(Luận văn thạc sĩ) effect of maturity stage and harvest location on chemical compositon and antioxidant capacity of extracts from different parts of musa balbisiana colla fruit

Introduction

Start of art

Nowadays, studies for foods containing natural compounds which is good for health becomes a new tendency and draws the scientists’concern.

In the plant, there are species containing noble secondary metabolits compounds like triterpenoids, carotenoids, alkaloids, one of which is phenolic compound Many researches have shown the relation between human’s health and the consumption of foods rich in polyphenols.

The phenolic compounds are well known for being antioxidants, actions in preventing cancer, reducing the risk of cardiovascular disease, increasing lifespan and helping prevent and cure some chronic diseases. Some researches showed that the intake of polyphenols helps reduce the risk of diabetes, slow down the aging Furthermore, phenolic compounds take action in curing neuro-degeneration, inhibiting oxidant stress and chronic inflammations (Williamson, 2005; Cicerale, 2012).

In the plants, polyphenols are typical coloring substances They protect plants againt UV ray, the penetration of microorganisms and other harmful creatures like insects In plant foods, polyphenols keep the main role of creating color, taste and smell Polyphenol compounds are classificed into phenolic acids, stilbene, flavonoids, lignans and ligins Among the phenolic compounds, stilbenes are known for their biological activitie like antioxidant, preventing cancer and cardiovascular disease and anti-inflammatory properties Therefore, the stilbene compounds have a lot attention of reseacher in the recent years Besides resveratrol, a stilbene which is popular in red wine, piceatanol is lately drawing many scientists’ huge interest In comparison with resveratrol, piceatannol has higher biological activity because it has one more hydroxyl group in its structure Studying the exploitation of stilbene, in general, or resveratrol and piceatannol, in particular from the nature is very necessary

“Chuoi hot” (Musa balbisiana Colla) has been utilized in Vietnamese traditional medicine for a long time Every part of it is used for curing deseases The ripe fruits are eaten like normal bananas and are used to treat diseases of the digestion.The green ones and the seeds of ripen fruits are used in treating diabetes, kidney stone disease So far, the application of Musa balbisiana Colla in traditional medicine has been taken mostly based on folk experiences The scientific explanation is very little There are not much information about its chemical composition Musa balbisiana Colla contains flavonoids (leuco-anthocyanins), coumarins, tanins, phytosterols (β- sitosterol) and stilbenes which are strong antioxidants (Huynh et al., 2002) Our recent unpublished research shows that Musa balbisiana Colla contains ahigh content of piceatannol Therefore, Musa balbisiana Colla may be a new source of piceatannol as well as of other phenolic compounds for application in food and drug industries

Many studies show that the stage of maturation has influence on the accumulation of antioxidant polyphenol content and profile For example, a ripe banana has lower content of tannin than a green one but has higher content of anthocyanins Besides, the environmental condition such as light intensity, kind of soil, nutritious supplying also affect the accumulation of secondary metabolits in the fruits Therefore, to have the scientific data on phenolic antioxidant content of seedy banana which helps best maturity for harvesting the bananas rich in polyphenols, especially in stilbene piceatannol, we determine “Effect of maturity stage and harvest location on chemical composition and antioxidant capacity of extracts from different parts of Musa balbisiana Colla fruit”

Objectives

To evaluate the effects of maturity stage and harvest location on physical-chemical properties, total polyphenol content, piceatannol content and antioxidant capacity of Musa balbisiana Colla.The result gives possibility to find out the right time and condition for harvesting the bananas with best biological activity. 1.2.2 Specific objective

- To determine the effect of maturity stage and harvest location on physical-chemical properties including mass percentage of each parts in fruit (peel, flesh and seed), hardness and sugar content of flesh.

- To determine the effect of maturity stage on chemical composition:total polyphenol content, the antioxidant capacity of the banana’s parts and the content of piceatannol in the seeds at different stage of maturity.

Literature review

Characteristics and classification

“Chuoi hot” (seedy banana) has latin name of Musa balbisiana Colla and belonge to Musa genus, Musaceae family, Scitaminae class (Borborah et al., 2016).

Musa balbisiana Colla is a herbaceous plant with big root (banana root) The upper stem is a bunble of huge succulent leaves wrapping tightly layer by layer The stem’s high is 2-4 m The leaf is 1-1.5 m in length with stout spout-shaped stalk, big middle vein convex to the under side and parallel extra veins The fruit is succulent and big with 5 edges, contains 4-5 mm black ball-shaped seeds whose embryo is white (Pham Hoang To, 2014).

Figure 1 Trees, branch fruit and seeds of “ chuoi hot” source : http://data.abuledu.org/wp/?LOM024 and http://www.bananas.org

Most of consumed banana varieties are hybridizations of 2 wild species called Musa acuminata Colla and Musa balbisiana Colla (Stover and Simmonds, 1987) The differences between these 2 species are listed in the Table 2.1

Table2.1 Characters used in the clasiffication of banana though a taxonomic scorecard

Character Musa acuminata Musa balbisiana

Pseudostem color More or less heavily marked Blotches slight or absent with brown or black blotches Petiolar canal Margin erect or spreading, Margin inclosed, not winged with scarious wing below, below, clasping pseudostem not clasping pseudostem

