FUNCTIONALITY OF AZADIRACHTA INDICA A. JUSS (NEEM) IN BEVERAGES
A Doctoral Dissertation
Presented to
The Faculty of the Graduate School
At the University of Missouri
In Partial Fulfillment
Of the Requirements for the Degree
Doctor of Philosophy
By
Abhinandya Datta
Dr. Ingolf Grün, Dissertation Supervisor
December 2016
© Copyright by Abhinandya Datta 2011 ALL Rights Reserved
The undersigned, appointed by the dean of the Graduate School, have examined the dissertation entitled
FUNCTIONALITY OF AZADIRACHTA INDICA A. JUSS (NEEM) IN BEVERAGES
Presented by Abhinandya Datta
A candidate for the degree of Doctor of Philosophy And hereby certify that, in their opinion, it is worthy of acceptance.
Dr. Ingolf Grün, Food Science
Dr. Misha Kwasniewski, Grape and Wine Institute
Dr. Azlin Mustapha, Food Science
Dr. Philip Deming, Statistics
ACKNOWLEDGEMENT
First and foremost, I would like to thank my advisor Dr.
Ingolf Gruen, for being an understanding and inspiring guide. His patience and amiable nature has gone a very long way in helping me through the challenges of pursuing a Ph.D. I appreciate his time, ideas, effort and funding, all of which have contributed immensely towards making my academic experience very productive and stimulating.
I want to extend my sincerest thanks and gratitude towards Lakdas Fernando, technical assistant in our lab.
It was his training on various instruments that allowed me to carry out my research smoothly.
I would like to thank all my other committee members- Dr.
Azlin Mustapha, Dr. Misha Kwasniewski and Dr. Philip
Deming, for their useful and constructive inputs, which have helped in making my thesis well rounded. I am very honored to have them on my committee.
I would also like to thank Brad Alberts and Dr. Lada
Michaes, from the Department of Social Statistics, for their time and help with data analysis.
Last but definitely not the least, I am grateful to my parents, family and friends for their love, care, encouragement and support, without which all of this would not have been possible.
ii TABLE OF CONTENTS
ACKNOWLEDGEMENT…………………………………………………………………………………………………ii
LIST OF FIGURES…………………………………………………………………………………………………vii
LIST OF TABLES………………………………………………………………………………………………………x
ABSTRACT………………………………………………………………………………………………………………… xi
CHAPTERS
1. INTRODUCTION…………………………………………………………………………………………………………1
1.1 Background……………………………………………………………………………………………………1
1.2 Objectives……………………………………………………………………………………………………5
2. LITERATURE REVIEW
2.1 Headspace Solid-Phase Microextraction………………………7
2.2 Essential oils…………………………………………………………………………………13
2.3 Flavonoids and their health benefits………………………20
2.4 Limonoids and their removal for de-bittering…33
2.5 Milk-protein interaction………………………………………………………41
3.IDENTIFICATION AND QUANTIFICATION OF FLAVONOLS- MYRICETIN, QUERCETIN AND KAEMPFEROL, TOTAL POLYPHENOLIC CONTENT AND ANTI-OXIDANT ACTIVITIES IN AZADIRACHTA INDICA A. JUSS LEAVES COMMERCIALLY AVAILABLE IN THE U.S. BY HPLC-DAD-ESI-MS/MS
3.1Introduction………………………………………………………………………………………………49 3.2 Material and methods 3.2.1 Plant material………………………………………………………………………51 3.2.2 Reagents………………………………………………………………………………………52 3.2.3 Extractions………………………………………………………………………………52 3.2.4 UPLC-ESI-MS/MS for identification of flavonols……………………………………………………………………………………54 3.2.5 HPLC-DAD conditions for quantification of flavonols……………………………………………………………………………………55 3.2.6 Total phenolic content by Folin-Ciocalteau assay………………………………………………………………………………………………55
iii 3.2.7 Colorimetric estimation of total limonoids……………………………………………………………………………………56 3.2.8 Anti-oxidant activity determinations………………………………………………………………………58 3.2.9 Statistical analysis………………………………………………………59 3.3 Results and discussions 3.3.1 Identification of flavonol aglycones……………60 3.3.2 Content of flavonols as determined by HPLC- DAD……………………………………………………………………………………………………64 3.3.3 Total phenolics determined by Folin- Ciocalteau assay…………………………………………………………………67 3.3.4 Anti-oxidant activities- FRAP anti-oxidant assay & DPPH anti-oxidant activity…………………68 3.3.5 Total limonoids- limonoid glucosides and aglycone………………………………………………………………………………………71 3.3.6 Analysis of commercial samples……………………………72 3.4 Conclusions………………………………………………………………………………………………74
4.CHARACTERIZATION OF THE VOLATILE PROFILE OF NEEM (AZADIRACHTA INDICA A. JUSS.) LEAF AND BARK COMMERCIALLY AVAILABLE IN THE UNITED STATES USING HS-SPME LINKED WITH GAS CHROMATOGRAPHY-MASS SPECTROMETRY
4.1 Introduction……………………………………………………………………………………………75 4.2 Materials and methods 4.2.1 Plant material………………………………………………………………………77 4.2.2 Chemicals……………………………………………………………………………………77 4.2.3 HS-SPME procedure………………………………………………………………77 4.2.4 Extraction of Essential oil……………………………………78 4.2.5 GC-MS Analysis………………………………………………………………………79 4.2.6 Statistical Analysis………………………………………………………79 4.3 Results and discussions 4.3.1 Comparison of the volatile profile of neem dried leaf powder, dry leaf and fresh leaf…………………………………………………………………………………………………80 4.3.2 Comparison between neem bark and leaf powder volatiles……………………………………………………………………………………84 4.3.3 Comparison between HS –SPME and essential oil volatiles of dried neem leaf powder……………………………………………………………………………………………88 4.4 Conclusions………………………………………………………………………………………………89
5.EFFECT OF TWO ADSORBENT BASED DE-BITTERING PROCEDURES IN NEEM (AZADIRACHTA INDICA A. JUSS) TEA- EFFECT ON TOTAL PHENOLIC CONTENT, ANTI-OXIDANT CAPACITY, COLOR AND VOLATILE PROFILE
5.1Introduction……………………………………………………………………………………………110 5.2 Materials and methods
iv 5.2.1 Plant material……………………………………………………………………112 5.2.2 Chemicals…………………………………………………………………………………112 5.2.3 Tea preparation…………………………………………………………………113 5.2.4 De-bittering procedures……………………………………………113 5.2.5 Extraction of flavonols……………………………………………114 5.2.6 Determination of polyphenol content by the Folin- Ciocalteu assay………………………………………………115 5.2.7 Determination of antioxidant activities…115 5.2.8 Colorimetric determination of total limonoid glucoside and limonoid aglycones…………………………………………………………………………………117 5.2.9 HPLC Analysis for the estimation of flavonols…………………………………………………………………………………118 5.2.10 Analysis of Flavor Volatiles by headspace- solid phase microextraction (HS-SPME) by GC-MS…………………………………………………………………………………………119 5.2.12 Color Properties……………………………………………………………120 5.2.13 Statistical Analysis…………………………………………………121 5.3 Results and discussions 5.3.1 Effect of debittering procedure on flavonols, total polyphenols and limonoids…………………………………………………………………………………122 5.3.2 Effect of debittering procedure on anti- oxidant activity of neem tea………………………………124 5.3.3 Effect of debittering procedure on color properties………………………………………………………………………………125 5.3.4 Effect of debittering procedure on the volatile profile of neem tea………………………………127 5.4 Conclusions……………………………………………………………………………………………133
6.EFFECT OF TEA MATRIX AND TYPE OF MILK ON THE RECOVERY OF FLAVONOLS, TOTAL PHENOLIC CONTENT AND ANTI-OXIDANT ACTIVITY WITH AN APPLICATION TOWARDS READY TO DRINK BEVERAGES (RTD’S)
6.1 Introduction…………………………………………………………………………………………134 6.2 Materials and Methods 6.2.1 Samples………………………………………………………………………………………137 6.2.2 Chemicals and standards……………………………………………138 6.2.3 Sample preparation…………………………………………………………138 6.2.4 Preparation of flavonol standards and flavonol extraction………………………………………………………139 6.2.5 HPLC analysis………………………………………………………………………140 6.2.6 Total phenolic content by Folin-Ciocalteau assay……………………………………………………………………………………………140 6.2.7 DPPH Anti-oxidant activity……………………………………141 6.2.8 Statistical analysis……………………………………………………141 6.2.9 Analysis of commercial sample……………………………142
v 6.3 Results and discussions 6.3.1 Effect of tea matrix on in-vitro flavonols, total phenolic content and DPPH anti-oxidant activity………………………………………………………………………………………………143 6.3.2 Effect of different types of milk on flavonol binding, total phenolic content DPPH anti- oxidant activity…………………………………………………………………………150 6.3.3 Analysis of commercial sample………………………………………157 6.4 Conclusions………………………………………………………………………………………………………158
FUTURE DIRECTION OF RESEARCH……………………………………………………………………159
REFERENCES……………………………………………………………………………………………………………………164
VITA……………………………………………………………………………………………………………………………………190
vi
List of Figures
Number of papers based on Web of Knowledge search for years 2006–2011…………………………………………………………………………………………………………8
Application of SPME to different food matrices………………………8
SPME Fiber assembly………………………………………………………………………………………………9
Steps in Headspace SPME coupled with GC-MS…………………………………10
Different classes of compounds found in essential oils……………………………………………………………………………………………………………………………15, 16
Basic skeleton structure of flavonoids……………………………………………22
Different flavonoid classes…………………………………………………………………………23
DPPH anti-oxidant assay……………………………………………………………………………………31
FRAP assay………………………………………………………………………………………………………………………33
Structures of some limonoids………………………………………………………………………36
Metabolic pathway of limonin………………………………………………………………………39
Chromatogram shows us three well separated peaks at 6.61, 9.81 and 12.96 mins when the signal is recorded at 370 nm with a photo diode array detector…………………………………………………………60
Structures of investigated flavonols…………………………………………………61
MS/MS spectrum of myricetin…………………………………………………………………………62
MS/MS spectrum of quercetin…………………………………………………………………………63
MS/MS spectrum of kaempferol………………………………………………………………………63
Retro Diels Alder cleavage of the C ring………………………………………64
Total phenolic content in the ethanolic extract of various tea samples………………………………………………………………………………………………67
Total phenolic content in the ethanolic extract of various tea samples………………………………………………………………………………………………68
Co-relation between FRAP values and total phenolic content………………………………………………………………………………………………………………………………69
vii Co-relation between DPPH values and total phenolic content………………………………………………………………………………………………………………………………69
Distribution of flavonols in commercial neem capsules in ethanolic extract and infusion…………………………………………………………………73
Percentage (%) of different classes of compounds as identified from the headspace of various neem samples by SPME and GC-MS……………………………………………………………………………………………………………81
Score plot for dried leaf powder (DLP), dry leaf (DL) and fresh leaf (FL)…………………………………………………………………………………………………………82
Loading plot for variables of dried leaf powder, dry leaf and fresh leaf……………………………………………………………………………………………………………82
Number of mono- and sesquiterpenes in only bark, only leaves or found in both tissues………………………………………………………………82
Chromatograms of the various neem leaf and bark samples………………………………………………………………………………………………………………………85,86
Effect of DP on quercetin in neem tea……………………………………………122
Effect of DP on the total phenolic content in neem tea………………………………………………………………………………………………………………………………………123
Effect of de-bittering procedure on the limonoid content of neem tea………………………………………………………………………………………………………………………………………124
Effect of de-bittering procedure on the FRAP anti-oxidant assay in neem tea…………………………………………………………………………………………………125
Effect of de-bittering procedure on the DPPH anti-oxidant activities in neem tea……………………………………………………………………………………126
Principal Component Analysis (PCA)- Score Plot. This shows the clustering tendency of neem tea (control) and the two de-bittered (treated) samples……………………………………………128
Loading plot of the variables reveals the specific volatiles (variables) that help discriminate between neem tea (control) and the two de-bittered samples (treatments)………………………………………………………………………………………………………………129
HPLC chromatograms of control (above) and treatment (below) samples………………………………………………………………………………………………………144
Comparative reduction in myricetin due to bovine milk addition in different tea matrices……………………………………………………145
viii
Comparative reduction in quercetin due to bovine milk addition in different tea matrices……………………………………………………145
Comparative reduction in kaempferol due to bovine milk addition in different tea matrices……………………………………………………146
Reduction in total phenolic content on addition of bovine milk to different tea matrices………………………………………………………………148
Reduction in DPPH anti-oxidant activity on addition of bovine milk to different tea matrices……………………………………………150
Comparative reduction in myricetin due to the addition of different types of milk to green tea………………………………………………152
Comparative reduction in quercetin due to the addition of different types of milk to green tea………………………………………………152
Comparative reduction in kaempferol due to the addition of different types of milk to green tea………………………………………153
Reduction in total phenolic content on addition of different types of milk to green tea………………………………………………154
Reduction in DPPH anti-oxidant activity after the addition of different types of milk to green tea………………………………………………………………………………………………………………………………………155
ix
List of Tables
Means of individual flavonols in ethanolic extract and infusion of various tea samples………………………………………………………………65
Means of anti-oxidant activities – FRAP and DPPH activity in the ethanolic extract of various tea samples……………………70
Means of anti-oxidant activities – FRAP and DPPH activity in the infusion of various tea samples……………………………………………70
Limonoid content in neem leaf and bark samples in the ethanolic extract……………………………………………………………………………………………………72
Limonoid content in neem leaf and bark samples in the infusion……………………………………………………………………………………………………………………………72
Volatile compounds in the different samples of neem as identified and quantified (%RA= %Relative Abundance) by GC-MS……………………………………………………………………………………………………………………………………91
Volatiles, unique to essential oil, as identified and quantified (%RA= %Relative Abundance) by GC-MS……………………105
The difference in color parameters of neem and de- bittered neem tea…………………………………………………………………………………………………126
Volatile compounds in NT and the two de-bittered samples identified on a DB-5 MS column………………………………………………………………130
Amount of flavonols-myricetin, quercetin and kaempferol in tea samples…………………………………………………………………………………………………………143
The total phenolic content of various tea samples as determined by the Folin’s assay……………………………………………………………147
The DPPH anti-oxidant activity of various tea samples…149
Nutritional differences between skimmed milk, soya milk and almond milk………………………………………………………………………………………………………151
x FUNCTIONALITY OF AZADIRACHTA INDICA A. JUSS (NEEM) IN BEVERAGES
Abhinandya Datta
Dr. Ingolf Grün, Dissertation supervisor
ABSTRACT
A significant increase in the health consciousness of people all around the world has rekindled interest in age old medicinal systems such as Ayurveda and Unani. The various trees, herbs and shrubs used in these ancient practices are being incorporated today into foods and beverages to meet the health expectations of consumers from their diet. There is an intense curiosity among analytical chemists to identify the compounds that contribute to the health benefits. Azadirachta indica A.
Juss is one such medicinal tree, indigenous to the Indian sub-continent that has found a revered place among village folks for its medicinal properties in being able to cure gastro-intestinal, dental and skin problems.
Modern day research on cancer cell lines and animal models have shown neem to possess excellent anti- inflammatory, anti-cancer and anti-diabetic properties.
In the first section, we analyze commercially available neem in the United States for its bio-active potential and contrast it with traditionally consumed teas- green and black tea. We found that the total polyphenols and anti-oxidant activities in green and black tea are far
xi higher than in neem, possibly due to the presence of flavan-3-ols in these teas. However, we used LC-ESI-MS/MS to identify specific flavonols- myricetin, quercetin and kaempferol in neem leaves, which were present in greater quantities in neem than in these teas. These flavonols have been known to impart neem its anti-diabetic property and therefore, its identification and quantification was crucial. In the second study, we examine the volatile profile of various neem leaf samples- powdered, dried and fresh, through solid phase microextraction (SPME) and essential oil extraction and analyze the constituents using gas chromatography-mass spectrometry. Fresh leaves contain organosulfur compounds that are absent in other samples. There is a preponderance of sesquiterpenes found in dried leaves and leaf powder. Diterpenes and acids were found to be major distinguishing factors between the
HS-SPME and essential oil volatile composition of dried neem leaf powder. The study reveals information about the aroma profile of different neem samples besides lending credence to its health properties as some of the volatiles identified are known to possess health properties. In the third section, we explore the area of
Ready to Drink beverages (RTD’s) and the consequences of adding milk to tea. We observe, that while tea matrix- green, neem and black tea, does not affect the decrease in flavonols-myricetin, quercetin and kaempferol, the
xii overall in-vitro phenolic content and anti-oxidant activity is reduced more markedly in green and black tea.
Among the different added milks, soya milk appeared to have the least effect on flavonols, phenolic content and anti-oxidant activity, in contrast with bovine and soya.
Although, protein-flavonoid interactions are important, the change in protein content of milk did not explain the changes in-vitro effect on phenolic compounds and consequent anti-oxidant activity. In the final section, we see explore the effect of two adsorbents based de- bittering strategies on the bioactive potential and organoleptic properties of neem tea. While both the solid phase extraction (SPE) and Amberlite XAD-16 (AMB) are successful in reducing the bitterness, both lead to a reduction in flavonol, total polyphenol, limonoid glucoside and anti-oxidant activity. On comparison, the reduction in SPE- treated neem tea is more than the AMB- treated, although both treatments lead to the removal of sesquiterpenes from the volatile profile. Given our results, the approach of using polyadsorbent resins for de-bittering purposes can be pursued further.
xiii CHAPTER 1
INTRODUCTION
1.1 BACKGROUND
Neem (Azadirachta indica A. Juss) is an evergreen, medicinal tree indigenous to South-East Asia
(Bhattacharyya and Sharma 2004). It is also found in tropical and sub-tropical regions of Africa, America and
Australia (Schmutterer 1990). Neem has been regarded for centuries as the centerpiece of natural healing in the villages of ancient Indian sub-continent. The taxonomic position of the Neem tree is as follows:
Order: Rutales; Family: Meliaceae (mahogany family);
Genus: Azadirachta; Species: indica.
A repository of abundant medicinal compounds, which have treated a multitude of diseases, neem has earned the respectable title of ‘sarvaroga nivarini’ or the panacea for all diseases (Arora and others 2008). All its parts including the leaf, stem, bark, twig, seed and flowers have been used for treating a wide variety of diseases.
Today, neem and its extracts have been commercialized into tea, soaps, shampoos, toothpaste and other cosmetic products. With a renewed interest in the age-old medical wisdom of our ancestors and the discovery of new analytical techniques, there is inquisitiveness in the scientific world to understand the applications of neem
1 towards treating some of the most challenging diseases of our times like cancer and diabetes.
There are several scientific papers elucidating the therapeutic role of neem extracts in cancer cell lines and model systems (Dasgupta and others 2004; Kumar and others 2006a; Roy and others 2007; Gunadharini and others
2011), in diabetes (Dholi and others 2011; Ponnusamy and others 2015; Mukherjee and Sengupta 2013), and its efficacy of anti-fungal and anti-bacterial activity
(Gupta and Bhat 2016; Raghavendra and Balsaraf 2014;
Akpuaka and others 2013). Although there are quantitative reports of bioactive limonoids, flavonoids and total phenolics in neem, the amount of information regarding specific compounds in these chemical groups is limited.
The studies on neem volatiles are comparatively fewer in number. The volatile composition of neem leaves and seeds has been studied by dynamic headspace extraction and solid phase microextraction, respectively. The bark volatiles have not been studied yet, although the gastro- protective and anti-microbial effects of its extracts have been elucidated (Bandyopadhyay and others 2002b; De and Ifeoma 2002; Tiwari and others 2010). There is literature documenting the anti-fungal (Zeringue and
Bhatnagar 1994) and pesticidal activity (Pathak and
Krishna 1991; Koul 2004; Balandrin and others 1988)of neem leaf and seeds. Shivashankar and others (2012)
2 identified organosulfur compounds such as 2,5 dimethyl thiophene; 3,4 dimethyl thiophene and 1,3 dithiane in neem seeds whereas Zeringue and Bhatnagar (1994) observed that the volatile profile of neem leaf was dominated by ketones, which accounted for 43% of the total area followed by alcohols, which occupied 23% of the headspace.
A growing consumer consciousness about the health value of their food choices has spawned the growth of a category of food products called functional foods. These foods and beverages aim to go beyond meeting the basic everyday nutritional needs and provide us protection from chronic diseases (Bigliardi and Galati 2013). Ready to drink beverages, which comprise of bottled or canned ice tea, coffee, fruit or vegetable smoothies, energy drinks, yogurt drinks, are an important market segment of this category.
Given the surge in popularity of tea as a health beverage, it has been combined with milk to produce ready to dink beverages that combine the health benefits of both. However, this complex mixture can pose challenges for food scientists given the interaction of milk proteins with various tea components affecting the anti- oxidant activity and bioavailability of polyphenols.
Complex formation of protein and phenolics results from hydrogen binding and hydrophobic interactions (Prigent
3 and others 2003; Yuksel and others 2010). The scientific community is split on the effect of milk addition on the anti-oxidant activity and bioavailability of tea polyphenols. While there are authors who observed that the addition of milk to tea leads to a decrease in polyphenol bioavailability and hence reduces the anti- oxidant activity (Serafini and others 1996; Langley-Evans
2000; Ryan and Petit 2010; Arts and others 2002; Xiao and others 2011)there are other authors, who suggested that milk addition has no effect on the anti-oxidant activity
(Leenen and others 2000). Hollman and others (2001), and
Kivits and others (1998) suggested that the in-vivo bio- availabilities of polyphenols are not affected in tea by milk addition.
The rich content of phytonutrients, especially limonoids leads to an extremely unpleasant bitter taste in neem leaf. This poses a huge problem for the food industry, as bitterness usually has an inverse impact on consumer acceptability of food products and beverages. While several de-bittering methods have been employed in the food industry for orange juice (Fernández‐Vázquez and others 2013), grapefruit juice (Lee and Kim 2003), legumes (Jiménez‐Martínez and others 2009) and other bitter foods and beverages, they can often lead to a loss of nutritional and sensorial properties that is
4 undesirable. Therefore, it is important to opt for a de-
bittering procedure, which is able to reduce the
bitterness without affecting the bioactive profile and
organoleptic properties adversely.
1.2 OBJECTIVES
In our study, we aimed to further support the medicinal
claims of Azadirachta indica by identifying and
quantifying polyphenols and volatile compounds
responsible for its health value. We further analyzed the
challenges a food scientist may face in incorporating
neem into food products, specifically working with neem
tea. In this regard, we analyzed the effect of various
milks (bovine, soy and almond) on the in-vitro measure
amounts of neem flavonols (polyphenols) with an
application in ready to drink beverages (RTD’s). Since
neem is rich in limonoids that impart bitterness, we
attempted to come up with adsorbent based de-bittering
strategies. We further investigated the impact of these
de-bittering strategies on neem tea with regards to its
polyphenolic, anti-oxidant and sensory properties.
Specific objectives of this study are:
1. To identify and quantify select flavonoids in neem leaf
extracts (ethanolic and water) and compare them to green
and black through HPLC-DAD-ESI- MS/MS.
5 2. Study neem volatiles as extracted by headspace solid
phase microextraction (HS-SPME) and hydrodistillation
and analyzed by gas chromatography-mass spectrometry.
3. Study the interaction of milk proteins and flavonoids,
its effect on anti-oxidant capacity and its potential
application in Ready to Drink beverages (RTD’S).
4. Assess the impact of two adsorbent based de-bittering
procedures on the polyphenolic, anti-oxidant and
organoleptic properties of neem tea.
6 CHAPTER 2
LITERATURE REVIEW
2.1 Headspace Solid-Phase Microextraction (HS- SPME)
Since its introduction in the 1990’s, solid phase microextraction (SPME) has emerged as the preferred tool for the extraction of volatiles in the analytical chemistry world. Invented by Pawliszyn and Arthur in
1990, SPME has found extensive usage for extraction of flavor volatiles in the food industry. It has several advantages over traditional extraction techniques, such as solvent assisted flavor evaporation (SAFE), liquid- liquid extraction (L/L E), high vacuum transfer (HVT), and has become the extraction technique of choice (Jeleń and others 2012). Fig 1 A. shows the dramatic increase in the number of publications involving SPME compared to other methods during the period of 2006-2011. Besides being cheap, sensitive, highly reproducible and offering fast sampling times, SPME is solventless and provides a high degree of enrichment of the analytes of interest
(Wardencki and others 2004). SPME can be automated but the manual form is also convenient to use, offering cheap, reusable holders in which the fiber can be replaced (Jeleń and others 2012). A literature search on
SPME reveals that is has an abundance of applications
7 with it being used for fruits and vegetables second only to wine, as depicted in Fig 1.B.
Fig 1. A. Number of papers based on Web of Knowledge search for years 2006–2011(Jeleń and others 2012)
B. Application of SPME to different food matrices (Jeleń and others 2012)
The SPME fiber assembly consists of a fiber holder and a cylindrical shaped fused silica fiber inside a stainless- steel needle, as shown in Fig.2. The needle is connected to a syringe so that the coated fiber can be protruded outside or drawn-in when needed. The fused silica fiber
8 is coated with a relatively thin film (in the order of microns) of various polymeric stationary phases.
Fig. 2 SPME Fiber assembly (Vas and Vekey 2004)
The SPME fiber is placed either in contact with the sample matrix (direct immersion) or into the headspace above (Pragst and others 2001)for a predetermined amount of time. The analyte(s) are extracted directly from an aqueous, gaseous or headspace above the solid or liquid samples onto a stationary phase. Then, the extracted analytes are desorbed either by thermal means or by using a solvent and analyzed by gas chromatography or high pressure liquid chromatography.
9 Fig. 3 Steps in Headspace SPME coupled with GC-MS
(Kataoka and others 2000a)
The choice of fiber in the SPME apparatus is crucial in determining the nature of the volatiles trapped (Kataoka and others 2000a). So far, there have been six types of fibers developed to address different applications. The coatings can be broadly classified into two groups- the pure liquid polymer coating such as polydimethylsiloxane
(PDMS) or polyacrylate (PA) and the mixed film, containing liquid polymer and solid particles such as
Carboxen-PDMS, Divinylbenzene (DVB)-PDMS, Carbowax-DVB and DVB-Carboxen-PDMS. The mixed films combine the absorption properties of the liquid polymer with the adsorption properties of porous particles. PDMS by nature is hydrophobic while polyacrylate is currently the most polar coating available. Hence, the former is used for trapping environmental pollutants such as poly aromatic
10 hydrocarbons(PAH) while the latter coating is used for analyzing fatty acids and reduced sulfur compounds.
Carboxen is a carbon molecular sieve containing macro-, meso- and micropores and is used in combination with
PDMS. The pore size does not allow the bigger molecules to enter the micropores (where the interactions are the strongest), so that the combination of Carboxen and PDMS improves the extraction for small molecules (Popp and
Paschke 1997; Azodanlou and others 1999). The divinylbenzene solid polymer has larger pores than
Carboxen and is thus better adapted for the extraction of bigger molecules such as aniline derivatives (Müller and others 1997; DeBruin and others 1998).
The DVB-Carboxen-PDMS fiber extracts a very wide spectrum of analytes varying in polarity and size. The first layer is made of PDMS/ Carboxen and is covered with a second layer made of PDMS/DVB. The small molecules, having a higher diffusion coefficient, reach the inner layer faster where they are adsorbed onto the Carboxen. The heavier molecules are retained in the outer of DVB layer.
Desorption is also facilitated with this configuration.
The Carbowax-DVB is the most polar fiber of the second group.
The affinity of the fiber for an analyte depends on the principle of “like dissolves like” (Kataoka and others
2000a). Thus, the polarity of the fiber coating and the
11 nature of the target compounds intended to be extracted from a food sample should be kept in mind. The fiber thickness can also affect the nature and the amount of the target analyte adsorbed onto the fiber (Kataoka and others 2000a). The extraction efficiency of analytes is also dependent upon, and can be improved, by heating the sample, saturating the sample with salts, or agitating the sample using a magnetic stir bar (Vázquez and others
2008; Psillakis and Kalogerakis 2001). The addition of salt increases the ionic strength of the solution leading to a “salting out” effect, which decreases the solubility of the target analyte thereby, improving the extraction efficiency. The attainment of equilibrium between the sample and fiber is necessary to achieve maximum sensitivity during SPME extraction (Jeleń and others
2012). After equilibrium is attained between the sample matrix and the filament, the mass of compound extracted by the coating is given by the relationship (Pawliszyn,
1999)- n = (Kfs. Vf. Vs. Co) / (Kfs. Vf +Vs)
Where n =mass of compound extracted by the coating, Kfs = fiber coating/sample matrix distribution constant, Vf = fiber coating volume, Vs =sample volume, Co =initial concentration of a given compound in the sample.
However, SPME is a non-exhaustive process and precise analysis does not require achievement of full
12 equilibration (Jeleń and others 2012) .This is because of the linear relationship between the amount of analyte adsorbed by the SPME fiber and its initial concentration in the sample matrix under non-equilibrium conditions
(Kataoka and others 2000b) .
2.2 Essential Oils
Essential oils (EO) are volatile, natural, complex compounds characterized by a strong odor and are formed by aromatic plants as secondary metabolites (Bakkali and others 2008). They are also called volatile or ethereal oils (Guenther 1948). While they can be extracted by expression, fermentation, enfleurage or extraction, the method of steam distillation is most commonly used for commercial production of essential oils. Essential oils have been used for centuries in medicine, perfumery, cosmetic, and have been added to foods as part of spices or herbs.
They contain hydrocarbons, such as monoterpenes, sesquiterpenes and diterpenes, and oxygenated organic compounds, such as alcohols, esters, ethers, aldehydes, ketones, lactones, phenols and phenol ethers (Guenther
1972). The composition varies with respect to plant species and geographical areas in which they are cultivated (Zygadlo and others 2003). They can be extracted from different parts of the plant such as from
13 flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits and roots (Sánchez-González and others 2011).
Essential oils protect plants through their insect repellant activity (Jaenson and others 2006; Govere and others 2000), because many monoterpenes, sesquiterpenes and diterpenes (Kiran and others 2007; Jaenson and others
2006; Sukumar and others 1991; Odalo and others 2005) have been associated with insect repellant activity. For example, beta–caryophyllene, a sesquiterpene found in several essential oils has been found to have strong insect repellant activity against A. aegypti (yellow fever mosquito) (Gillij et al., 2008). Moreover, the oxygenated compounds phenylethyl alcohol, b-citronellol, cinnamyl alcohol, geraniol, and alpha-pinene, isolated from the essential oil of Dianthus caryophyllum, showed strong repellent activities against ticks (I. ricinus)
(Tunón and others 2006).
Given the myriad of chemical compounds found in EOs, the mechanism of anti-microbial action cannot likely be attributed to a single mechanism (Skandamis and Nychas
2001; Carson and others 2002).
Since essential oil components are hydrophobic in nature, they can partition into the cell membrane lipid layer and disrupt it allowing the leakage of important cell components and ions (Sikkema and others 1994; Oosterhaven and others 1995; Carson and others 2002; Skandamis and
14 Nychas 2001). EOs possessing a high concentration of phenolic compounds such as thymol, carvacrol and eugenol have been found to be very effective against food-borne pathogens (Dorman and Deans 2000; Juliano and others
2000; Lambert and others 2001).
Fig.4 Different classes of compounds found in essential oils (Bakkali and others 2008)
15
The structural configuration of EO components also affect the efficiency of anti-microbial action. The change in position of the hydroxyl group in the phenolic ring in carvacrol and thymol, were found to affect how they acted against gram-positive and gram-negative species (Dorman and Deans 2000). The lack of the phenolic ring itself
(destabilized electrons) seems to have an impact on the anti-microbial activity as seen with menthol compared to carvacrol (Ultee and others 2002). The addition of an acetate moiety, such as in the conversion of geraniol to geranyl acetate, increased the anti-microbial activity
16 (Dorman and Deans 2000). In non-phenolic compounds, the saturation of the alkyl groups seems to affect the activity against micro-organisms as seen in the case of limonene ((1-methyl-4-(1-methylethenyl)-cyclohexene), which was found to be more effective than p-cymene (1-
Methyl-4-(1-methylethyl) benzene)(Dorman and Deans 2000).
Another possible mechanism by which EO components act on cell membranes is by affecting proteins located in the bilipid layer (Knobloch and others 1989). Two probable ways in which cyclic hydrocarbons in EOs work is by either accumulating in the bilipid layer and disrupting the lipid–protein interaction or by direct interaction of the lipophilic compounds with hydrophobic parts of the protein (Juven and others 1994). Cinnamon oil and its components have been shown to inhibit amino acid decarboxylases in Enterobacter aerogenes by possibly directly binding with the protein (Wendakoon and
Sakaguchi 1995).
In the context of food borne pathogens and food spoilage bacteria, EOs have been found to be more effective against gram-positive than gram-negative bacteria. This is because of the presence of a cell wall in gram negative bacteria (Ratledge and Wilkinson 1988), which restricts diffusion of hydrophobic compounds through its lipopolysaccharide covering (Vaara 1992).
17 The phenolic and terpenoid compounds in EO’s make them potent anti-oxidants, which has been measured by chemical assays (Bektaş and others 2016; Yassa and others 2015).
One of the organelles attacked by the EO seems to be the mitochondria. As proposed by Bakkali and others (2008),
EOs disrupt the mitochondrial membrane. When this happens, there are changes in the electron transport chain, leading to the production of free radicals which then damage DNA, proteins and lipids (Van Houten and others 2006). Moreover, some of the phenolic constituents of EOs react with reactive oxygen species (ROS) to produce highly reactive phenoxyl radicals which cause further damage. These types of radical reactions are dependent on and enhanced by the presence of cell transition metal ions such as Fe++, Cu++, Zn++, Mg++ or
Mn++ (Stadler and Fay 1995; Sakihama and others 2002;
Jiménez‐Martínez and others 2009; Azmi and others 2006).
Essential oils, being volatile, are being widely used in aromatherapy. Lavender essential oil, with its two main components linalool and linalyl acetate, has been successfully shown to have a sedative effect (Buchbauer and others 1991). Essential oils of lavender, rose, orange, bergamot, lemon, sandalwood, clary sage, Roman chamomile and rose scented germanium have found extensive usage in reduction of anxiety, stress and depression
(Setzer 2009).
18 The crude essential oils classified as GRAS by FDA include, amongst others, clove, oregano, thyme, nutmeg, basil, mustard, and cinnamon (Carson and others 2002).
There are regulatory limitations on the accepted daily intake of essential oils or essential oil components. So, before they can be used in food products, a daily intake survey should be available for evaluation by FDA
(Hyldgaard and others 2012). However, the research into the use of essential oils in food preservation has yielded encouraging results. Singh and others (2002) used thyme oil treatment followed by aqueous chlorine dioxide/ozonated water, or ozonated water/aqueous chlorine dioxide and saw that it caused a significant
3.75 and 3.99 log, and 3.83 and 4.34 log reduction in E. coli O157:H7, when applied on lettuce and baby carrots, respectively.
Chouliara and others (2007) observed an additional preservation effect on fresh chicken breast meat, when oregano essential oil (0.1% and 1% w/w) and modified atmosphere packaging (MAP) (30% CO2/70% N2 and 70% CO2/30%
N2) were applied in combination. In yet another study, the addition of 0·8% (v/w) oregano essential oil to beef meat fillets resulted in an initial reduction of 2–3 log of the majority of the bacterial population
(Tsigarida and others 2000).
19 Despite these positive results, there are limitations to the use of essential oils in food systems because of their intense aroma which may alter the sensory profile of the food, even at low concentrations (Lv and others
2011). The fact that essentials oils possess anti- microbial activity only at high concentrations limits its usage.
Furthermore, there are food matrices in which the EO constituents are rendered ineffective due to their interaction with fat (Rattanachaikunsopon and
Phumkhachorn 2010), starch (Gutierrez and others 2008) and proteins (Kyung 2012).
In spite of the fact that a considerable number of EO components are GRAS and/or approved food flavorings, some research data indicate irritation and toxicity, for example, with eugenol, menthol and thymol in root canal treatments (Gómez-López 2012).
Some EOs and their components have been known to cause allergic contact dermatitis in people who use them frequently (Carson and Riley 2001). Splasmogenic properties have been seen in essential oils used in aromatherapy and paramedicine, although it was not possible to link it with a specific component (Madeira and others 2002). It is recommended that more safety studies be carried out before EOs are more widely used or at greater concentrations in foods that at present.
20 2.3 Flavonoids and their health benefits
Flavonoids are secondary plant metabolites, nearly ubiquitous in plants and are recognized as the pigments responsible for the colors of leaves (Middleton and others 2000). Over 5000 structurally unique flavonoids have been identified in plant sources (Xia and others
2013). Flavonoids possess low molecular weights and comprise of the basic structure of fifteen carbon atoms, arranged in a C6–C3–C6 configuration (Ignat and others
2011). They contain two benzene rings (A and B) linked through a heterocyclic pyran or pyrone (with a double bond) ring (C) in the middle (Middleton and others 2000), as shown in Fig. 1. The aromatic ring A is derived from the acetate/malonate pathway, while ring B is derived from phenylalanine through the shikimate pathway (Merken and Beecher 2000). Variations in the substitution patterns of ring C results in the major flavonoid classes, i.e., flavonols, flavones, flavanones, flavanols
(or catechins), isoflavones, flavanonols, and anthocyanidins (Hollman and others 1999) of which flavones and flavonols are the most widely occurring and structurally diverse (Harborne and others 1999), as depicted in Fig. 2. Substitutions to rings A and B give rise to different compounds within each class of flavonoids (Pietta 2000). These substitutions may include
21 oxygenation, alkylation, glycosylation, acylation, and sulfonation (Balasundram and others 2006).
Fig 1. Basic skeleton structure of flavonoids
(Hammerstone and others 2000)
On average, the daily USA diet was estimated to contain approximately 1 g of mixed flavonoids expressed as glycosides (Kühnau 1976) .The flavonoid consumed most was quercetin, and the richest sources of flavonoids consumed in general were tea, onions, and apples (Hertog and others 1993). Recent evidence indicates that flavonoid- glycosides are much more readily absorbed than the aglycones by humans (Hollman and others 1999). Flavonoids have important effects in plant biochemistry and physiology, acting as antioxidants, enzyme inhibitors, precursors of toxic substances, and pigments and light screens (McClure 1975). They affect synthesis of plant growth hormones and growth regulators, the control of
22 respiration, photosynthesis, morphogenesis, and sex determination, as well as defense against infection
(Smith and Banks 1986). Its anti-inflammatory, anti- oxidant, hepatoprotective, antiviral, and anti- carcinogenic activities (Fu and others 2013; Huang and others 2015; Sirovina and others 2013; Romagnolo and
Selmin 2012; Liu and others 2008b) have been well documented. A renewed interest in traditional folk medicine, along with the development of analytical methodologies, has rekindled interest in the flavonoids and the need to understand their interaction with mammalian cells and tissues.
Fig 2. Different flavonoid classes (Ignat and others
2011)
23
Flavonoids are synthesized in the cytosol but are transported and found accumulated in plant vacuoles
(Kitamura 2006). Extraction is a very important step in the isolation, identification and use of flavonoids, but there is no standard extraction method. Solvent (Baydar and others 2004; Bucić-Kojić and others 2007), microwave– assisted (Gao and others 2006; Chen and others 2008), super critical fluid, ultrasonic- assisted and high hydrostatic pressure extractions have been used in the past. Out of these, solvent extraction is the most popular method of extraction (Routray and Orsat 2012).
24 The extraction process usually involves a pre-treatment step aimed at increasing the contact surface area between the solvent and the sample. These pretreatment steps lead to the breakdown of cellular structures, which further enhances the yield of the bioactive compounds. Some of these steps include maceration, centrifugation, vortexing, homogenization, grinding, milling, or drying
(generally freeze drying to prevent degradation of flavonoids)(Routray and Orsat 2012; Merken and Beecher
2000).
The extraction efficiency depends on several factors such as a time, temperature, nature of solvent, liquid-solid ratio, flow rate and particle size. The most common solvents used are water, methanol or ethanol (Fiamegos and others 2004; Wang and Helliwell 2001; Wang and others
2003; Chu and others 2000).
Flavonoids exist in both glycoside and aglycone forms (Lv and others 2015). For ease of analysis, the glycosides are usually hydrolyzed into the common aglycone form.
Both acidic and alkaline hydrolysis are done. Although reaction times and temperatures for the acidic and alkaline hydrolysis conditions vary a great deal, this general method involves treating the plant extract or food sample itself with inorganic acid (HCl) (Escarpa and
González 2001)or NaOH (1-2 M) (Shahrzad and Bitsch
25 1996)at reflux or above reflux temperatures in aqueous or alcoholic solvents.
High Pressure Liquid Chromatography (HPLC) has emerged as the analytical tool of choice for the separation, identification and quantification of flavonoids (Wang and
Helliwell 2001; Ooh and others 2015; Tomás-Barberán and others 2001). The chromatographic mode is, almost exclusively, reverse phase performed on a C18 column. The mobile phase usually consists of a binary solvent system, with gradient elution, containing acidified water
(solvent A) and a polar organic solvent (solvent B).
UV/VIS diode array detector (DAD)(Sakakibara and others
2003), mass or tandem mass spectrometry (Ali and Alan
2015; Qiao-Hui and others 2016) have been the detectors of choice.
For complex matrices, pre-concentration steps such as solid phase extraction (SPE) (Lalaguna 1993;
Michalkiewicz and others 2008) and divinylbenzene styrene resins such as XAD 4 OR XAD-16 (Liu and others 2008b; Li and others 2005) are used to remove interfering components.
Flavonoids contain conjugated ring structures and hydroxyl groups that have the potential to function as antioxidants in vitro or in cell free systems by scavenging superoxide anion, singlet oxygen, lipid peroxyradicals, and stabilizing free radicals involved in
26 oxidative processes through hydrogenation or complexing with oxidizing species (Yao and others 2004).
Flavonoids are thought to mediate their anti-oxidant action by the following mechanisms:
Direct radical scavenging involves the acceptance of electrons by the flavonoid from free radicals to oxidize itself to form a less-reactive radical. This can be best described by the equation:
Flavonoid (OH) + R• > flavonoid (O•) + RH (Nijveldt and others 2001) where R• is a free radical and O• is an oxygen free radical. The protective action of flavonoids in preventing LDL (Low Density Lipoprotein) oxidation to prevent atherosclerosis is because of this free radical scavenging function (Hirano and others 2001; Fuhrman and
Aviram 2001).
Interfering with nitric oxide synthase activity is another mechanism through which flavonoids attenuate free radicals. The inorganic free radical nitric oxide (NO) has been implicated in physiological and pathological processes such as vasodilation, non-specific host defense, ischemia reperfusion injury, and chronic or acute inflammation (Matsuda and others 2003). Apigenin, diosmetin, tetra-O-methylluteolin and hexa-O- methylmyricetin were found to show potent nitric oxide synthase inhibitory activity (Matsuda and others 2003).
27 Nitric oxide released by activated macrophages reacts with free radicals to form peroxynitrite that has an oxidative effect on low-density lipoproteins (LDLs)
(Jessup and others 1992). Haenen and others (1997) found that the peroxynitrite scavenging activity of flavonoids was found to be 10 times more than the known peroxynitrite scavenger ebselen. This effect has a direct bearing on the beneficial effect of flavonoids and the incidence of coronary heart disease (Haenen and others
1997).
The flavonoids react with the free radicals to counteract the formation of peroxynitrite. Nitric oxide, although being a vasodilator, can on its own be regarded as a free radical. There are reports of flavonoids directly scavenging nitric oxide molecules.
In humans, xanthine oxidase is a flavoprotein enzyme responsible in catalyzing the oxidative hydroxylation of hypoxanthine and xanthine to produce uric acid and subsequent reduction of O2 at the flavin center with generation of reactive oxygen species, either superoxide anion radical or hydrogen peroxide (Boban and others
2014). Excessive uric acid deposits in joints and causes a painful disorder called gout (Martinon and others
2006). Furthermore, there is overwhelming acceptance that xanthine oxidase is associated with pathological conditions involving inflammation, metabolic disorders,
28 cellular aging, reperfusion damage, atherosclerosis, hypertension, and carcinogenesis (Dawson and Walters
2006; Pacher and others 2006). Flavonoids have shown to have an inhibitory effect on xanthine oxidase (Lin and others 2015; Cos and others 1998). The hydroxyl groups at
C-5 and C-7 and the double bond between C-2 and C-3 were found to be essential for a high inhibitory activity on xanthine oxidase (Cos and others 1998).
Several flavonoids have exhibited an iron and copper chelating activity (Mira and others 2002). In fact, the prominent anti-oxidant activity of the flavonoid quercetin is attributed to its iron chelating activity by which it is able to suppress DNA strand scission and cytotoxicity caused by tert-butylhydroperoxide (Sestili and others 1998). Quercetin, catechin and diosmetin have been implicated in prevention of iron induced lipid peroxidation of rat hepatocytes (Morel and others 1993).
Eicosanoids derived from arachidonic acid metabolism, including products from cyclooxygenase (COX)
(prostaglandins) and lipoxygenase (LOX) (leukotrienes), seem to also play a critical role in inflammation.
Flavonoids have been implicated in an anti- inflammatory role (MORONEY and others 1988; Ferrandiz and Alcaraz
1991). For example, cyanidin-3-glucoside has shown inhibitory effects on the production of several mediators during inflammation in the colonic carcinoma cell line
29 HT29 by down-regulating COX-2 (cyclooxygenase) expression. Quercetin glycoside, quercitrin when administered under 5mg/kg body weight showed anti- inflammatory response in different model systems
(Comalada and others 2005; de Medina and others 1996).
Oxidative damages to cells have been implicated in cancer
(Paz-Elizur and others 2008), liver disease(Preedy and others 1998), Alzheimer’s disease (Moreira and others
2005), aging (Liu and Mori 2005), arthritis (Čolak 2008), inflammation (Mukherjee and others 2007), diabetes (Rains and Jain 2011) and other diseases.
A biological antioxidant has been defined as “any substance that, when present at low concentrations compared to those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate” (Halliwell and Gutteridge 1999). The beneficial effects of polyphenols in fruits, vegetables and various plant products has been attributed to their anti-oxidant capacity. There are several methods to measure the anti-oxidant capacity of foods and beverages but each of them come with their drawbacks.
DPPH or 2,2- Diphenyl-1-picrylhydrazyl assay is a widely- used method of measuring anti-oxidant capacity of biological systems. 2,2- Diphenyl-1-picrylhydrazyl is a stable organic nitrogen radical, which has a purple color
(MacDonald‐Wicks and others 2006). The principle of the
30 assay is based on the decolorization of the DPPH radical due to the presence of anti-oxidants, which is measured at 515 nm (Moon and Shibamoto 2009)(Fig. 3). The decrease in the absorbance of the test sample is proportional to the concentration of anti-oxidants in the sample.
Fig. 3 DPPH anti-oxidant assay (Moon and Shibamoto 2009)
The assay tests the ability of compounds to act as hydrogen donors (Brand-Williams and others 1995). It is simple, rapid and can be performed with a spectrophotometer. However, the assay has several
31 drawbacks. Carotenoids, which have the same absorption maxima as DPPH radical of 515nm, often interfere with the assay (Nomura and others 1997). Steric accessibility is a crucial factor in the DPPH reaction since small molecules have a better access to the radical site than larger ones
(Xie and Schaich 2014). The radical site is protected inside a reaction cage formed by the two phenyl rings orthogonal to each other, and the picryl ring angled about 30° with its two nitro groups oriented above and below the radical site. DPPH also is decolorized by reducing agents as well as hydrogen transfer, which also contributes to inaccurate interpretations of anti-oxidant capacity. DPPH is a stable nitrogen radical that bears no similarity to the highly reactive and transient peroxyl radicals involved in lipid peroxidation. Many antioxidants that react quickly with peroxyl radicals may react slowly or may even be inert to DPPH due to steric inaccessibility and therefore at times, is not a realistic representation of anti-oxidant capacity of foods and beverages.
FRAP or ferric reducing anti-oxidant assay involves the transfer of electrons from anti-oxidants, which reduces ferric salt, Fe (III)(TPTZ)2Cl3 (TPTZ) 2,4,6-tripyridyls- triazine), to its reduced Fe(II) form, to form a blue colored complex whose absorbance is measured at 593 nm
(Fig.4). The time period of analysis has a bearing on the
32 FRAP results. Some polyphenols such as quercetin, caffeic acid, tannic acid, ferulic acid and ascorbic acid, react slowly with TPTZ (Pulido and others 2000)compared to others. Although, FRAP assay is simple, rapid and can be automated, it does not measure the anti-oxidant capacity of thiol based anti-oxidants and carotenoids. FRAP measures only the reducing capability based upon the ferric ion, which is not relevant to antioxidant activity mechanistically and physiologically.
Fig. 4 FRAP assay (Moon and Shibamoto 2009)
2.5 Limonoids and bitterness
A dramatic increase in the health consciousness of consumers around the world has fostered the commercial development of functional foods, whose health benefits go beyond providing just basic nutritional needs (Bigliardi
33 and Galati 2013). A better understanding of the relationship between diet and health has led to a boom in dietary health supplements, such as foods and beverages incorporating medicinal plant extracts. These functional foods contain bioactive compounds, such as anthocyanins, carotenoids, flavonoids, isoflavones and terpenes, which are targeted towards improving digestive health, child nutrition, weight management, obesity, diabetes, and beauty enhancement, to mention a few. Numerous consumer studies have pointed towards the primary role of taste as a factor, which directs consumers’ food choice in general
(Urala and Lähteenmäki 2003; Grunert and others 2000;
Richardson and others 1994). Also, in the specific case of functional foods, taste experiences have been reported as extremely critical factors when selecting this food category (Tuorila and Cardello 2002; Childs 1997; Gilbert
2000). Although increasing the functionality of the food should not necessarily change its sensory quality, there are instances where bitter, acrid, astringent or salty off-flavors often get incorporated with the use of bioactive compounds or plant-based phytonutrients (Urala and Lähteenmäki 2004). These off –flavors have led to a decrease in consumer liking and consumption of functional foods despite having convincing health claims, as shown in some studies (Drewnowski and Gomez-Carneros 2000;
Tuorila and Cardello 2002). Bitter taste is one of these
34 offensive sensorial modalities and has been the primary reason for the rejection of food products although there are certain foods such as coffee, beer, wine and bitter melon where a certain degree of bitterness is acceptable.
(Binello and others 2004; Binello and others 2008; Singh and others 2002a; Shaw and others 1984). Therefore, bitterness removal is one of the biggest hurdles for the functional food industry, in order to make functional foods more palatable to a larger consumer base.
Limonoids are highly oxygenated triterpenes found abundantly in the plant kingdom, in species belonging to the family Meliaceae and Rutaceae (Fig.1). Limonoids are found either in glycosylated or non-glycosylated forms.
While the glucosides are soluble in water and tasteless in nature (Hasegawa and others 1989), the aglycones are bitter in nature and contribute to the bitterness in neem and citrus fruits (Nathan and others 2005; Kita and others 2000). They are synthesized via the terpenoid biosynthetic pathway, which involves the cyclization of squalene to form a tetracyclic triterpene cation, euphane and tirucallane, two chemically similar compounds that are purportedly the precursors of limonoids.
Limonoid based bitterness removal has been studied extensively in the context of the citrus juice industry.
Several techniques have been employed to reduce the
35 content of bitter limonoids in citrus juice, such as a) adsorption b) encapsulation agents (e.g., cyclodextrins) c) bio-degradation by enzymes from microbial cells and d) post-harvest treatment of fruits.
Fig.1 Structures of some limonoids (Perez and others
2010)
36
Adsorption is a physico-chemical process that involves the mass transfer of a solute (adsorbate) from the fluid phase to the adsorbent surface till the thermodynamic equilibrium of the adsorbate concentration is attained, with no further net adsorption (Belter and others 1987;
Doran 2006). Some of the earliest successful attempts of using adsorbents to remove bitterness were made by
Chandler and Kefford (1968) with polyamides.
Consequently, a variety of adsorbents, such as cellulose acetate, nylon-based matrices, porous polymers, and ion exchangers have been explored to reduce bitterness and acidity in grapefruit juice (Johnson and Chandler 1982)
37 Synthetic polyadsorbent resins such as Amberlite neutral resins (XAD-4, XAD-7 and XAD-16), as well as natural adsorbents (activated diatomaceous earths, activated granular carbon), have been used for removal of limonin from orange juice (Ribeiro and others 2002). Ribeiro and others (2002) found that the synthetic resins are more effective than natural adsorbents at removing bitter principles in orange juice. In addition, it was selective in the removal process by causing minimal reduction in reducing sugars, vitamin C and proteins. In a comparative study between cyclodextrins and synthetic resins, Wilson and others (1989) found XAD- 16 resins to be more effective at bitterness removal than XAD- 4 and beta cyclodextrin. Kola and others (2010) tested the efficiency of an adsorbent resin (Amberlite XAD-16 HP) and an ion-exchange resin (Dowex Optipore L285) in
Washington Navel orange juices. Both the treatments were able to reduce bitterness satisfactorily. However, the ion exchange resin led to some undesirable properties such as reduced titratable acidity, increased soluble solids content and increased pH.
Cyclodextrins are cyclic oligosaccharides composed of
6(α), 7 (β) and 8 (γ) glucopyranose units joined together by glycosidic bonds (Del Valle 2004). They possess a unique, amphipathic bucket like structure where the outer side of the molecule is hydrophilic while the inner
38 cavity is hydrophobic (Szejtli 1998). The inner hydrophobic cavity can bind with lipophilic bitterness imparting compounds forming an inclusion complex (Szejtli and Szente 2005). This leads to a reduction in oral solubility of the bitter compound upon ingestion or a limited exposure to taste buds, thereby minimizing the perception of bitterness (Shaw and others 1984; Shin and
Lee 2015; Fajarika and Noor 2015). It has been used for debittering of navel orange juice (Shaw and others 1984), grapefruit juice (Shaw and others 1984; Shaw and Buslig
1986; SHAW and WILSON 1985)and tangerine juice
(Mongkolkul and others 2006) .
Use of enzymes from immobilized microbial masses to convert limonin into non-bitter metabolites, has been applied successfully in citrus juices by several authors.
It utilizes the pathway illustrated in Fig. 2.
Fig. 2 Metabolic pathway of limonin (Puri and others
1996)
39
Hasegawa, Vandercook and others (1985) observed that 81% of limonin and almost all nomilin were converted to non- bitter end products. The constitutively produced limonol dehydrogenase enzyme, converted limonin to limonol after navel orange juice serum was treated with Corynebacterium fascians cells immobilized in acrylamide gel, after a
24 h reaction time, in a packed bed column. Also of significance was the result that organic acids including citric, malic, and ascorbic acid, as well as sugars, i.e. fructose, glucose, and sucrose, which are important for organoleptic properties, were unaffected. A slightly lower 73% reduction in Limonin was seen when Arthrobacter globiformis cells were used under the same conditions
(Hasegawa and others 1983). A purified soil bacterium,
Acetinobacter sp. was identified by Vaks and Lifshitz
(198l) and used to treat early season (more bitter) juice. The bacterium was able to use limonin as a sole
40 carbon source and convert it into two non-bitter products- deoxylimonin and deoxylimonic acid. Ribeiro et al. (2003) utilized Acinetobacter calcoaceticus to de- bitter orange juice, in which limonin was converted into non-bitter products through the deoxylimonoid pathway, without affecting the sugar content of the juice. Cánovas and others (1998), used Rhodococcus fascians with synthetic orange juice at pH 4, and observed a 70% reduction in limonin. When these cells were immobilized in polyurethane foams, 85% limonin conversion was attained, after a 200 h reaction, in a continuous reactor.
Puri and others (1996) noted that the techniques of immobilization occupied the same loci as the ones that are useful for enzyme activity. As a result, the enzyme method of debittering exhibited slow kinetics, which made it impractical for scale up operations. Therefore, as a solution, use of free cells was undertaken. Inactivation of enzymes by particulate matter or clogged columns were the other drawbacks of the enzymatic method of de- bittering, as noted by Puri and others (1996).
Post-harvest treatment of naval orange, lemon, and grapefruit with 20 ppm ethylene accelerated limonoid metabolism and reduced bitterness to more palatable levels than untreated ones (Maier and others 1973). The bitterness reduction, which can be also achieved through
41 2-chloroethylphosphonic acid (CEPA), was a result of the destruction of Limonoate A Ring Lactone (LARL) that was prevented from converting itself to the bitter limonin.
Limonoids are quite bioactive in nature having insecticidal, anti-bacterial, anti-malarial, anti-fungal, anti-cancer, anti-viral and other pharmacological activities (Govindachari and others 1996; Nathan and others 2005; Abdelgaleil and others 2004; Poulose and others 2006; Balestrieri and others 2011; Zhang and others 2007; Rahman and others 2009). Therefore, their removal may lead to a significant decline in the health value of foods and beverages. The ideal method should aim to retain health-promoting compounds while improving or maintaining organoleptic properties.
2.5 Milk protein- flavonoid interaction
Bovine milk contains 3–3.5% (w/v) of proteins of which about 80% on average consist of caseins and the whey or serum proteins make up the remaining 20% (Bordin and others 2001). It consists of water-soluble globular proteins, main fractions of which are beta-lactoglobin, alpha-lactalbumin, bovine serum albumin and immunoglobulins (Haug and others 2007). Caseins are an important nutrient delivery system carrying calcium and phosphate (Xiao and others 2011). It is rich in proline residues (Kohmura and others 1989). Casein has a micelle structure with hydrophilic parts on the surface while the
42 interiors are hydrophobic in nature (Sahu and others
2008).
Milk consumption with tea is a part of daily practice.
The application of tea or tea extracts in dairy products is also becoming popular due to the antibacterial and bioactive properties of polyphenols found in tea
(Ferruzzi and Green 2006). Proteins interact with polyphenols (flavonoids) either reversibly or irreversibly. The reversible interactions include hydrogen bonding, van der Waal’s forces and hydrophobic bonding. The irreversible bonds are covalent in nature.
Yuskel and others (2010) studied the interaction between green tea flavonoids and milk protein through spectrofluorometric analysis and observed a decrease in protein surface hydrophobicity via quenching of tryptophan and tyrosine fluorescence, which indicated hydrophobic binding between milk proteins and green tea flavonoids. The binding enthalpies obtained from
Isothermal Titration Calorimetry (ITC) analysis also backed up his findings and showed that interaction was non-covalent between catechin and beta-casein. Ye and others (2013) provided further confirmation of hydrophobic interactions by observing fluorescence quenching of whole milk in green tea and black tea solutions. They also suggested the possibility of hydrogen bonding between the phenolic hydroxyl group and
43 the amide group of milk proteins, as evidenced by the increase of UV absorption intensity, upon milk addition.
Ye and others (2013) observed an alteration in the structure of proteins due to polyphenol-milk protein interactions. This results in altering the secondary structure of milk protein from random coils and large loops to alpha- helix, intra- beta sheet and turn structures, as revealed by FTIR data. The interactions of phenolic compounds and proteins are known to affect the structure of proteins, content of free polyphenols, antioxidant capacity and bioavailability of phenolic compounds in foods.
There are several factors that affect the binding of polyphenols with proteins. They are as follows:
1. Temperature: Temperature can affect hydrogen bonding and lead to the formation of hydrophobic bonds. Both
Sastry and Rao (1990), and Prigent and others (2003) observed a decrease in the binding affinity of proteins for 5-O- caffeoylquinic acid with an increase in the temperature. However, Hoffman and others (2006) concluded that the precipitation of bovine serum albumin with procyanidin derivatives was not affected by temperature.
Tsai and She (2006) on the contrary reported that the superoxide dismutase (SOD) activity from peas increased because of its increase in heat stability at higher temperatures because of protein-phenolic interactions.
44 2. pH: The highest precipitation of the protein- polyphenolic complex is seen at 0.3-3.1 pH below the isoelectric point of the protein (Naczk and others 2006).
Unlike temperature, pH only affected only the degree of binding not the binding affinity for the interaction between 5-O- caffeoylquinic acid and 11S protein from sunflower seeds (Sastry and Rao 1990). The lower pH facilitated the dissociation of oligomeric proteins exposing more binding sites for the polyphenol to bind.
The pH can affect the nature of bonding between polyphenols and proteins as shown by Prigent and others
(2003). The authors reported that while chlorogenic acid
(polyphenol) interaction with bovine serum albumin (BSA), lysozyme and α-lactalbumin was supported by non-covalent bonds at pH ≤ 7, the increasing pH produced radicals and quinones from auto-oxidation of proteins that led to covalent interactions with polyphenols. Contradictory to the studies above, Frazier and others (2006), and
Charlton and others (2002) failed to see an effect of pH on (-)-epicatechin- BSA interaction. They suggested that electrostatic interactions or non-covalent interactions are not a major factor in complex formation. They attributed increased protein-polyphenol precipitation close to isoelectric pH to limited protein solubility at this pH.
45 3. Types of proteins and protein concentration: The hydrophobicity, isoelectric point and amino acid composition of proteins affect its interaction with polyphenols (Prigent and others 2003). The authors observed that the binding of chlorogenic acid was higher with BSA as compared to lysozyme and α-lactalbumin.
The protein concentration also plays a role in its complex formation with polyphenols. At lower concentrations, no statistically significant difference
(p ≤ 0.05) was found between precipitation at 0.5 and 1.0 mg/ml BSA. This was however, not the case at concentrations higher than 1.0 mg/ml.
4. Types and structures of phenolic compounds: The size of polyphenol molecules and the presence/absence of carbohydrate, methyl, methoxy and hydroxyl groups affect its affinity with proteins. Dubeau and others (2010) reported that the large theaflavins, thearubigins polymers in black tea bound more than their respective catechin monomers. A stronger interaction was found between quercetin and BSA as compared to its glycosylated derivative, quercetin 3-O-β-D glucopyranoside (Martini and others 2008). Xiao and others (2011) observed a very slight increase between quercetin and its rhamnoside, quercitrin, in its interaction with bovine milk protein.
The increasing glycosylation of flavonoids, the authors suggest, leads to increasing steric hindrance that
46 weakens protein binding. Xiao and others (2011) also investigated the effects of methylation, methoxylation and hydroxylation of polyphenols and their affinities for milk proteins. They observed that while methylation of flavonoids leads to a decrease in affinity for milk protein, methoxylation produced little effect. For example, formononetin had a 14.79 times lesser affinity than its non-methylated form, daidzein.
The effect of hydroxylation of flavones on milk protein binding depended on the ring that was hydroxylated. While hydroxylation of ring A of flavones increased binding affinity, the hydroxylation of ring C did not produce any effect. Hydroxylation of ring B produced a mixed effect.
For example, the affinity of apigenin (5, 7, 3) for milk protein was found to be 4.27-times higher than that of chrysin (5,7) while the affinity of apigenin (5, 7, 3) for milk protein was the same as that of luteolin
(5,7,3,4).
The hydroxylation of flavonol A and B rings slightly enhanced the binding affinity for milk protein. The hydroxylation of position 3 of the B ring of kaempferol to form quercetin enhances the binding affinity by 1.41 times. This affinity further increases to 2.09 times when quercetin is converted to myricetin by the addition of a hydroxyl at the 5th position of B ring. In flavanones too, the hydroxylation of ring A leads to a highly
47 significant increase in binding affinity up to 104.71 times.
5. Addition of extraneous chemicals: Addition of salt (Na
Cl) or sodium sulfate (Na2SO3) inhibited the dissociation of oligomeric proteins and hence reduced the number of binding points (Sastry and Rao 1990). This led to a decrease in the quantum of binding not the binding affinity of polyphenols.
So far, there have been a multitude of studies in the field of flavan-3-ol (catechin and its derivatives) and milk protein interactions and its effect on anti-oxidant capacity. But these studies have revealed contradictory results. Three types of results have been observed.
Firstly, a non-masking effect of milk or milk proteins was seen by some authors (Kyle and others 2007; Richelle and others 2001; Leenen and others 2000), in which the anti-oxidant potential remained the same. Secondly, certain authors (Stojadinovic and others 2013; Xiao and others 2011; Sharma and others 2008; Arts and others
2002; Serafini and others 1996) observed a masking effect of flavonoids that led to a reduction in anti-oxidant activity of the tea-milk mixture. Thirdly, Dub eau and others (2010) observed a dual effect in which, upon milk addition, the ABTS+ anti-oxidant capacity of teas was reduced but the chain breaking anti-oxidant capacity, determined by lipid peroxidation method, increased.
48 All these variable results, have led to a confusion in understanding the effect of milk addition on the bioavailability of polyphenols and anti-oxidant potential of tea.
Chapter 3
49
Identification and quantification of the flavonols myricetin, quercetin and kaempferol, total polyphenolic content, total limonoids and anti-oxidant activities in Azadirachta indica A. Juss leaves commercially available in the United States
3.1 Introduction
Flavonoids are plant secondary metabolites whose putative health values include anti-inflammatory, anti-oxidant, hepatoprotective, anti-viral, and anti-carcinogenic effects (Liu and others 2008a; Huang and others 2015;
Sirovina and others 2013; Romagnolo and Selmin 2012).
With better analytical techniques and an increase in popularity of alternative medicines, there has been a renewed interest in studying them.
Neem (Azadirachta indica A. Juss) is an evergreen tree cultivated in various parts of the Indian sub-continent
(Biswas and others 2002). It has been in use in Indian folk medicine for centuries because of its purported therapeutic value (Kaushik and others 2012). Given the prominent role it has played in curing diseases of Indian villagers, it has been hailed as a “divine” tree, a
“village dispensary” and “nature’s drugstore” (Maithani and others 2011). Today, extensive research has shown that it may have anti-cancer (Subapriya and Nagini 2003), anti- diabetic (Khosla and others 2000), anti- inflammatory (Schumacher and others 2011), anti-
50 ulcerogenic (Dorababu and others 2006) and anti-microbial effects (Badam and others 1999). Taking advantage of its reputation in rural India, Neem has been commercialized into products in the form of tea, soaps, facewashes, creams, and toothpastes.
While there are several encouraging reports on the in- vitro therapeutic effects of Neem in literature, studies characterizing its bioactive profile are not as numerous.
Existing studies have used colorimetric methods to determine total phenolics, total flavonoids and total anti-oxidant activity (Nahak and Sahu 2010; Ghimeray and others 2009; Hismath and others 2011) but fail to isolate and identify specific compounds. Others studies that have identified specific flavonoids in neem (Pandey and others
2014; Chakraborty and others 1989) use older analytical methods, such as thin layer chromatography, and do not provide quantitative information. We have used neem leaf and bark samples commercially available in the United
States for our analysis and, to our knowledge, this is the only study profiling its polyphenols, limonoid content and anti-oxidant activities.
Our aims and objectives for this research were four-fold:
A) To identify and quantify specific flavonoids, i.e.;
flavonols, namely myricetin, quercetin and kaempferol,
using HPLC-DAD-ESI-MS/MS in neem leaves and bark.
51 B) To quantify the total phenolics and the associated anti-oxidant activities.
C) To quantify the total limonoids in neem leaf and bark
due to their abundance in members of the Meliaceae
family (Taylor 1984).
D) To analyze the distribution of flavonols in neem
capsules available from different vendors in the United
States.
To place the bio-active profile of neem in context, we have compared it with more conventionally consumed teas such as green and black tea leaves, which are well known for their health promoting properties.
3.2 Materials and methods
3.2.1. Plant materials
Three different lots of Neem (Azadirachta indica) leaf powder and tea cut leaves were purchased from Neem Tree
Farms Inc. (Brandon, FL., USA) while Great Value green tea and Schnucks 100% Natural Orange Pekoe & Pekoe Cut black tea were purchased from local Walmart and Schnucks super markets, respectively.
Commercial samples of neem leaf capsules were bought online from three different vendors, Nature’s way,
PipingRock and Vitacost.
3.2.2. Reagents
52 Myricetin, quercetin, and kaempferol were purchased from
Cayman Chemicals (Ann Arbor, MI, USA). HPLC-grade acetonitrile, water, DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent, TPTZ (2,4,6-Tripyridyl-s-triazine, gallic acid, as well as trifluoroacetic acid and Folin’s reagent were purchased from Sigma Aldrich Co. (St. Louis, MO, USA).
Absolute ethanol was bought from the University of
Missouri chemical store (Columbia, MO, USA). Limonin glucoside was purchased from Abcam Biochemicals
(Cambridge, MA, USA) while limonin was bought from LKT
Laboratories Inc. (St Paul, MN, USA).
3.2.3. Extractions
The method of Wang and Helliwell (2001) was followed for extracting flavonols, with the addition of an anti- oxidant. Briefly, 1 g of neem leaf powder, neem tea cut leaves, neem bark powder, green and black tea leaves and were suspended in 40 mL of 60% ethanol and 5 mL of 6M HCl together with 40 mg of ascorbic acid as the antioxidant.
This mixture was refluxed at 95° C for two hours to allow the hydrolysis of the flavonol glycosides into the respective aglycones. The hydrolyzed solution was cooled, vacuum filtered using P8 Fisher Scientific filter paper and made up to 50 mL in a volumetric flask using 60% ethanol. This solution was filtered through a 0.45 μm nylon filter (EMD Millipore, Billerica, MA, USA) and 20
μL was injected into the HPLC.
53 Neem infusions were made according to the method shown in the Neem information booklet provided by the supplier.
Neem leaf powder, Neem tea-cut leaves, bark powder, black or green tea leaves (1.25 grams) were added to 250 mL of boiling water, and the aqueous extraction was allowed to take place for 5 mins. This solution was filtered and made up to volume in a 250-mL volumetric flask using de- ionized water. 16 mL of this solution was mixed with 24 mL of absolute ethanol to produce a concentration of 60 % ethanol. 40 mg of ascorbic acid and 5 mL of 6 M HCl was added to this solution. The solution was refluxed, filtered and made up to a volume of 50 mL as described for the dry tea leaves extraction.
For the purpose of extracting polyphenols and subsequently measuring their anti-oxidant activities, 200 mg of neem leaf powder, neem tea cut leaves, bark powder, green and black tea leaves were extracted in 20 mL 60% ethanol or water, for 2 hours at 100° C. The samples were vacuum filtered and made up to 25 mL using 60% ethanol or water.
3.2.4. UPLC-ESI-MS/MS for identification of flavonols
The identity of the flavonol aglycones were confirmed by a H-class Waters Xevo QTOF MS UPLC with PDA detector set at 370 nm. Separation of flavonols was performed on a
54 Acquity UPLC BEH C18 column (1.7 um pore size, 2.1 x 100 mm), whose temperature was set at 40º C. A gradient elution system with a flow rate of 0.5 mL/min was used and consisted of mobile phase A: 5% acetonitrile in water containing 0.01 % formic acid and mobile phase B: 100% acetonitrile containing 0.01 % formic acid. The gradient program was as follows: mobile phase A was held at 95%
(B=5%) for 1 min and then reduced to 0% from 1 to 8 mins and held at that concentration till 11 mins.
Electron spray ionization (ESI) was performed in the positive mode and mass spectra was collected over a range of 50-1000 m/z. In addition to MS, we collected MS/MS data for m/z ions 318, 303, 287 (representing the molecular ions for myricetin, quercetin and kaempferol).
The Q-TOF (Quadrupole Time of flight) parameters were:
Collison energy ramp: 6-50 V, Capillary Voltage:1.00 Kv, sampling core: 30, extraction core:4, Source temperature:
125°C, Desolvation temp: 550°C, Cone gas 50 L/hr, desolvation gas flow: 800 L/hour.
3.2.5. HPLC-DAD conditions for quantification of flavonols
HPLC analysis was carried out on an Agilent 1100 series liquid chromatographic system with a diode-array detector
(DAD), with wavelength set at 370 nm. A Kinetex 250 X 4.6
55 mm, 5 μ C 18, 100 Å (Phenomenex Inc., Torrance, CA) column was used for the separation of the flavonol aglycones. A gradient elution system comprising of two mobile phases, namely mobile phase A, 0.1 % trifluoroacetic acid in water and mobile phase B, 0.1% trifluoroacetic acid in acetonitrile, was used. The gradient used was 80% A and 20% B at 0 min which was gradually reduced to 60% A at 20 mins and held for another 5 mins. A 5-min post time was added for the mobile phase concentrations to come back to initial levels. The flow rate was 1 mL/min and the column temperature was set at 40° C.
Myricetin, quercetin and kaempferol standards were dissolved in 60% ethanol and external standard curves were developed for quantification purposes within the appropriate concentration range of the sample. The myricetin and kaempferol standard curves were developed within the range of 0-0.04 mg/mL respectively, while the quercetin standard curve was developed within the range of 0-0.25 mg/mL.
3.2.6. Total phenolic content by Folin-Ciocalteau assay
The amount of total phenolics was determined using the
Folin–Ciocalteau assay adapted from (Devi and others
2009). 50 μL of the ethanolic or aqueous extract (diluted to fit the range of the standard curve) was added to a mixture of 250 μL of Folin's reagent (1:3 diluted with
56 distilled water), 750 μL 7% sodium carbonate and 3 mL distilled water. The solution was incubated at room temperature for 2 hours and absorbance readings were taken at 725 nm using a Varian Cary® 50 UV-VIS spectrophotometer (Agilent Technologies Inc., Santa
Clara, CA). A calibration curve of gallic acid (ranging from 0.1 to 0.7 mg/mL) was prepared and the results, determined from regression equation of the calibration curve (y = 30.657x + 0.0041, R2 = 0.99), were expressed as mg gallic acid equivalents per gram of the sample.
3.2.7. Colorimetric estimation of total limonoids
The method of Breksa and Ibarra (2007) was used with minor modifications to quantify limonoids in neem leaf and bark.
The extraction of limonoid glucosides was done using C 18
SPE cartridges which were conditioned using 5 mL methanol and consequently with 5 mL of de-ionized water. Then 5 mL of neem leaves and bark extract (aqueous and 60% ethanol) was loaded onto the cartridge. The limonoid glucosides trapped in the column were eluted with 5mL of methanol and evaporated under a steady flow of nitrogen gas. The sample was reconstituted in 3 mL of 30% acetonitrile.
The aglycones were extracted by chloroform. Neem tea was mixed with chloroform in a 1:2 ratio (2.5: 5mL) and vortexed. Once the phases separated, the upper phase was discarded and the lower phase (chloroform) was evaporated
57 to dryness under a steady flow of nitrogen gas. The sample was re-constituted in 3 mL of 100 % acetonitrile and the colorimetric assay was performed exactly according to the procedure of limonin glucoside determination quantified at 470 nm.
Briefly, 2 mL of limonin glucoside standard or neem/ SPE/
AMB treated samples was mixed with 1.65 mL of DMAB indicator. To this, 1.65 mL of stock acid solution is added. The test tubes were incubated for 30 minutes at room temperature and the color developed was measured spectrophotometrically at 503 nm. Total limonoid glucoside in the sample was determined in terms of limonin glucoside equivalents. This was done using a calibration curve generated from limonin glucoside made up in 30% acetonitrile (0- 200 μg/mL, y = 2.1269x +
0.001, R2=0.99) while the limonin standard curve developed with limonin (0-40 μg/mL, y = 5.676x + 0.0039,
R2=0.99).
The colorimetric quantification was based on the formation of red to orange colored derivatives resulting from the treatment of limonin glucoside, or the neem extract with 4- dimethylamino benzaldehyde (DMAB) in the presence of perchloric and acetic acids.
3.2.8 Anti-oxidant activity determinations
A) FRAP assay
58 The FRAP assay was performed according to the method of
Thaipong and others (2006) with minor modifications. The
FRAP reagent was prepared by mixing 300mM acetate buffer
(pH 3.6), 10 mM TPTZ (2,4,6-Tripyridyl-s-triazine) and 20 mM ferric chloride in the ratio of 10:1:1 ratio respectively along with 24 mL of distilled water. 150 μL of the sample (diluted appropriately to fit the range of the standard curve) was added to 2850 μL of FRAP reagent.
The samples and standards were incubated for 30 mins at room temperature in the dark. The absorbance of the colored product [ferrous tripyridyltriazine complex] was measured spectrophotometrically at 593 nm. The standard curve was developed using gallic acid as a standard within the concentration range of 0.01-0.04 mg/mL (y=
18.149x - 0.0084, R2=0.99). Results are expressed in mg
GAE/g of the sample.
B) DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay
DPPH anti-oxidant assay was performed according to the method of Thaipong and others (2006) with modifications.
Stock solution was prepared by dissolving 20 mg DPPH in
100mL methanol and it was then stored at -20° C until used. The working solution was obtained by mixing 20mL of stock solution with 80 mL methanol.
59 To 2ml of de-ionized water, 200 μL of sample (of appropriate dilutions) or blank was added and was allowed to react with 2800 μL of the DPPH solution. Then the decrease in absorbance was measured at 515 nm after 90 minutes. Methanol was used as blank and 60% ethanol (or water for tea infusion) was treated as control . Anti- oxidant activity was expressed as percentage inhibition of the DPPH radical and was determined by the following equation as reported by (Yen and Duh 1994).
%퐴퐴 = (퐴푏푠표푟푏푎푛푐푒푐표푛푡푟표푙 − 퐴푏푠표푟푏푎푛푐푒푠푎푚푝푙푒) ∕ 퐴푏푠표푟푏푎푛푐푒푐표푛푡푟표푙 × 100
The percentage DPPH anti-oxidant values were converted to vitamin C equivalent anti-oxidant capacity using a standard curve, which was found to be linear between
0.0176 -0.1408 mg/mL (y= y = 693.12x - 3.0024, R2=0.98).
The results were expressed in mg/g of vitamin C equivalent anti-oxidant capacity (VCEAC).
3.2.9 Statistical analysis
Three different batches of Neem leaf powder, Neem tea-cut leaves, green and black tea were bought and each batch was subjected to triplicate analysis. Analysis of variance (ANOVA) was done to test for differences in flavonol, phenolic and anti-oxidant activities while the
Tukey’s range test was used to subsequently separate the means if the ANOVA was significant, using Minitab 16 statistical software (Minitab Inc., PA, USA).
60
3.3 Results and discussions
3.3.1 Identification of flavonol aglycones
The three flavonols are separated clearly on the reverse phase C18 column eluting in the order of myricetin, quercetin and kaempferol at approximately 8, 12 and 18 mins (Fig. 1). The structures of the flavonols is shown in Fig. 2.
Fig. 1. Chromatogram shows us three well separated peaks at 6.61, 9.81 and 12.96 mins when the signal is recorded at 370 nm with a photo diode array detector.
Fig. 2. The structures of the investigated flavonols (Huck et al., 2001)
61
The mass spectrum was collected for each peak. They were found to contain the molecular ion [M+H] + peaks for myricetin, quercetin and kaempferol with m/z ions 319,
303 and 287, respectively (Sun, Chen, Lin, & Harnly,
2011; Stecher, Huck, Popp, & Bonn, 2001; Bertoncelj,
Polak, Kropf, Korošec, & Golob, 2011). For further identification, we induced the fragmentation of the molecular ions (MS/MS) (Figs. 3A, 3B and 3C), and compared the resulting daughter ion spectrum with previously published literature. The MS/MS spectrum for the suspected quercetin peak ([M+H] +=303) yields the characteristic daughter/ product ion of 153 and 229 m/z, which is in agreement with a study by Häkkinen and
Auriola (1998) and is providing strong confirmation. The ion at m/z 229 is produced by the dehydration followed by
62 sequential loss of two CO ions at the C ring
(Tsimogiannis and others 2007; March and Miao 2004).
Further degradation of the C ring by retro Diels-Alder cleavage produces the product ion of 153 m/z. The breakup of the kaempferol molecular ion, yields the daughter ions with 153 and 165 m/z (Tsimogiannis and others 2007). Fig.
4 shows the production of m/z 153 and m/z 165 product ions.
Fig. 3. MS/MS spectrum of – A) Myricetin
B) Quercetin
63
C) Kaempferol
64 Fig. 4. Retro Diels Alder cleavage of the C ring (Tsimogiannis et al. 2007)
3.3.2 Content of flavonols as determined by HPLC-DAD
The neem leaf powder ethanolic extract yielded the highest amount of flavonols followed by green and black tea with 12.79 ± 0.80 mg/g, 5.92 ± 0.49 mg/g and 5.82 ±
0.40 mg/g, respectively (Table 1). This may be attributed to the larger particle size of green and black tea as compared to the finely powdered Neem leaves. A similar phenomenon was observed by Wang and Helliwell (2001) in their study. The ANOVA results show that quercetin, which was the pre-dominant flavonol, was significantly (p<
0.05) higher in neem powder as compared to green and black tea. The neem bark samples were not found to contain any of the flavonols under investigation. This
65 contradicts results of Sultana and Anwar (2008), who found quercetin and kaempferol at levels of 31.9 ± 1.3 and 0.5 ± 0.1 mg/kg in neem bark, respectively.
Table 1. Means of individual flavonols in ethanolic extract and infusion of various tea samples.
Ethanolic extract Infusion
Samples Myricetin Quercetin Kaempferol Myricetin Quercetin Kaempferol
Neem leaf powder 1.90 ± 0.19a 9.33 ± 0.46a 1.56 ± 0.15a 2.07 ± 0.32a 8.31 ± 0.42a 1.36 ± 0.19a
Green tea 1.67 ± 0.03a 2.99 ± 0.16b 1.25 ± 0.16a 1.80 ± 0.12a 2.92 ± 0.15b 1.11 ± 0.11a
Black tea 0.56 ± 0.08b 3.67 ± 0.26b 1.59 ± 0.10a - 3.61 ± 0.38b 1.40 ± 0.08a
Comparison of means is done between flavonols in the same row between samples (n= 3 S.D.)
In the tea infusions or aqueous extracts (Table 1), the total flavonols followed a similar pattern to that of the ethanolic extracts, with neem powder showing significantly higher amounts than green and black tea.
Again, similar to the ethanolic extractions, quercetin was the flavonol present in the highest concentration for all samples.
It is also seen, irrespective of the extraction solvent used, that the content of myricetin is lower in black tea as compared to green tea. This is to be expected because of the fermentation step exclusive to black tea and the
66 fact that myricetin is the most susceptible of the three flavonols (McDowell and others 1990).
Wang and Helliwell (2001) studied the three flavonols – myricetin, quercetin and kaempferol in four different varieties of green tea and two different varieties of black tea. Consistent with our results, they found that, except for the Longjing variety of green tea and the
Qimen variety of black tea, quercetin was the predominant flavonol. However, they also found that myricetin was the least abundant flavonol across all varieties and types of tea. This is a contradiction to our results, where although our dry black tea sample yielded more kaempferol than myricetin, the levels were reversed in green tea.
As reported by Wang and Helliwell (2001), when extracted with ethanol, the total flavonols in dry tea leaves range from 0.83 to 3.31 mg/g for green tea and 0.24 to 2.31 mg/g for black tea. Our results indicate much higher values for both, which can be explained by differences in tea variety, their geographical location and agricultural conditions in which they were cultivated. Another possible reason to explain the higher values could be the use of an anti-oxidant, ascorbic acid that we adapted from Nuutila and others (2002) which could have had a protective effect on flavonol oxidation, during the hydrolysis procedure.
67 The higher yield of flavonol aglycones in neem is significant. Chakraborty and others (1989) had attributed the anti-diabetic potential of an aqueous extract of neem to the presence of various glycosides of myricetin, quercetin and kaempferol.
3.3.3 Total phenolics determined by Folin-Ciocalteau assay
In the ethanolic extracts and infusions, we observed that green tea had the highest phenolic content followed by black tea, Neem powder (Fig. 5A and B). Neem bark and neem leaf samples, interestingly were found to have similar phenolic contents, although neem bark doesn’t contain any flavonols. This indicates the presence of other flavonoids such as flavan-3-ols, flavones, and flavanones in neem bark, which need to be explored.
Fig. 5. Total phenolic content of various tea samples
A) Ethanolic extract 180 a 160 140 120 b 100 80
(GAE) (GAE) c 60 c 40 20
mg/g mg/g Gallic acidequivalent 0 Neem leaf Neem bark Greeen tea Black tea powder powder
Comparison of means is done between flavonols in the same row between samples (n= 3 S.D.)
68 B) Infusion 160 a 140 120 100 b
80 (GAE) 60 c c 40 20
mg/g mg/g Gallic acidequivalent 0 Neem leaf Neem bark Greeen tea Black tea powder powder
Comparison of means is done between flavonols in the same row between samples (n= 3 S.D.)
The higher phenolic levels in green tea, as compared to black tea, can be attributed to the loss of catechins during processing of black tea involving fermentation
(Zuo and others 2002).
3.3.4 Anti-oxidant activities- FRAP anti-oxidant assay & DPPH anti-oxidant activity
The results of the FRAP and DPPH assay co-relate strongly with that of the Folin’s assay used for the determination of total phenolics (Fig. 6A and B). This in agreement with other authors (Zhao and others 2014; Bhoyar and others 2011; Dudonné and others 2009).
69 Fig. 6. Co-relation between-
A) FRAP values and total phenolic content
90 80 R² = 0.9532 70 60 50 40 30 20
FRAP FRAP mg/gGAE 10 0 0 50 100 150 200 Total polyphenols (mg/g)
B) DPPH values and total phenolic content
350 300 R² = 0.9061 250 200 150 VCEAC 100
50 DPPH DPPH activity mg/g 0 0 50 100 150 200 Total polyphenols (mg/g)
With the ethanolic and aqueous extracts, the FRAP and
DPPH values show the order of: green tea> black tea> neem bark> neem leaf (Table 2 and 3). We do not find any significant differences between the neem leaf and bark powder.
70 Table 2. Means of anti-oxidant activities – FRAP and DPPH activity in the ethanolic extract of various tea samples
Samples FRAP (mg/ g GAE) DPPH activity (mg/g VCEAC)
Neem leaf powder 6.87 1.68c 42.81 5.20c
Neem bark powder 10.92 2.83c 54.18 8.24c
Green tea 80.36 9.02a 294.70 13.35a
Black tea 34.11 2.96b 221.51 12.66b
Comparison of means is done between samples in the same row (n=3 S.D.)
Table 3. Means of anti-oxidant activities – FRAP and DPPH activity in the infusion of various tea samples
Samples FRAP (mg/ g GAE) DPPH activity (mg/g VCEAC)
Neem leaf powder 6.73 0.65c 16.59 0.04c
Neem bark powder 7.88 1.15c 16.43 0.06c
Green tea 69.62 3.16a 167.32 1.89a
Black tea 25.43 1.40b 150.22 9.76b
Comparison of means is done between samples in the same row (n=3 S.D.)
The high anti-oxidant activity of green and black tea is because of its high polyphenolic content (Arts and others
2002). Neem contains a significantly lower (p < 0.05) content of polyphenols which may explain its lower anti- oxidant activity. Neem is rich in a group of tetranortriterpenoids called limonoids (Champagne and others 1992) which is the main contributor to its bio-
71 activity (Huang and others 2004; Nathan and others 2005).
But these limonoids possess far lesser anti-oxidant activity than polyphenols because of the lack of hydroxyl groups which get oxidized (Yu and others 2005).
3.3.5 Total limonoids- limonoid glucosides and aglycones
Table 4 and 5 shows the content of limonoids- glucosides and aglycones in neem leaf and bark in ethanolic and aqueous extracts. The limonoid glucosides extracted by neem leaf and bark in both the ethanolic extract and the infusion is similar. While more limonoid aglycones are extracted in neem leaf than neem bark in the ethanolic extract, we did not find any significant differences
(p<0.05) in the infusion. Neem, belonging to the
Meliaceae family, is a rich repository of limonoids(Roy and Saraf 2006), and that is supported by the content of limonoids extracted in our experiments. These values are much higher than in Washington Navel oranges and Rio star grapefruit where LE were found to be 0.002 ± 0.00 and
0.01 ± 0.00 mg/g, and the limonoid glucoside content was found to be 0.14 ± 0.00 and 0.21± 0.01 mg/g LGE, respectively.
72 Table 4. Limonoid content in neem leaf and bark samples in ethanolic extract
Samples Limonoid glucoside Limonoid aglycone (*LGE (mg/g) (*LE mg/g)
Neem leaf powder 0.58 0.13a 7.59 2.38a
Neem bark powder 0.5 0.16a 3.32 0.5b
Comparison of means is done between limonoids in the same row between samples (n= 3 S.D.) *LGE= Limonin Glucoside Equivalent LE= Limonin Equivalent
Table 5. Limonoid content in neem leaf and bark samples in the infusion
Samples Limonoid glucoside Limonoid aglycone (*LGE (mg/g) (*LE mg/g)
Neem leaf powder 6.19 1.12a 3.88 1.30a
Neem bark powder 5.99 1.41a 3.19 1.77a
Comparison of means is done between limonoids in the same row between samples (n= 3 S.D.) *LGE= Limonin Glucoside Equivalent *LE= Limonin Equivalent
3.3.6 Analysis of commercial samples
The presence of myricetin, quercetin and kaempferol in the three commercially available neem capsules were confirmed by LC-ESI-MS/MS. The ethanolic extract shows that quercetin is the pre-dominant flavonol among all three samples, in accordance with our previous results.
The neem capsules bought from Nature’s way and Vitacost showed similar levels of the three flavonols while the
73 ones bought from Piping Rock contained significantly
(p<0.05) lower levels (Fig.7)
Fig.7. Distribution of flavonols in commercial neem capsules
A) Ethanolic extract
10.00 a 9.00 a 8.00 7.00 6.00 5.00 b 4.00 a a 3.00
Flavonol Flavonol content (mg/g) a a 2.00 b b 1.00 0.00 Nature's way Vitacost Piping Rock
Myricetin Quercetin Kaempferol
Comparison of means is done between the same flavonol of different samples. Means with different letters are significantly different (p<0.05)
B) Infusion
0.6 a a 0.5
0.4
0.3 b
0.2
Quercetin content content Quercetin (mg/g) 0.1
0 Nature's way Vitacost Piping Rock
Comparison of means is done between the same flavonol of different samples. Means with different letters are significantly different (p<0.05)
74
Quercetin was the only flavonol extracted in the infusion. Similar to the ethanolic extract, the level of quercetin was significantly (p<0.05) lower Piping rock compared to samples obtained from the other two vendors.
3.4 Conclusions
Our study provides nutritional information about the health value of neem leaf and bark to U.S. consumers that would help them in making a better purchasing decision while choosing between a medicinal tea i.e.; neem and more traditional beverages such as green and black tea.
While green and black tea proved to be much richer repositories of polyphenols and hence, possess significantly more anti-oxidant activity than neem leaf and bark, a closer look at the levels of specific flavonoids, i.e.; flavonols-myricetin, quercetin and kaempferol provides encouraging reasons for the use of neem leaf. These flavonols and high quantities of limonoids, might be the basis of the many health benefits of neem.
75
Chapter 4
CHARACTERIZATION OF THE VOLATILE PROFILE OF NEEM (AZADIRACHTA INDICA A. JUSS.) LEAF AND BARK COMMERCIALLY AVAILABLE IN THE UNITED STATES USING HS-SPME LINKED WITH GAS CHROMATOGRAPHY-MASS SPECTROMETRY
4.1 Introduction
Medicinal trees have formed an integral part of the remedial measures various civilizations have adopted over the years to counter a multitude of diseases. These trees have acted as an alternative to traditional western medicine and have found consumer acceptability because of being a ‘natural source’, in various parts of the world
(Bussmann and others 2007). More recently, with a better understanding of the relationship between diet and health, consumers have been especially attracted to herbs and the functional foods incorporating them, for a healthier lifestyle.
Azadirachta Indica A. Juss. (Meliaceae) is one such medicinal tree, whose leaves, bark, seed pulp, flower, fruit and twig have been used since times immemorial in the Indian sub-continent as a popular household remedy
(Subapriya and Nagini 2005). Neem tree is considered as
76 ‘sarvaroga nivarini’ (the panacea for all diseases) and has also been hailed as ‘heal all’, ‘divine tree’,
‘village dispensary’ and ‘nature’s drugstore (Subapriya and Nagini 2005). Today, neem and its extracts are being commercially sold as tea, soaps, cosmetic creams, toothpastes and shampoos.
Neem has been experimentally shown to have anti-cancer
(Kumar and others 2006b), anti-diabetic (Perez-Gutierrez and Damian-Guzman 2012a), anti-inflammatory (Akihisa and others 2011), anti –ulcerogenic (Bandyopadhyay and others
2002a), anti-microbial (SaiRam and others 2000) and contraceptive (Raghuvanshi and others 2001) properties.
Much of these beneficial properties have been attributed to the non-volatile flavonoids and limonoids in neem.
Although in general, headspace volatiles and volatiles from essential oils from various botanicals have received considerable attention in the medical community, literature regarding neem leaf volatiles is scarce except for Zeringue and Bhatnagar (1994), while it is totally missing in the case of bark.
This study aims to bridge the gap by elucidating a comprehensive profile of the volatiles in different samples of neem leaf and neem bark powder by using an efficient, non-invasive and solvent-less technique
(Jelen, Majcher, & Dziadas, 2012) called solid phase microextraction coupled with gas chromatography–mass
77 spectrometry. Furthermore, we also aim to report the extraction and constituents of the essential oil derived from neem leaf, previously never reported.
With this information, we seek to find justification for the application of neem leaf and bark as a medicinal resource in functional food products and beverages.
4.2 Materials and methods
4.2.1 Plant material
Neem leaf powder, fresh leaf, dried leaf and bark powder was bought from Neem Tree Farms Inc., Brandon, Florida.
Samples from two different commercial batches of each sample type were used for triplicate analysis producing a total sample size of six (6) for every sample type.
4.2.2 Chemicals
Volatile chemical standards – alpha-ionone, beta caryophyllene, beta –ionone and D-limonene were bought from Bedoukian chemicals (Bedoukian Research, Inc., CT,
USA) and prepared in hexane.
4.2.3 HS-SPME procedure
2 grams of neem leaf /bark powder in 4 mL water were placed with 4 mL water into a 10-mL glass vial with an open center screw cap and a Teflon/Silicon septum
(Supelco, Bellefonte, USA). The sample was equilibrated for 4 hours at 60°C in a water bath followed by SPME
78 fiber exposure of 1 hour, allowing the volatiles to be adsorbed on the fiber.
For analyzing fresh and dried neem leaf samples, a small vial was unusable for analysis. Therefore, a larger glass beaker with a modified cap and a rubber septum was used as the extraction vessel. 5 grams of leaf samples were placed into 200 mL of water and were treated the same way as the neem leaf and bark powders, using identical equilibration time, exposure time and temperature. A
Stable Flex fiber (Sigma –Aldrich, St. Louis, MO) coated with 50/30 μm Divinylbenzene/Carboxen on
Polydimethylsiloxane was used for this analysis.
4.2.4 Extraction of Essential oil
25 g of Neem leaf powder was subjected to a Soxhlet extraction for 4 hours with Petroleum ether (BP 35˚-60˚C) to get the total hydrophobic extract. The solvent was evaporated using a rotary evaporator. The greenish residue that collected was then subjected to hydrodistillation (Clevenger apparatus) for 3 hours. The steam volatile fraction, i.e., the essential oil was collected by trapping it in hexane. To the hexane extract, a pinch of sodium sulfate was added to remove any water. It was then concentrated down to 0.5 mL using nitrogen gas; the remaining extract was stored in sealed vials at 4˚C until further analysis.
79 4.2.5 GC-MS Analysis
A Varian GC 3400CX (Varian, Walnut Creek, CA, USA) equipped with a 1078 programmable injector connected to a
Varian Saturn 2000 Mass spectrometer with an ion trap detector was used for GC-MS analysis. Volatiles were separated by using a DB-5MS UI, 30 m X 0.25 mm, 0.25 um film thickness (Agilent J & W Columns) fused silica capillary column. Helium carrier gas flow rate was kept at 1ml/min and injector, transfer line and ion trap temperatures were 250, 250, 1500C, respectively. The analysis was done in the splitless mode and the post desorption split flow was 100 ml/min. The column temperature program was: 350C held for 5 min. and linear temperature program from 35 to 2500C at 30C/Min.
Identification of chemical compounds was established using mass spectra comparison with the NIST 1992 and
Wiley 5 libraries, retention indices of standards, and literature values.
4.2.6 Statistical Analysis A multivariate analysis in the form of Principal
Component Analysis (PCA) was performed on three different neem samples- leaf powder, dried leaf and fresh leaf to visualize their grouping tendencies and identify variables (volatiles) influencing their variability. The composition data matrix of three samples (103 variables x
18 samples= 1854 data points) was analysed using
80 Microsoft Excel software 2011 (Microsoft Corp., Redmond,
WA, USA) with the help of XLSTAT software add-in.
Eigenvalues were calculated using a co-variance matrix among 103 chemical compounds as input, and the score and loading plots were generated using sample type and volatile constituents, respectively.
4.3 Results and discussion
4.3.1 Comparison of the volatile profile of neem dried leaf powder, dry leaf and fresh leaf
Forty-two (42), thirty-seven (37) and seventy-one (71) compounds were detected in neem dried leaf powder, dry leaf and fresh leaf, respectively. In all these samples, the sesquiterpenes were the most significant class of compounds, that made up 534.8%, 56.8% and 18.3% of the total number of compounds extracted, respectively.
Caryophyllene oxide at 17.04 ± 3.58%, unidentified sesquiterpene 21 at 35.77± 6.78 % and 2,6 nonadienal (E,
Z) at 8.27 ± 6.00% relative abundance (Table 1), made the highest contribution towards the headspace of neem dried leaf powder, dry leaf and fresh leaf, respectively. The other major classes of compounds were monoterpenes, aldehydes, ketones and alcohols as shown in Fig. 1.
The three sample types were clearly differentiated by
Principal Component Analysis. Fig.2 and Fig. 3 show the score and loading plot, for samples and variables, respectively.
81 Fig. 1. Percentage (%) of different classes of compounds as identified from the headspace of various neem samples by SPME and GC-MS.
60
50
40
30 Dried leaf powder
extracted extracted (%) 20 Dry leaf
10 Fresh leaf Percentage Percentage oftotal volatiles 0
The first two principal components were able to explain more than 75% of the total variability (F1= 52.5%,
F2=19.95%) between the three samples. We observed, that the dry leaf and dried leaf powder samples were separated from the fresh leaf samples on F1. F2 not only separated the dry leaf samples from the dried leaf powder samples.
The variation between the dry leaf and dried leaf powder, and between the two fresh leaf batches is primarily due to the variability in monoterpenes, pyrroles and aromatic compounds and to a lesser extent due to esters and ethers, as is evident from the loading plot (Fig.3). Fig.
3 also shows us that alcohols, sulfur compounds and furans contribute most to the separation of samples by F1 while aldehydes, esters, ethers and ketones load both on
F1 and F2 thus contributing to differentiation of samples
82 by the two main principal components. Some of the important compounds in each sample type have been shown in the chromatograms in Fig. 4.
Fig. 2. Score plot for dried leaf powder (DLP), dry leaf (DL) and fresh leaf (FL)
Fig. 3. Loading plot for variables of dried leaf powder, dry leaf and fresh leaf
83 One of the distinguishing factors of the volatile profile of fresh leaf is the presence of organosulfur compounds in the headspace. Organosulfur compounds such as di-n- propyl and n-propyl-1-propenyl di-, tri- and tetra sulfides and their importance have been reported before in neem seeds (Balandrin, Lee, & Klocke, 1988).
Shivashankar et al. (2012) also identified 2,5 dimethyl thiophene; 3,4 dimethyl thiophene and 1,3 dithiane in neem seeds via HS-SPME. The protective action of organosulfur compounds against Aedes aegypti, Heliothis virescens, Heliothis zea (Balandrin et al., 1988) and
Asian citrus psyllid (Rouseff, Onagbola, Smoot, &
Stelinski, 2008) have been elucidated.
Zeringue and Bhatnagar (1994) collected fresh neem leaf volatiles by purging them with air and trapping the volatiles in a Tenax trap, before thermally desorbing them into a GC. The volatile profile was dominated by ketones which accounted for 43% of the total area followed by alcohols which occupied 23% of the headspace.
This is in sharp contrast to our observations, where we found that the headspace of fresh neem leaves had a high proportion of sesquiterpenes (~50%).
Several organosulfur compounds (10) were also identified, as mentioned above, that were missing in the study of
Zeringue and Bhatnagar (1994). Two contributing factors to this variation might be the different geographical
84 origin of our neem sample and /or the different extraction technique used in our study.
4.3.2 Comparison between neem bark and leaf powder volatiles
Courtois et al. (2012) from an extensive study of fifty- five (55) tropical tree species, showed that the terpene volatiles released by the leaves of a species are very different from the ones released by the bark. Considering that leaves and bark are attacked by different communities of insect herbivores (Novotny & Wilson,
1997), some qualitative differences were found in the released volatiles, indicating that some terpenes may be specialized to ward off non-overlapping communities of herbivores. A similar phenomenon was observed in our study. The commonalities and differences have been elucidated in Fig.4.
Fig.4. Number of mono- and sesquiterpenes in only bark, only leaves or found in both tissues
60
50
40 Only Bark 30 Commmon 20 Only Leaf
Number Number compounds of 10
0 Monoterpenes Sesquiterpenes
85
Fig.5. Chromatograms of the various neem leaf and bark samples
A) Neem dried leaf powder
B) Neem dry leaf
86
c) Neem fresh leaf
D) Neem essential oil
Table 1 compares the headspace volatiles extracted by HS-
SPME between neem bark and leaf powder. Sixty-seven (67) of the sixty-seven (67) compounds detected in neem bark
87 powder by HS-SPME analysis were identified. The volatile profile of the bark was dominated by sesquiterpene volatiles (35 compounds), which accounted for 51.4 % of the total volatiles, much like for the leaf. Alcohols, ketones and monoterpenes constituted 9 % of the total volatiles. Aldehydes and aromatic compounds made up 4.4 % while alkanes made up 2.9 %. Diterpenes, esters, ethers, furans and alkenes formed a miniscule percent of the extracted volatiles at 1.4 %.
The highest contribution to the headspace of bark came from naphthalene, a polycyclic aromatic compound that was unique to bark in our analysis, as opposed to caryophyllene oxide, a sesquiterpene, for leaf. This might be surprising as naphthalene is usually associated with anthropogenic activity related to the mineral oil and coal industry. However, they have been found on the antennae of stem borers (lepideptorus insects) and are thought to have semiochemical activity (attractant), being released from Napier grass, Pennisetum purpureum, and Sudan grass, Sorghum sudanensis (Khan, Pickett, Berg,
Wadhams, & Woodcock, 2000).
Naphthalene’s relative abundance was 25.43 ± 6.98% while that of caryophyllene oxide was 17.04 ± 3.58 %. Other major contributions in neem bark from the sesquiterpene class came from beta caryophyllene, alpha copaene, acoradiene, caryophyllene alcohol and alloaromadendrene
88 with relative abundance of 8.57 ±5.35 %, 3.14 ± 1.62%,
2.76± 1.13% ,2.52 ± 1.73% and 2.39 ± 1.85 %.
4.3.3 Comparison between HS –SPME and essential oil volatiles of dried neem leaf powder
Table 1 compares the qualitative aspects of the volatiles extracted by HS-SPME and in essential oil. Sixty-four
(64) compounds were identified in neem essential oil. The essential oil produced a very low yield of ~0.0001 % and was rich in sesquiterpenes much like the headspace volatiles extracted by SPME, accounting for 46.1 % of the total volatiles extracted. Ketones and monoterpenes were the second and third most abundant group with 12.5 % and
10.9 % of the total volatiles respectively. Esters made up 9.2%, which was in sharp contrast with the headspace volatile composition where esters made up merely 2.3%.
Alcohols, aldehydes and diterpenes made up 4.7 % of the volatile composition. The presence of diterpenes and acids are major distinguishing factors between the headspace and essential oil volatile composition. In addition to the compounds shown in the Table 1, compounds unique to essential oil allowing us to distinguish between it and different samples are listed in Table 2.
Some of the major contributors to the volatile composition were caryophyllene oxide with 12.88 ± 6.29%, an unknown sesquiterpene with 4.56± 0.48% and spathulenol with 3.88 ± 1.31% of the relative abundance, all
89 belonging to sesquiterpenes. The carboxylic acid, n- hexadecanoic acid made up a significant 12.66 ± 7.56 % of the total while the diterpene phytol contributed 4.67 ±
2.96%.
Thus, caryophyllene oxide is most important contributor to both, the headspace and essential oil volatiles. This provides further evidence of neem as a tree with tremendous medicinal benefits as caryophyllene oxide has been implicated for its anti-inflammatory role in lymphoma and neuroblastoma cells (Sain et al. 2014) and is a major constituent of various essential oils with health beneficial effects (Tung, Chua, Wang, & Chang,
2008; Chang, Chen, & Chang, 2001; Baratta, Dorman, Deans,
Biondi, & Ruberto, 1998). Some of the important compounds in the essential oil are shown below in Fig. 6.
4.4 Conclusions Neem (Azadirachta indica A. Juss) has always been considered a tree with great medicinal properties by people of the Indian sub-continent. Our study adds valuable information in elucidating the chemicals that may be responsible for neem’s effects. We observe, that the process of drying the fresh leaves removes some of the alcohols and furans while eliminating organosulfur volatiles. The process of mechanical crushing to further convert the dried leaf into powder, leads to either a complete or partial loss of compounds such as- octanal;
90 2,4 heptadienal (E, E), ethyl hexanol, benzeneacetaldehyde, geranyl acetone and specific sesquiterpenes. The profile of the dried leaf and bark powder is important in the beverage context, as they are being marketed and sold as medicinal teas. Neem essential oil can be incorporated into foods as a natural oxidant given the high load of beneficial compounds it contains.
However, an optimum method to improve its extraction yield must be figured out before any commercial application can be undertaken.
The high proportion of sesquiterpenes in all the neem leaf samples and bark, whose role as anti-carcinogenic and anti-microbial chemicals is already well established, supports the image of neem as a very effective medicinal plant that could play a significant role in the functional foods industry.
91
Table 1. Volatile compounds in the different samples of neem as identified and quantified (%RA= %Relative Abundance) by GC-MS.
d Compounda RIb RILc ID Dried Bark Dry Fresh Essential leaf powder leaf leaf oil powder
2 Ethyl furan - - - 0.35 - -
3 Methyl butanal - - 0.14 - 0.88 -
91 1-(2- - - 0.12 - - -
propenyloxy), butane
1 Methyl 1 H - 2.09 - 0.68 0.85 - pyrrole
Toluene - - 0.26 - - -
Butyl, - - 0.38 - - - cyclobutane
Hexanal 809 808 MS, RIL 0.30 - 0.26 0.50 -
d Compounda RIb RILc ID Dried Bark Dry Fresh Essential leaf powder leaf leaf oil powder
2,5 Dihydro, 3,4 827 - MS - - - 1.04 - dimethyl furan
1,1, 3 Trimethyl 844 - MS 1.35 - 2.37 2.08 - cyclopentane
2,4 Dimethyl 872 878 MS, RIL - - - 0.30 - thiophene
Styrene 887 893 MS, RIL - - - 0.19 -
2 Heptanone 891 895 MS, RIL - 0.31 - - -
3,4 Dimethyl 899 - MS - - - 1.50 -
thiophene 92
Heptanal 902 903 MS, RIL - - - 0.33 -
Methoxy benzene 916 918 MS, RIL - 0.09 - 3.74 -
Dl propyl sulfide 918 - MS - - - 0.15 -
1,3 Dithiane 934 - MS - - - 0.33 -
Benzaldehyde 960 960 MS, RIL 2.54 1.73 0.66 6.12 -
Heptanol 974 973 MS, RIL - 0.59 - - -
d Compounda RIb RILc ID Dried Bark Dry Fresh Essential leaf powder leaf leaf oil powder
Octen-3-ol 983 979 MS, RIL - 0.25 - - -
Sulcatone 987 985 MS, RIL 1.93 0.79 0.52 0.39 0.12
2 Pentyl furan 991 993 MS, RIL - 1.37 - 1.68 -
Isolimonene 998 998 MS, RIL - - - 4.17 -
Octanal 1001 1001 MS, RIL - - 0.38 0.68 -
2,4 heptadienal 1008 1006 MS, RIL - - 0.49 0.82 0.40
93 (E, E)
2 Methyl 4 1020 - MS - - - 0.53 - octanone
p Cymene 1023 1027 MS, RIL - 1.37 - - -
D- Limonene 1028 1030 MS, RIL, 0.26 1.32 0.60 0.14 0.01 RIS
2 Ethyl hexanol 1031 1032 MS, RIL - 0.44 0.87 0.78 -
Benzeneacetaldehy 1043 1044 MS, RIL - 0.31 0.78 3.65 - de
d Compounda RIb RILc ID Dried Bark Dry Fresh Essential leaf powder leaf leaf oil powder
Butanoic acid, 1059 1059 MS, RIL - - 0.20 - pentyl ester
Acetophenone 1062 1062 MS, RIL - - 0.19 -
3,5 Octadien-2- 1066 1066 MS, RIL 0.84 - - 0.22 0.09 one (E, E)
Octanol 1074 1078 MS, RIL - 1.12 - - -
p Cymenene 1089 1090 MS, RIL - 0.33 - 3.00 - 94
2 Nonanone 1093 1091 MS, RIL - 0.54 - 2.98 -
Linalool 1101 1100 MS, RIL 5.86 0.41 1.62 0.67 1.53
Nonanal 1107 1104 MS, RIL 1.79 0.63 - 1.13 0.24
Unidentified 1117 - MS - - - 0.36 - sulfur compound 1
Unidentified 1123 - MS - - - 0.56 - sulfur compound 2
Unidentified 1129 - MS - - - 0.38 - sulfur compound 3
d Compounda RIb RILc ID Dried Bark Dry Fresh Essential leaf powder leaf leaf oil powder
Isodurene 1148 1137 MS, RIL - 4.96 - - - 95
Unidentified 1150 - MS - - - 0.70 - sulfur compound 4
2,6 Nonadienal 1153 1154 MS, RIL - - - 8.27 - (E, Z)
2 Nonenal (E) 1160 1162 MS, RIL - - - 1.72 -
1,3 Dimethoxy 1167 1164 MS, RIL - 0.17 - - - benzene
2,4 Dimethyl 1172 1178 MS, RIL 0.34 - - 0.51 - benzaldehyde
Nonanol 1175 1171 MS, RIL - 0.60 - - -
Naphthalene 1183 1179 MS, RIL - 25.43 - - -
Methyl salicylate 1191 1191 MS, RIL 1.12 0.17 - - -
Safranal 1194 1197 MS, RIL 0.90 1.04 0.47 -
d Compounda RIb RILc ID Dried Bark Dry Fresh Essential leaf powder leaf leaf oil powder
Beta cyclocitral 1218 1219 MS, RIL 1.05 0.22 1.37 2.23 -
(Z) Citral 1234 1238 MS, RIL, 0.54 - - - 0.68
RIS
9 6
Unidentified 1247 - MS 5.61 - - - - monoterpene 1
Beta 1252 - MS - - 0.45 - homocyclocitral
(E) Citral 1265 1269 MS, RIL, 0.68 - - - 1.15 RIS
Alpha ethylidene 1265 1268 MS, RIL - - - 0.31 - benzene acetaldehyde
Cyclohexene, 5 1279 - MS - - - 0.88 - methyl-3- (1 methylethenyl), trans (-)
2 Undecanone 1295 1291 MS, RIL - 0.65 - - -
d Compounda RIb RILc ID Dried Bark Dry Fresh leaf powder leaf leaf powder
4 hydroxy-3- 1302 1323 MS, RIL - 0.42 - - - methylacetophenon e
2-(3-Isopropyl-4- 1316 - MS - 0.49 - - - methyl- pent-3- en-1-ynyl)-2- methyl- cyclobuta
none 97
Unidentified 1326 - MS 0.68 - - - - sesquiterpene 1
Delta elemene 1330 1337 MS, RIL 1.05 - 0.33 - -
Unidentified 1330 - MS - - - 2.46 - sulfur compound 5
Unidentified 1331 - MS - - 1.07 - - sesquiterpene 2
Unidentified 1339 - MS - - - 4.07 - sulfur compound 6
Compounda RIb RILc IDd Dried Bark Dry Fresh Essential leaf powder leaf leaf oil powder
Unidentified 1342 - MS 0.58 - - - - sesquiterpene 3
1H indene, 1350 - MS - - - 0.38 - ethylideneoctahyd ro-7a methyl (1Z, 3a.alpha, 7a.beta)
98 (-)- 1352 1378 MS - 0.37 - - -
Isolongifoline
Bicyclo [5.2.0] 1368 1458 MS - 0.48 - - - nonane, 4- methylene-2, 8,8- trimethyl-2- vinyl-
2H-2, 4a- 1373 - MS - 0.72 - - - Ethanonaphthalene , 1,3,4,5,6,7- hexahydro-2, 5,5- trimethyl-
b c d Compounda RI RIL ID Dried Bark Dry Fresh Essential leaf powder leaf leaf oil powder
Alpha copaene 1375 1377 MS, RIL 1.07 3.14 3.91 0.60 -
Beta bourbonene 1378 1380 MS, RIL 3.22 - 0.98 - -
Beta elemene 1389 1393 MS, RIL 2.21 0.59 1.24 - -
Unidentified 1404 - MS - 0.74 - - - sesquiterpene 4
Unidentified 1412 - MS - 0.80 - - - sesquiterpene 5
Alpha cedrene 1415 1415 MS, RIL - 0.81 - - - 99
Beta 1415 1418 MS, RIL, 5.28 8.57 5.54 0.33 0.32 caryophyllene RIS
Alpha ionone 1418 1422 MS, RIL, 0.53 - 0.97 0.78 0.29
RIS
Unknown 1423 - MS - 0.88 - sesquiterpene 6
Gamma elemene 1424 1425 MS, RIL 3.32 - 5.28 0.61 -
d Compounda RIb RILc ID Dried Bark Dry Fresh Essential leaf powder leaf leaf oil powder
Trans alpha 1428 1432 MS, RIL 0.95 0.81 0.90 - - bergamotene
Di-epi-α-cedrene 1430 1427 MS, RIL - - - 0.46 -
Beta patchoulene 1442 1380 MS - - - 0.73 -
Alpha himachalene 1444 1447 MS, RIL 4.94 - - - -
Geranyl acetone 1449 1448 MS, RIL - 1.09 1.11 0.49 -
Humulene 1455 1455 MS, RIL 1.31 1.27 1.75 0.25 -
Alloaromadendrene 1459 1460 MS, RIL - 2.39 - - - 100
Beta acoradiene 1466 1466 MS, RIL - 2.76 - - -
Unidentified 1470 - MS 2.65 - - - - sesquiterpene 7
Beta ionone 1473 1477 MS, RIL, 3.29 - 5.92 4.05 1.99 RIS
(+)-Epi 1476 1482 MS, RIL - 1.70 7.48 - - bicyclosesquiphel landrene
d Compounda RIb RILc ID Dried Bark Dry Fresh Essential leaf powder leaf leaf oil powder
Beta ionone 1479 - MS - - - - - isomer
Beta Selinene 1483 1485 MS, RIL 0.96 - - - -
Unidentified 1486 - MS 0.85 - - - - sesquiterpene 8
Unidentified - MS 1.79 sesquiterpene 9
Valencene 1487 1490 MS, RIL 1.57 - 0.80 0.49 0.38
Unidentified 1488 - MS - 0.67 - - -
sesquiterpene 10 101
Unidentified 1491 - MS - 1.19 -
sesquiterpene 11
Delta selinene 1493 1493 MS, RIL - 0.58 -
Alpha muurolene 1494 1499 MS, RIL 2.35 0.33 - -
Beta himachalene 1494 1499 MS, RIL 2.04 - - 0.24 0.71
d Compounda RIb RILc ID Dried Bark Dry Fresh Essential leaf powder leaf leaf oil powder
Butylated hydroxy 1497 1504 MS, RIL 1.06 - - - 0.31 tolouene
Tau cadinene 1504 1505 MS, RIL - 0.58 - -
Unidentified 1507 - MS 0.43 - - - - sesquiterpene 12
Unidentified 1508 - MS - 0.65 - - - sesquiterpene 13
Trans gamma 1515 1513 MS, RIL 3.37 1.32 2.15 0.40 - cadinene
Unidentified 1516 - MS - 2.17 - - -
102 sesquiterpene 14
Delta cadinene 1518 1524 MS, RIL - 2.35 - - 0.55
Cis-Calamenene 1522 1521 MS, RIL 0.28 1.24 - - -
Unidentified 1531 - MS 1.55 - - - - sesquiterpene 15
Unidentified 1533 - MS - - 0.80 - - sesquiterpene 16
d Compounda RIb RILc ID Dried Bark Dry Fresh Essential leaf powder leaf leaf oil powder
3,7, (11) 1537 1532 MS, RIL - - 0.82 - - Selinadiene
Unidentified 1537 - MS - 0.51 - - - sesquiterpene 17
Unidentified 1541 - MS - 0.82 - - - sesquiterpene 18
Unidentified 1546 - MS - 1.12 - - -
sesquiterpene 19 103
Alpha cedrene 1550 - MS - 1.16 - - - oxide
Unidentified 1559 - MS 10.76 0.40 35.77 - 0.36 sesquiterpene 20
Spathulenol 1571 1575 MS, RIL 0.79 - 1.96 - 3.78
Caryophyllene 1576 1568 MS, RIL - 2.52 - - - alcohol
Caryophyllene 1582 1581 MS, RIL 17.04 1.51 2.42 - 12.88 oxide
d Compounda RIb RILc ID Dried Bark Dry Fresh Essential leaf powder leaf leaf oil
powder
Unidentified 1594 - MS - 1.07 - - - sesquiterpene 21
Unidentified 1631 - MS - 1.32 - - - sesquiterpene 22
1 Gamma eudesmol 1657 1651 MS, RIL - 1.57 - - - 04 Cadalene 1673 1674 MS - 0.72 - - -
Heptadecene 1682 1676 MS - 0.39 - - -
Abietatriene >1714 2051 MS - 0.54 - -
Methyl (z) >1714 2274 MS - - - 2.58 - 5,11,14,17
eicosatetraenoate
a Compounds are listed in order of elution from a DB-5MS column. b Linear retention index on DB-5MS column, experimentally determined using homologous series of C8-C20 alkanes. c Relative retention index according to Adams, 1995 for DB 5 capillary columns. d Identification methods: MS, by comparison of the mass spectrum with the NIST 1992 and Wiley 5 libraries; RIL, by comparison of RI with those reported in the literature (Adams, 1995); RIS, by matching retention times of commercial standards with compound peaks in the samples. Compounds identified by only MS are done so tentatively.
Table 2. Volatiles, unique to essential oil, as identified and quantified (%RA= %Relative Abundance) by GC-MS.
b c d Compounda RI RIL ID %RA
2,3 Dimethyl 2 butanol - - MS 0.25
3 Methyl 3 pentanol - - MS 0.09
2 Hexanone - - MS 0.06
Alpha terpineol 1196 1198 MS, RIL 0.27
3 methyl -3-(4 methyl 3 1233 MS 0.34
105 pentenyl)
Oxiranecarboxaldehyde
Linalyl acetate 1252 1257 MS, RIL 0.57
Unidentified sesquiterpene 1348 - MS 0.41 1
2 Methyl propanoic acid, 3 1373 1365 MS, RIL 0.51 hydroxy 2,4,4 trimethyl pentyl ester
Geranyl acetate 1381 1383 MS, RIL 0.37
b c d Compounda RI RIL ID %RA
Longifolene 1450 1448 MS, RIL 1.47
Beta ionone epoxide 1483 - MS 1.01
Unidentified sesquiterpene 1517 - MS 0.75 2
Unidentified sesquiterpene 1526 - MS 1.19
106 3
Unidentified sesquiterpene 1538 - MS 0.35 4
Unidentified sesquiterpene 1542 - MS 0.32 5
Unidentified sesquiterpene 1559 - MS 0.36 6
E-Nerolidol 1564 1564 MS, RIL 0.35
Lauric acid 1574 1571 MS, RIL 1.93
Unknown sesquiterpene 7 1591 - MS 1.28
Unknown sesquiterpene 8 1605 - MS 0.58
b c d Compounda RI RIL ID %RA
2,5,9 trimethylcycloundeca- 1611 - MS 3.24 4, 8 dienone
Unknown sesquiterpene 9 1614 - MS 0.91
Unidentified sesquiterpene 1618 - MS 0.69
10 107
7 R, 8R-8hydroxy-4- 1624 1696 MS 1.27 isopropylidene-7- methylbicyclo undecene
Unidentified sesquiterpene 1639 - MS 0.95 12
Alpha elemene 1649 - MS 1.33
Unidentified sesquiterpene 1657 - MS 2.02 13
Aromadendrene oxide II 1672 1678 MS, RIL 2.45
Gamma costol 1683 - MS 1.48
b c d Compounda RI RIL ID %RA
6 Isopropenyl 4,8a dimethyl 1692 1690 MS, RIL 0.92 octahydro naphthalen-2-ol
2 Octyl benzoate 1710 - MS 1.71
Unidentified sesquiterpene 1714 - MS 2.32 14
3 Isopropyl 6,7 1745 - MS 1.10 dimethylcyclo decane-9, 10 diol
108 Benzyl benzoate 1769 1762 MS, RIL 1.29
7R, 8R-8-Hydroxy-4- 1772 - MS 3.41 isopropylidene-7- methylbicyclo[5.3.1] undec- 1-ene
Neophytadiene 1841 1838 MS, RIL 1.88
Hexadecanoic acid methyl 1929 1927 MS, RIL 2.25 ester
Isophytol 1951 1956 MS, RIL 0.54
b c d Compounda RI RIL ID %RA
n Hexadecanoic acid 1971 1972 MS, RIL 12.66 109
8 Octadecanoic acid, methyl >1971 2110 MS 1.33
ester (E)
Phytol >1971 2122 MS 4.67
Linolein >1971 - MS 2.23
Cis-9-eicosenol >1971 - MS 0.82
a Compounds are listed in order of elution from a DB-5MS column. b Linear retention index on DB-5MS column, experimentally determined using homologous series of C8-C20 alkanes. c Relative retention index according to Adams, 1995 for DB 5 capillary columns. d Identification methods: MS, by comparison of the mass spectrum with the NIST 1992 and Wiley 5 libraries; RIL, by comparison of RI with those reported in the literature (Adams, 1995). Compounds identified by only MS are done so tentatively.
Chapter 5
Comparison of two adsorbent based de-bittering procedures for neem (Azadirachta indica A. Juss) tea- Effect on polyphenols, anti-oxidant capacity, color and volatile profile
5.1 Introduction
Bitterness reduction is a huge challenge for the food industry. Human beings have an inherent liking for sweet taste
(Myers and Sclafani 2003) but bitterness, generally, leads to consumer rejection of food products (Stein and others 2003).
This is especially of concern for functional foods and medicinal extracts, which target digestive health, weight management, cancer and diabetes (Cencic and Chingwaru 2010).
The phytonutrients in these food products and extracts are usually associated with a bitter and acrid off flavors, which leads to a negative consumer experience (Drewnowski and Gomez-
Carneros 2000). Therefore, to improve the commercial viability of such health promoting products, counteracting the bitterness becomes a vital aspect of product development.
Neem (Azadirachta indica A. Juss.) is an evergreen tree cultivated in various parts of the Indian sub-continent (Ray and others 1996). It has been in use over centuries in Indian folk medicine for its therapeutic value. Its bioactivity and health promoting properties, have to a large extent, been attributed to a group of bitter tetranortriterpenoids called limonoids, in addition to various polyphenolic substances
110 (Champagne and others 1992). Today, extensive research has shown that neem may have anti-cancer (Wu and others 2014), anti- diabetic (Perez-Gutierrez and Damian-Guzman 2012b), anti-inflammatory (Schumacher and others 2011), anti- ulcerogenic (Dorababu and others 2004), and anti-microbial effects (El-Mahmood and others 2013). Neem leaves are sold today as ‘medicinal’ tea to health-conscious consumers all over the world. But the high content of limonoids make it extremely bitter and unpalatable.
The area of bitterness reduction has been researched extensively in orange juice using various strategies such as cyclodextrin polymers (Wilson III and others 1989), polyadsorbent resins (Ribeiro and others 2002) and enzymatic reactions (Cánovas and others 1998). One of the compounds responsible for orange juice bitterness is limonin, which belongs to the larger class of compounds called limonoids. The bitterness principle of neem also constitutes limonoids, azadirachtin being one of the primary ones being reported
(Parida and others 2002). Therefore, one could expect the methods adopted to minimize bitterness in orange juice to work effectively for neem tea as well. Solid phase extraction (SPE) has been used previously to isolate limonoids in citrus juice
(Widmer 1991) and neem extracts (Jarvis and Morgan 2000; Lee and others 2013) and therefore we hypothesized, that it could be an effective method for removing limonoids and reducing bitterness. In addition, there are several examples of the use
111 of cross linked divinylbenzene- styrene resins as effective agents of bitterness removal (Ribeiro and others 2002; Stinco and others 2013; Fernández‐Vázquez and others 2013).
While adsorption based removal of bitterness through polymeric resins has the advantage of being a low energy processes, they have elicited a “flavor scalping” effect (Fayoux and others
2007) and changes in color properties (Lee and Kim 2003) due absorption of pigments by resins, which may have an adverse effect on sensorial properties. Our aim is to apply adsorbent based DPs to Neem tea infusion and observe the changes linked to its purported health promoting. The results of this study are useful for the food and beverage industry in the product development process. The results can help functional food developers specifically, to come up with de-bittering procedures that can eliminate the bitterness and off-flavors of phytonutrients while minimizing the loss in bio-active compounds.
5.2 Materials and methods
5.2.1 Plant material
Three batches of neem tea-cut leaves (A. indica) with different lot numbers were bought online from Neem Tree Farms
(Brandon, FL). These leaves were imported from Mexico and then dried in a solar heated drying room, according to information provided by the vendor.
5.2.2 Chemicals
112 Analytical grade ethanol was bought locally from the chemical stores of the Department of Chemistry, University of Missouri,
Columbia. HPLC grade water, ascorbic acid, acetic acid, chloroform, acetonitrile and Amberlite XAD-16 hydrophobic, macro-reticular resin were bought from Thermo Fisher
Scientific (Waltham, MA, USA). Folin-Ciocalteau reagent, perchloric acid (70%) DPPH (2,2-diphenyl-1-picrylhydrazyl) were bought from Sigma-Aldrich (St. Louis, MO, USA) while the
C18 solid-phase extraction cartridges (100 mg, 1.5mL) were bought from Grace Alltech (Columbia, MD, USA). The standard for quercetin was bought from Cayman Chemical Co. (Ann Arbor,
MI, USA), and the standards for limonin glucoside and limonin were bought from Abcam (Cambridge, MA, USA).
5.2.3 Tea preparation
To 100 mL of boiling water, 0.5 grams of tea was added and the aqueous extraction was allowed to take place for 5 mins. This solution was filtered using Fisherbrand P8 filter paper and made up to volume in a 100-mL volumetric flask with distilled water.
5.2.4 De-bittering procedures (DPs)
The DP’s were optimized for their bitterness reduction capacity through bench top testing. The volume of tea to bed volume (adsorbing material) ratio was noted.
A) Solid Phase Extraction (SPE) de-bittering procedure
20 mL of Neem tea was passed through each Grace™ Alltech™
Prevail™ C18 SPE column and the eluent was collected into two
113 10-mL glass tubes. The cartridges were sold pre-conditioned; therefore, no preconditioning step with any organic solvent was required. The volume ratio of tea: bed was 200:1.
B) Amberlite (AMB XAD-16) de-bittering procedure
The polymeric, macroreticular, hydrophobic, adsorbent resin was washed overnight with distilled water to clear away the anti-microbial sodium chloride and sodium carbonate salts that it ships with. After drying in an oven, 2 grams of the bead- like resin, was added to 50 mL of the Neem tea and stirred at
600 rpm with a magnetic stirrer in a batch operation. The volume ratio of tea: bed was 25:1)
5.2.5 Extraction of flavonols
In order to quantify the amount of flavonols, the hydrolysis method of Wang and Helliwell (2001) was followed with slight modifications. Briefly, 8 mL of the neem tea was added to 12 mL of absolute (99.99%) ethanol in 100-mL round bottom flasks.
To this mixture, 5 mL of 6 N HCL and 40 mg of ascorbic acid
(anti-oxidant) (Nuutila and others 2002) was added and the solution was refluxed at 95° C for two hours. The hydrolyzed solution was filtered and made up to 25 mL in a volumetric flask, using 60% ethanol. It was filtered through a 0.45 μ nylon filter (EMD Millipore, Billerica, MA, USA) and injected into the HPLC.
A stock solution of the flavonol standard, quercetin, was prepared by dissolving 10 mg of the pure standard in 25 mL of
114 dimethyl sulfoxide (DMSO). The standard curve was prepared within the concentration range of 0-0.04 mg/mL.
5.2.6 Determination of polyphenol content by the Folin- Ciocalteu assay
Total phenolics content was determined using the Folin–
Ciocalteau spectrophotometric assay adapted from Dudonné and others (2009). A calibration curve of gallic acid (ranging from 0 to 0.05 mg/mL) was prepared and the results, determined from regression equation of the calibration curve (y = 17.715x
- 0.0387, R² = 0.99117), were expressed as mg gallic acid equivalents per mL of the sample. In this method, 400 μL of tea extract undiluted or diluted 2 times with deionized water
(to obtain absorbance in the range of the prepared calibration curve) was mixed with 1.6 mL of 7% sodium carbonate solution.
2 mL of Folin–Ciocalteau phenol reagent (diluted 10 times) was added to the mixture and shaken thoroughly. The mixture was allowed to stand for 30 min and the blue color formed was measured at 765 nm using a Varian Cary® 50 UV-VIS spectrophotometer (Agilent Technologies Inc., Santa Clara,
CA).
5.2.7 Determination of antioxidant activities
A) 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay
The DPPH anti-oxidant assay was performed according to the method of Thaipong and others (2006) with modifications. The stock solution was prepared by dissolving 24 mg DPPH with 100 mL methanol and then stored at -20 ° C until needed. The
115 working solution was obtained by mixing 20 mL of stock solution with 80 mL of methanol.
Briefly, 2800 µL of DPPH working solution was added to 200 µL of sample and the solution was diluted with 2 mL of de-ionized water. The samples or standard was incubated for 30 mins at room temperature in the dark. The decolorization was measured spectrophotometrically at 515 nm using a Varian Cary® 50 UV-
VIS spectrophotometer (Agilent Technologies Inc., Santa Clara,
CA). Anti-oxidant activity was expressed as percentage inhibition of the DPPH radical and was determined by the following equation as reported by Yen and Duh (Yen and Duh
1994).
%퐴퐴 = (퐴푏푠표푟푏푎푛푐푒푐표푛푡푟표푙 − 퐴푏푠표푟푏푎푛푐푒푠푎푚푝푙푒) ∕ 퐴푏푠표푟푏푎푛푐푒푐표푛푡푟표푙 × 100
The percentage (%) anti-oxidant activity was converted to ascorbic acid equivalents using an ascorbic acid standard curve found to be linear between 0.0176-0.1408 mg/mL (y =
693.12x - 3.0024
R² = 0.98683). The results were expressed in mg of “vitamin C equivalent anti-oxidant capacity” (VCEAC) per gram of the sample.
B) Ferric ion-reducing antioxidant power (FRAP) assay
The FRAP assay was performed according to the method of
Thaipong and others (2006) with minor modifications. The FRAP reagent was prepared by mixing 300 mM acetate buffer (PH 3.6),
10 mM TPTZ and 20 mM ferric chloride in the 10:1:1 ratio. 150
μL of the sample or standard was added to 2 mL of de-ionized
116 water and 2850 μL of FRAP reagent. The samples and standards were incubated for 30 mins at room temperature in the dark.
The absorbance of the colored product [ferrous tripyridyltriazine complex] was measured at 593 nm using a
Varian Cary® 50 UV-VIS spectrophotometer (Agilent Technologies
Inc., Santa Clara, CA). The standard curve was developed using gallic acid as a standard and found to be linear between
0.0025-0.045 mg/mL (y=12.198 x + 0.0378, R2= 0.99582). