of the oxidation

HPLC analysis for iridoids:

The method SCAN (reverse phase high performance liquid chromatography) using methanol can beused for separation and analysis of iridoids compounds The standard calibrating curve of geniposide,shanzhiside methyl, gardenoside, and geniposidic acid are established (Fig 1.9 and Annex 5) Theretention time of the peaks of the standard of geniposide, shanzhiside, gardenoside and geniposidicacid are 12.7, 11.9, 10.3 and 9.6 min, respectively Geniposidic acid is the most polar and geniposide

is the less polar

Inthe profile of HPLC of crude extract of gardenia fruit therear~two major peaks with retentiontime: 12.66 min and 11.63 min at 240 nm (Fig 1 10) The main peak with retention time 12.66min is determined as geniposide (12.7 min) by the standard geniposide in the same HPLC conditions(Fig 1 II) The co-chromatography of crude extract and standard geniposide were also carried outand the results is present in Fig.I.12 There is only peak at 12.6 min on co-chromatogram of standardgeniposide and geniposide of crude extract of gardenia Geniposide content was determined by HPLC(Table 1.2) (see also Annex 6)

TableI 2:Result of analysis of geniposide from gardenia fruit

Geniposide content=5.76 ± 1.01 [% w of dried fruit]

% geniposide in total iridoids=72.71 + 0.60 %

This result was somewhat different from that analyzed by Oshima et al (1988), i.e 4-6 %.[22]

Fig L9:Standard curve of geniposide

Column 0055:4.6mm1.0 x 250mm, photodiode arraydetector: 200nm-500nm, flow rate: ImVmin,solvents:A:water,B:MeOH, 0-10min:0 % B to 50%B,10-40min:50%B to loo%B, 40-50min: loo%B, 50-65min:O%A

11:21:C6

I): 21:47 11:21:)1

S~.ple S ~h In' I,d SCil! H' Yial Jiojed

Tlpe ~ue ~~o~nt Hsocnt Fitter Inj Hr. YIJI IJl

Puk ~eteiltion ~m llli9~t hik F'eiik

I 0.2000

Absor"banc:e

I

o.mo

in the region UV: IRRITESTl: crude extract with standard geniposide, IRRITEST2: standardgeniposide

E 1.20c

" 0.20