Peduncle Usually downy or hairy Glabrous

Ovules Two regular rows in each Four irregular rows in each loculus loculus

Bract shoulder Usually high ( ratio 0.30) Bract curling Bract reflex and roll back Bracts lift but no roll

After opening Bract shape Lanceolate or narrowly Broadly ovate, not tapering ovate, tapering sharply from sharply the shoulder

Bract color Red, dull purple or yellow Distinctive brownish-purple outside; pink, dull purple or outside; bright crimson inside yellow inside

Color fading Inside bract color fades to Inside bract color continuos to yellow towards the base base

Bract scars Prominent Scarcely prominent

Free tepal of male flower Variably corrugated below Rarely corrugated tip Male flower color Creamy white Variably flushed with pink Stigma color Orange or rich yellow Cream, pale yellow or pale pinkSource: Simmonds and Shepherd (1955)

Musa balbisiana Colla grows mostly in South-east Asia and the south of China In Vietnam, they grow in the north mountainous areas like Yen Bai, Lao Cai, Lang Son and Hoa Binh province.

Musa balbisiana Colla is a hydrophyte and has stronger vitality than other species It can handle shade and compete with other plants.

Therefore, in order to protect land, people usually plant it in the corner of the garden, under other fruit trees’ shadow or even next to the bamboos.

Every year, there are 1-3 new trees growing from one mother stem The seeds also have strong germinant ability (Pham Hoang To, 2014).

2.1.3 Nutritious compositon and bioactive compounds

According to the scientific literature, banana is a source of carbohydrates, minerals, protein, fiber and important vitamins in human’s diet It contents 10 essential amino acids for human body Hence, banana is very appropriate to add to diet of children and the old people The carbohydrate composition of banana chang hugely during matuaration Beside, banana is an important source of vitamins and minerals, specially kali, vitamin B6, vitamin C and fatty acid (palmitic, linoleic, lonolenic, oleic acid) which help improve health and re-create energy (Pothavorn et al., 2010) Banana’s flesh and peel contain β-carotene with high concent of 40- 4960àg/100g (Mohapatra et al., 2010)

Table 2.2: Nutritious composition in banana flesh

Component Without peel (g/100g banana With peel (g/100g banana powder ) powder )

Source: Hasilinda et al (2009) Banana contains many powerful antioxidants which are good for health, 2 serotinin and nore-pinephrin, dopamine and catecholamine Dopamine is an important neurotransmitter in brain In “chuoi hot”, dopamine plays role of a powerful antioxidant which present in feel with concentration of 80-.560mg/100g and 2.5-100g in fresh (Emaga et al., 2008b; Kanazawa and Sakakibara, 2000) Some flavonol glycoside substances such us rutin (242.2– 618.7lg/g of dry weight), antioxidant tannins are found in flesh and peel very good for health (Mohapatra et al., 2010; Tsamo et al., 2014) In addition, some researchers have reported that there are leucocyanidin in banana flesh which help to prevent gastric ulcer (anti-ulcerogenic) (Lewis et al., 1999).

Some studies have been done on different parts of Musa balbisiana Colla fruits The studies showed that there are anthocyanin in bracts with two main anthocyanins being delphinidin and cyanidin (Horry and Ray, 1987) Japanese researchers indicate that some phytoalexins including 1,2,3,4 - tetrahydro -6,7 – dihydroxy-1-(4’-hydroxycinnamyliden)naphathalen-2-on; 2-(4’-methoxyphenyl)

- 1,8 - naphthalic anhydrid; 2 - phenyl - 1,8 - naphthalic anhydride are present in the banana fruits (Kamo et al., 1998).

In India, a published research reported the persence of three Neo – clerodanditerpenoids separated from Musa balbisiana seeds called musa balbisian A, B, C (Ali, 1991) At Ho Chi Minh City University of Medicine and Pharmacy, Nguyen Thi My Hanh and Bui My Linh determined chemical composition of the “chuoi hot” Their result showed that there are saponin, coumarin, tannin, flavonoid anthocianosid and uronic compound, essential oil, phytosterol in the seeds However, it has to emphasize that the tests used in their study were qualitative The identification and quantification of individual compounds were not done.

Several studies found out that the resin (Musa babisiana) contains caffeoylquinic acid, myricetin-3-O-rutinosid and myricetin glycoside.Some other phenolic compounds were also discovered in banana such as dopamine, N-acetylserotonin, kaempferol-3-O-rutinoside, quercetin-3-O- rutinoside, naringenin glycoside, apigenin glycoside I, naringenin glycoside II; naringenin glycoside II by absorption spectral analysis at280-320 nm wavelength (Pothavorn et al., 2010).

In Thailand, 6 anthocyanins are identified in the banana flowers by HPLC-MS method: delphinidin-3-rutinoside, cyanidin-3-rutinoside, petunidin-3-rutinoside, pelargonidin-3-rutinoside, peonidin-3-rutinoside, and malvidin- 3-rutinoside Musa babisiana fruit contains delphinidin-3- rutinoside and cyanidin-3-rutinoside (Kitdamrongsont et al., 2008).

Studies in flesh and peel of 13 variaties of banana showed that they contain many phenolic compounds including: caffeic acid-hexoside, ferulic acid –hexoside, sinapic acid-hexoside, ferulic acid–dihexoside, mycricetin deoxyhexose-hexoside (high in pulp), ferulic acid, sinapic acid, querceetin- deoxyhexose- hexoside (high in pulp), methymycricetin-deoxyhexose- hexoside, querceetin- hexoside, isorhamnetin-3-O-rutinoside (Tsamo et al., 2015)

In Spain, a research group separated a fatty ester of phytol, a fatty ester of n-alkanol, ò - sitosterol and stigmasta - 5, 22 E - dien - 3ò – ol from chloroform extract of “chuoi hot” From acetone extract, the group separated a (+) – epiafzelechin compound This compound is tested its ability to prevent Cryptolestes pusillus Schocherr-an insect harmful to cereals (Pascual - Villalobos and Rodríguez, 2007).

The studies on chemical composition of 3 Musa species at 3 ripeness levels show that in the flesh of these Musa species there are alkaloids, saponins, glycosides, flavonoids and tannin with different contents at each ripeness level (Obiageli A et al., 2016).

Researcher at National Science and Technology center have studied preliminarily the composition of the “chuoi hot” inVietnam The result showed the presence of 2 compounds which are cyclomusalenon [(24S)-24 methyl-29- norcycloart -25-en-3-on] and stigmasterol Stigmasterolis a very common sterol in nature Cyclomusalenon is a 5-cycle triterpen containing cyclopropan cycle with 3-oxo-29-norcycloar rarely seen in nature (Tran et al., 2003)

Phenolic compounds

Phenolic compounds are aromatic compounds with hydroxyl groups attached directly to the benzene ring Molecules with many hydroxyl groups attached directly to the benzene ring are called polyhydroxylphenol (monomer), many monomers bound together called polymers (Le Ngoc Tu, 2003)

The wide variety of structures, functions and widespread distribution of polyphenols in plants lead to different ways to classify them Polyphenols can be classified by their origin, biological functions, and chemical structures Due to the structure depending on the structure of the carbon cycle, phenolic compounds are classified into different groups shown in Figure 2.3

Figure 2.3 Classification and structure of major phenolic compound

Source: Adapted from Han et al (2007)

Phenolic acids appear to be abundant in the plant Depending on their structure characteristics, they are subdivided into two subgroups: hydroxybensoic acid and hydroxycinnamic acid with carbon chain of C 3 -C 6 and

C 1 -C 6 respectively Hydroxycinnamic acids contain many hydroxyl and methyl groups in the struture They are materials for lignin synthesis and many other compounds Hydroxybensoic acids are found with low content in edible plants In plants, they are the raw material for the synthesis of lignin and hydrolysis tannin

Flavonoid is a secondary metabolite product of plants, with a carbon chain of C 6 -C 3 -C 6 Depending on the characteristics of the carbon chain(containing double bonds or containing the hydroxyl group), flavanoid compounds can be grouped into small groups including flavonol, flavanol,flavone, isoflavone, flavanone and anthocyanin They are abundant in plants(Robards and Antolovic, 1997) They have strong antioxidant capacity.Besides, some flavonoids have anti-inflammatory, anti-allergy, anti- inflammatory, antibacterial properties (Middleton et al., 2000; Chrisnos, 2008)

Ligin is special polymeric compounds of plants, usually found in many wood tissues, which are cell adhesives, which increase mechanical strength, waterproofing the xylem cell wall, preventing infiltration of pathogenic microorganisms Lignin is the condensation product of phenylpropanes Two phenylpropanes coalesce to form lignan They are abundant in linseed (up to 3.7g/kg dry matter) Lignin and its derivatives are of interest in research because they are thought to be feasible for use in the treatment of cancer and other diseases (Salee, 2005).

Tanin is a mixture of C 6 - C 1 and C 6 - C 1 - C 6 (gallic acid and diagallic acid in free form and glucose - conjugated form) Tanin compounds are common in plants and classified into 2 types:

Tannins are popular in some trees such as guava, banana, persimmons, etc , Tannin content is very different in different parts of the plant

Stilbene is a small molecular weight compound (MW = 210 ÷ 270), which is a natural secondary compound that protects plants against bacteria, preventing bad effects from ultraviolet light and some serious diseases Stilbene is synthesized via the phenylpropanoid route The synthesis of stilbene synthesized by plants mainly depends on the stimulation of the environment The five most common stilbene compounds in nature include: resveratrol, piceatannol, pinosylvin, rhapontigenin and pterostilbene (Roupe et al., 2006) Among these give stilbenes, resveratrol and piceatannol are well studied by researchers

Piceatannol(3,5,3',4'-tetrahydroxystilbene;5-[2-(3,4dihydroxyphenyl) ethenyl] benzene-1,3-diol is derirative of to resveratrol.

Figure 3.4 Structure of Piceatannol and Resveratrol Piceatanol is has molecular formula of C 14 H 12 O 4 Piceatannol is a white powder, has a melting point at 223 0 C-226 0 C, molecular weight of 244.24 This compound is insoluble in water but soluble in ethanol and dimethyl sulphoxide Spectral analysis of piceatannol in ethanol showed that piceatannol absorbed up to 322 nm, while trans- resveratrol absorbed maximum at 308 nm (Rossi et al, 2008).

Piceatanol is proven to be highly bioactive, anti-oxidant, anti- inflammatory, anti-obesity and diabetes, anti-cancer, cardiovascular (Piotrowska et al, 2012) Using foods containing high resveratrol and piceatannol helps to reduce the risk of cardiovascular disease, to prolong longevity and to enhance human health (Roup et al., 2006).

Piceatannol is found in many plants, in which the most important source in human diet is red wine and grapes with concentration lower than that of resveratrol The amount of piceatannol and resveratrol in grapes are 0.78 and 3.18 μg/g respectively, although the amount ofg/g respectively, although the amount of piceatannol in red wine is higher about two times than resveratrol (908 and 208 μg/g respectively, although the amount ofg/g) (Cantos et al., 2000) According to our present study, the piceatanol content in the sim is 2.3 mg/g dry matter, 1000-2000 times greater than the one of red grape (Lai et al., 2013) In addition, piceatannol is also found in lemon creeper, Asian beans, peanut, and so on.

2.2.2 Biological activity of phenolic compound

Antioxidant activity is the most studied property of phenolic compounds Antioxidants, in general, and most phenolic compounds, in particular, can slow down or inhibit the oxidative process generated by ROS (reactive oxygen species) and RNS (reactive nitrogen species) in excess.

ROS and RNS are well recognised as being both deleterious and beneficial species At low or moderate concentrations, they have physiological roles in cells, for example, in the defence against infectious agents (Valco et al., 2007). Their level is controlled by endogenous antioxidants including enzymes and antioxidant vitamins (i.e., vitamins E and C) However, various agents such as ionising radiation, ultraviolet light, tobacco smoke, ozone, and nitrogen oxides in polluted air can cause “oxidative stress” characterised by an over production of ROS and RNS on one side, and a deficiency of enzymatic and non-enzymatic antioxidants on the other ROS and RNS in excess can damage cellular lipids, proteins,or DNA, and there by inhibit their normal functions (Valco et al., 2007) Phenolic compounds are strong dietary antioxidants that reinforce,together with other dietary components (carotenoids, antioxidant vitamins),our antioxidant system against oxidative stress (Tsao, 2010) The antioxidant mechanisms of phenolic compounds are now well understood (Nijveldt et al., 2001; Amic et al., 2003), and include:(i) direct free radical scavenging, (ii) chelation with transition metal ions, and (iii) inhibition of enzymessuch as xanthine oxidase, catalysing the radical formation.

Cardiovascular diseases are the leading cause of death in the United States, Europe, and Japan, and are about to become one of the most significant health problems worldwide In vivo and ex vivo studies have provided evidence supporting the role of “oxidative stress”in leading to severe cardiovascular dysfunctions Increased production of ROS may affect four fundamental mechanisms contributing to atherosclerosis, namely:(i) oxidation of low density lipoproteins (LDL) to oxidised-LDL,(ii) endothelial cell dysfunction,(iii) smooth muscle cell migration and proliferation as well as matrix metalloproteinase release, and (iv) monocyte adhesion and migration as well as foam cell development due to the uptake of oxidised-LDL (Bahorun et al., 2006) Phenolic compounds in fruits (Burton-Freeman et al., 2010), cocoa powder, dark chocolate (Wan et al., 2001), and coffee (Natella et al., 2007) were reported to inhibit the oxidation of LDL, hence reducing cardiovascular risk Green tea consumption reduced total and LDL cholesterol, and inhibited the susceptibility of LDL to oxidation, and was therefore associated with decreased risks of stroke and myocardial infarction (Alexopoulos et al., 2010) Resveratrol and piceatannol, two stilbenes detected in red wine, were shown to elicit a number of cardioprotective activities, including inhibition of LDL oxidation, mediation of cardiac cell function, suppression of platelet aggregation, and attenuation of myocardial tissue damage during ischemic events (Roupe et al., 2006) Moderate consumption of red wine rich in these stilbenes has been linked to the “French Paradox” observation described by Renaud and De Lorgeril in 1992, i.e an anomaly in which southern French citizens, who smoke regularly and enjoy a high-fat diet, have a very low coronary heart mortality rate (Roupe et al., 2006)

Inflammation is a dynamic process that is elicited in response to mechanical injuries, burns, microbial infection and other noxious stimuli (Shah et al., 2011) It is characterised by redness, heat, swelling, loss of function, and pain Redness and heat result from an increase in blood flow, swelling is associated with increased vascular permeability, and pain is the consequence of activation and sensitisation of primary afferent nerve fibers A huge number of inflammatory mediators,includingkinins,platelet-activating factors, prostaglandins, leukotrienes, amines, purines, cytokines, chemokines, and adhesion molecules, have been found to act on specific targets, leading to thelocal release of other mediators from leucocytes and the further attraction of leucocytes, such as neutrophils, to the site of inflammation Under normal conditions, these changes in inflamed tissues serve to isolate the effects of the insult and thereby limit the threat to the organism However, low-grade chronic inflammation is considered acritical factor inmany diseases including cancers,obesity, type II diabetes, cardiovascular diseases, neurodegenerative diseases, and prematureaging (Santangelo et al., 2007) Phenolic compounds have been reported to display marked invitro and invivo antiinflammatory properties via various mechanisms of action including:

(i) inhibition of the arachidonic acid pathway, (ii) modulation of the nitric oxide synthetase family, and (iii) modulation of the cytokine system as well as of the nuclear factor kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) pathways (Santangelo et al., 2007).

Meterials and methods

Sample and chemical

“Chuoi hot” was harvested at Namdinh and Yenbai province In each province, we harvested 3 bunches with the same biological maturity At least

30 fruits were collected from middle hands of each bunch and allowed to ripen at room temperature in cardbroad The next steps, they were separated them in five maturity stages including: green, green more than yellow, yellow and green end, yellow and yellow with brown spots For each ripening stage,

3 fruits were removed from the cardboard and weighted.

Figure 3.1 Five maturity stages of “chuoi hot”

For freeze dried sample preparation

Each fruit was cut on the length and across the width into four quarters Diagonally opposite quarters were put together to form two groups. For each group, samples from the three fruits were pooled The pulps, peels and seeds of one group were separately freeze-dried and then vacuum sealed in polypropylene plastic bags They were ground to powder and the samples of freeze dried powder were stored at -20 o C until analysis.

Figure 3.2 All part of “chuoi hot”

Sodium carbonate (Na 2 CO 3 ); acetone (C 3 H 6 O,100%); acetonitrile (C 2 H 3 N, 99.8%); 2,2-diphenyl-1-picrylhydrazyl (DPPH); 3,4,5-Trihydroxybenzoic acid monohydrate (gallic acid, monohydrate); Folin-Ciocalteu’s reagent; 6- hydroxyl-2,5,7,8- tetramethylchroman-2-carboxylic acid ( Trolox)

Heat dried oven (Memmet, Germany)

Centrifugation (Mikro 220R, Mikro 200R, Hettichzentrifugen, Germany)

Vortex mixer ( JK-VT-F JINGKI SCIENTICIN, China)

Freeze drying (FR-Drying Digital unit -Thermo, USA)

Method

Total dry matter was ditermined by drying method to a constant weight at 105 0 C.

The stiffness of “chuoi hot” was determined by DIGITAL FIRMNESS TESTER machines in Idian.

Figure 3.3 Stiffness machine 3.2.3 Determination sugar profiles

Briefly, 0.3 g of freeze dried sample was weighted into 15ml fancol and mixed with 9ml distilled water by using vortex and then centrifuge 12000 rpm,

4 0 C for 10 min The supernatant was taken for analysis by HPLC equipment.

Preparation standard of sugar profiles

0.1 g each of sugar was weighted into 2 ml micro tube, added with 1 ml distilled water and mixed throunghly by using votex The sugar solutions 10% were continuced to do dilution with difference concentrations 0.25%, 0.5%, 1%, 1.5%.

Quantification of sugar profile was performed by HPLC using a Shimadzu system (Japan) equipped with a DGU-20A3 degasser, LC-10Ai pumps, a CBM-20A Monitor and a RID detector A 20 àl aliquot of the extract was injected onto SUPELCOSILN LC – NH 2 (25 cm x 4.6 mm, 5àm particle size) equipped with a guard column of the same type (Supelco, Japan) The mobile phase was acetonitrile 80%. The flow rate was 1 ml/min, and the column temperature was 30 0 C. mV(x100)

Figure 3.4 Chromatography of glucose and fructose at concentration of 0.5%

Concentration (%) Figure 3.5 Standard curves of glucose and fructose

3.2.4.Determination total polyphenol content and antioxidant capacity Extraction of phenolic compounds

Phenolic compounds in different part of “chuoi hot” were extarcted by using a protocol previously optimized by our research group Briefly, approximately 0.13 g of freeze-dried sample was mixed with 4 ml of acetone 60% in a water bath and shakened for 60 min at 40 0 C After centrifugation at 6000 rpm for 10 min at 4 0 C The supernatant was collected and evaporated to dryness with a rotary evaporator at

35 0 C The residue from the evaporation was added methanol 70% and analysed for total phenolic contant, antioxidant capacity and piceatannol content

The total phenolic content of the extract was determined by the Folin–Ciocalteu method (Singleton, L and Rossi, 1965).

Briefly, 500μg/g respectively, although the amount ofl of sample solution diluted to the appropriate concentration, mixed thoroughly with 250àl of Folin–Ciocalteu reagent 1N for 5 min, followed by the addition of 1250àl Na 2 CO 3 7,5% The mixture was allowed to stand for a further 30 min in the dark, and absorbance was measured at 755nm The total phenolic content was calculated from the calibration curve (Figure 3.6), and the results were expressed as mg of gallic acid equivalent per g dry weight (mg GAE/g DW).

Figure 3.6 Gallic standard curve Determination of antioxidant capacity

Scavenging activity of DPPH radical was assessed according to the method of Larrauri, Sanchez-Moreno and Saura-Calixto (1998) with some modification.

Briefly, 0.1 ml of diluted sample solution was mixed with 2.9 ml of 0.1 mM DPPH methanol solution After the solution was incubated for 30 min at

25 0 C in drank, the decrease in the absorbance at 517nm was measured Control contained methanol instead of the antioxidant solution while blanks contained methanol instead of DPPH solution The inhibition of DPPH radicals by the sample was calculated according to the following equation:

Figure 3.7 Trolox standard curve 3.2.5 Determination of piceatannol content

Quantification of the piceatanol was performed by HPLC using a Shimadzu system (Japan) equipped with a DGU-20A3 degasser, LC- 10Ai pumps, a CBM-20A Monitor and a SPD-M20A Diode array detector (DAD) A 20 àl aliquot of the extract was injected onto Kinetex 5u EVO C18 column (150x4.6mm i.d; 5 àm particle size) equipped with a guard column of the same type (Phenomenex, Netherlands) The mobile phases were A (H 2 0 with 0.1% formic acid) and B (acetonitrile with 0.1% fomic acid) The flow rate was 1 ml/min, and the column temperature was 30 0 C The 42 min gradient was as shown in Table 3.1.

Figure 3.8 Chromotogaphy of piceatannol standard at concentration of 100 àg/ml re a

Concentration (àg/ml) Figure 3.9 Piceatannol standard curve

Data were analysed using the statistical software Minitab 16.0 Analysis of variance was carried out using a Generalised Linear Model (GLM) procedure to determine the effect of the havest location, maturity stage and their interactions onanalysed index The model configuration was Yi = a + b1*X1+b2*X2 + b12*X1*X2 (Y: the analysed index; X1: haverst location and X2:maturity stage).Tukey test were used to determine the differences among the means p-values < 0.05 were considered to be significantly different.

Results and discussions

Effects of the maturity stage and harvest location on physical-chemical

4.1.1 The ratio of each part in “chuoi hot”

The propotion of each part in “chuoi hot” fruit were changed during ripening.The result was showed in Figure 4.1

Pe rc en ta ge 60

Pe rc en ta ge 60 pulp

Figure 4.1 Impact of the maturity stage of the “chuoi hot” fruit harvested in Namdinh and Yenbai on the propotion of different part.

Statistics analysis result showed that harvest location and maturity stage had significant effect on percentage of peel (p=0.000, p=0.003) while interaction between location and maturity stage had no signifficant effect (p=0.636) The propotion of peel in Yenbai was 34.71 ± 3.96%, while the pecentage of peel in Namdinh was lower about 3.45 times, with value of 10.04 ± 0.96% Concerning maturity stage, the pecrentage of peel decreased dramatically from the first maturity to the firth maturity At Namdinh province, the figure for peel decreased gradually from 12.8 ± 0.53% to 7.97 ± 1.34 %, coresponding a fall of about 1.6 time from the 1 st to the 5 th stages maturity At Yenbai province, the peel with the propotion reduced from 40.42 ± 2.85% to 31.28 ± 7.35 %

Statistics analysis result indicated that harvest location significantly effected percentage of pulp (p = 0.000) Maturity stage and interaction between

75.13± 1.39% in fruit, while the for the one in Yenbai was lower,with value of 43.16± 5.23%.The percentage of pulp were increased lightly between the 1 st and

5 th maturity stage At Namdinh province, there was a small increase in the precent of pulp from 70.47 ± 1.63 % to 80.1 ± 2.04 % for the 1 st and 5 th maturity stage At Yenbai provice, the propotion of pulp showed a rise from 40.57 ± 6.53% to 43.42 ± 4.98 %, a rise of about 1.07 time within 5 maturity stages

Harvest location significantly effected the percentage of seed (p

= 0.005) while maturity stage and interaction between harvest location and maturrity did not significantly effect to percentage of seed stage (p

= 0.893, p = 0.378) The propotion of seed in Yenbai (22.13 ± 8.62 %) was higher than Namdinh (14.83 ± 1.37 %) The percentage of seed in 5 maturity stages were simillar in 2 harvest location.

Regarding in two havest location, overall, we saw that the propotion of pulp was a highest in the “chuoi hot” fruit In comparion with pulp, the propotion of peel was lower and the seeds was lowest Besides, natural conditions such as climate, temperature, environment conditions, nutritionmay effect propotion of fruit in different harvest location.Compared to other different banana such as “chuoi tieu”, “chuoi su” and “chuoi bom” whose the proportion of pulp were 65%, 72% and 73%, respectively The percentage of pulp in “chuoi hot” harvested at Namdinh was higher but that of the one harvested in Yenbai was lower (Huynh Nguyen Thai Duy, 2013). 4.1.2 Hardness of fruit

Hardness is one of the important indicators to evaluate maturiy stage. Green fruit has high hardness and ripen fruit has low hardness.

Maturity stage signifficantly effected hardness of pulp (p=0.000) while harvest location, interaction between harvest location and maturity stage did not effect hardness(p = 0.103, p = 0.329).

Among 2 harvest location, hardness of “chuoi hot” harvested in Yenbai was higher than that harvested in Namdinh.The hardness decreased dramatically between the first matuarity to the last matuarity The hardness of

“chuoi hot” harvested in Namdinh reduced sharply from 9.32 ± 0.76 kg/cm 2 to0.489 ± 0.2kg/cm 2 and for Yenbai from 10.31± 1.24kg/cm 2 to 0.39± 0.04kg/cm 2

Specially, the Figure 4.2 showed a rapid reduce of hardness from the 1 st to 2 nd maturity stage of fruit pulp for all harvest locations The fall for Namdinh is from 9.32 ± 0.76 kg/cm 2 to 1.92 ± 0.58 kg/cm 2 and Yenbai being from 10.306 ÷

1.24kg/cm 2 to 2.70 ÷ 0.56kg/cm 2 , coresponding to a decrease of 4.84 and 3.81 times,respectively It was explained that during maturity time pectine was decomposed by ezyme pectinase, cells was spradic,water content was increased In fact that green banana is harder than ripen banana.

Figure 4.2 Impact of the maturity stage of the “chuoi hot” fruit harvested in Namdinh and Yenbai on pulp hardness

Many studies showed that hardness was effected by maturity stage. Research on the biochemical of fruit changes with maturity had showed that: the hardness of a kind banana decreased 9 times, the mango fail 9 times, and the papaya reduced to 60 times (Bui Quang Huy and Pham Quang Hung, 2009) Research on quatity assessment of tomato posthravest saw that hardness of tomato decreased gradually with maturity stages (Nguyen Minh Thuy and Nguyen Thi Kim Quyen, 2009). 4.1.3 Changing of sugar content of banana pulp harvest in 2 locations

The result of HPLC analysis shown that the composition of

Figure 4.3: Sugar ptofile of “chuoi hot pulp at 5 th matyrity”

Maturity stage Maturity stage Glucose

Figure 4.4 Impact of the maturity stage of the “chuoi hot” fruit harvested in Namdinh and Yenbai on sugar content

Statistic results shown that harvest location and matuarity stages inificantly glucose content (p=0.027, p=0.000).The interaction between harvest location and maturity stage did not have effect on glucose content of pulp (p= 0.305) Glucose content in Namdinh was higher than Yenbai which accounted for 31.24 ±3.55 % and 26.46 ± 5.55% Glucose content in pulp increased from the 1 st to the 5 th maturity stages.

31 glucose content was 2.87 ± 3.93% DW However, the 5 th maturity stage it increased about 13.8 times, reached the value of 39.81 ± 1.63

%DW in Namdinh “chuoi hot” There was a rapid increase of glucose content in Yenbai from just around 2.00 ± 1.59 %DW to 39.08 ± 1.9

%DW of the 1 st and the 5 th maturity stages, respectively.

Statistic results showed that harvest location, matuarity stage sinificantly effected (p=0.044, p=0.000) fructose content in pulp while interaction between harvest location and maturity stage did not have any significant effect (p=0.520) Average fructose content of “chuoi hot” harvested in Namdinh was of 22.78 ± 3.96% higher than 19.15 ± 3.9% which was fructose content of “chuoihot” in Yenbai.There was a dramatic increase of fructose content in all harvest locations when from just the 1 st to the 5 th maturity stage

Sugar content rich a highest value from the 1 st maturity to 2 sd and became stable when “chuoi hot” changed from 2 nd to 5 th maturity stage Sugar content in fruit can explain that ripening fruit is sweeter than green one In compariton with fructose, glucose content in “chuoi hot” was higher Sugar content of “chuoi hot” harvested in Namdinh was higher than the one harvested in Yenbai

According to this study, during maturation, the total suluble solid in the fruit increased due to the strongly hydrolysis reaction. For example, Starch and tanin content were reduced to form single sugar, and lipid content were joined hydrolysis reaction This rule is true for raspberries and strawberries (Wang et al., 2009).

4.2 EFFECT OF MATURITY STAGE TO TOTAL POLYPHENOL CONTENT OF“CHUOI HOT”

Total polyphenol of different part of “chuoi hot” changed during maturation.

Statistics analysis result showed that maturity stage significantly effected total phenolic content of peel (P=0.000) while harvest location, interaction between harvest location andmaturity stage had not significant effect to Total phenolic content of peel.

The average of total phenolic content of peel harvested in Namdinh was 19.74± 2.52mg GAE/g DW This value was not significantly higher than the one harvested in Yenbai (17.86 ± 5.63mg GAE/g DW) The total polyphenol content of peel in 2 harvest locations dramatically decreased from the 1 st to

5 nd maturity stages At Namdinh, the phenolic content fell from 30.68 ± 1.12 mg GAE/g DW to 11.27 ± 4.51mg GAE/g DW and in Yenbai from 34.58 ± 13.61mg GAE/g DW to 8.94 ± 3.16mg GAE/g DW, corresponding to a reduction of 2.7 times and 3.8 times for Namdinh and Yenbai, respectively Papaya peels had a gradual fall from 471.97 to 358.67 mg GAE/100g FW, coresponding a decline of 1.31 time during ripening (Sancho et al., 2010).

Figure 4.5 Impact of the maturity stage of the “chuoi hot” fruit harvested in Namdinh and Yenbai on total phenolic content of peel

Statistics analysis result showed that harvest location, maturity stage and interaction between harvest location andmaturity stage significantly effected TTP of pulp (p =0.000, p = 0.000, p = 0.000).

The average of total phenolic content of pulp in Yenbai was higher than the one in Namdinh which accounted for 33.15 ± 5.83mg GAE/g DW, 10.31 ± 1.31 mg GAE/g DW respectively Among maturity stages, the 3 rd maturity stage had the highest total phenolic content while the 1 st stage had the lowest

There was a dramatically drop about 2.4 times in total polyphenol content of pulp in Namdinh from the 1 st (19.33 ± 1.78 mg GAE/g DW) to the 2 nd (8.87 ±

Conclusion and recommendation

Conclusion

In this study, the detailed of physical-chemical properties and chemical composition including total polyphenol, antioxidant capacity and piceatannol content of Musa babilsiana were determined The results shown that:

In “chuoi hot”, the propotion of pulp was the highest and the one of seed was the lowest.

During maturation, the ratio of peel decreased, the portion of pulp increased, while the percentage of seed did not change.

The hardness of pulp fall during maturation.

“Chuoihot” harvested in Namdinh and Yenbai contented two monosaccharids including glucose and fructose The sugar content increased during maturity Harvest location and maturity stage effected phenolic content and antioxidant capacity of pulp, peel and seed of “chuoi hot” Peel, pulp and seed harvested in Namdinh had a decrease of total phenolic content and antioxidant capacity during maturation “Chuoi hot” harvested in Yenbai had different tendency of change in phenolic content and antioxidant capacity of peel, pulp and seed

For the first time, piceatannol, a stilbene with many potent biological activity was found in “chuoi hot” This compound was present only in the seed of “chuoi hot” with high concentration Maturity stage did not effect the piceatannol content in seed but harveste location had.This finding suggested a new source of health-promoting compound, piceatannol,which need to be studied, exploited and applied in the future.

Recommendation

‘Chuoi hot’ had high phenolic antioxidant content Until now, only piceatannol was identified in this fruit In the near future, the identification of phenolic compounds in this fruit need to be done The resultcould explain the use of “chuoi hot” in traditional medicine of Vietnam and also open new window for the development of this wild fruit in the future.

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Welcome to Minitab, press F1 for help.

General Linear Model: % seed versus province, stage

Factor Type Levels Values province fixed 2 ND, YB stage fixed 5 1,2,3,4,5

Analysis of Variance for % seed, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P province 1 400.32 400.32 400.32 9.90 0.005 stage 4 44.01 44.01 11.00 0.27 0.893 province*stage 4 179.93 179.93 44.98 1.11 0.378

Obs % seed Fit SE Fit Residual St Resid

R denotes an observation with a large standardized residual.

Grouping Information Using Tukey Method and 95.0%

Means that do not share a letter are significantly different.

Confidence province stage N Mean Grouping

General Linear Model: % pulp versus province, stage

Analysis of Variance for % pulp, using Adjusted SS for Tests

Obs % pulp Fit SE Fit Residual St Resid

General Linear Model: % peel versus province, stage

Analysis of Variance for % peel, using Adjusted SS for Tests

Obs % peel Fit SE Fit Residual St Resid

General Linear Model: Hardness versus province, stage

Analysis of Variance for Hardness, using Adjusted SS for Tests

Obs Hardness Fit SE Fit Residual St Resid

General Linear Model: Glucose versus province, stage

Analysis of Variance for Glucose, using Adjusted SS for Tests

Obs Glucose Fit SE Fit Residual St Resid

General Linear Model: Fructose versus province, stage

Analysis of Variance for Fructose, using Adjusted SS for Tests

Obs Fructose Fit SE Fit Residual St Resid

General Linear Model: PPT peel versus province, stage

Analysis of Variance for PPT peel, using Adjusted SS for Tests

Unusual Observations for PPT peel

Obs PPT peel Fit SE Fit Residual St Resid

General Linear Model: PPT pulp versus province, stage

Analysis of Variance for PPT pulp, using Adjusted SS for Tests

Unusual Observations for PPT pulp

Obs PPT pulp Fit SE Fit Residual St Resid

General Linear Model: PPT seed versus province, stage

Analysis of Variance for PPT seed, using Adjusted SS for Tests

Unusual Observations for PPT seed

Obs PPT seed Fit SE Fit Residual St Resid

General Linear Model: DPPH peel versus province, stage

Analysis of Variance for DPPH peel, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P province 1 481 481 481 0.29 0.599 stage 4 109160 109160 27290 16.16 0.000 province*stage 4 521 521 130 0.08 0.988

Unusual Observations for DPPH peel

Obs DPPH peel Fit SE Fit Residual St Resid

General Linear Model: DPPH pulp versus province, stage

Analysis of Variance for DPPH pulp, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P province 1 493 493 493 0.32 0.581 stage 4 23583 23583 5896 3.77 0.019 province*stage 4 94413 94413 23603 15.11 0.000

Unusual Observations for DPPH pulp

Obs DPPH pulp Fit SE Fit Residual St Resid

General Linear Model: DPPH seed versus province, stage

Analysis of Variance for DPPH seed, using Adjusted SS for Tests

Unusual Observations for DPPH seed

Obs DPPH seed Fit SE Fit Residual St Resid

General Linear Model: Piceatannol versus province, stage

Analysis of Variance for Piceatannol, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P province 1 0.076924 0.076924 0.076924 29.52 0.000 stage 4 0.006202 0.006202 0.001550 0.59 0.670 province*stage4 0.004477 0.004477 0.001119 0.43 0.786

General Linear Model: TTP versus Part, stage

Part fixed 3 peel, pulp, Seed stage fixed 5 1,2,3,4,5

Analysis of Variance for TTP, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P

Obs TTP Fit SE Fit Residual St Resid

Seed 4 6 56.03 A B C peel 1 6 32.98 B C D pulp 3 6 29.63 C D pulp 2 6 27.22 D peel 2 6 21.25 D pulp 4 6 19.28 D pulp 5 6 17.12 D peel 3 6 16.06 D pulp 1 6 15.39 D peel 4 6 13.60 D peel 5 6 10.11 D

Tiêu đề	Effect Of Maturity Stage And Harvest Location On Chemical Composition And Antioxidant Capacity Of Extracts From Different Parts Of Musa Balbisiana Colla Fruit
Tác giả	Ngo Thi Huyen Trang
Người hướng dẫn	Dr. Lai Thi Ngoc Ha
Trường học	Vietnam National University of Agriculture
Chuyên ngành	Food Science and Technology
Thể loại	thesis
Năm xuất bản	2017
Thành phố	Hanoi

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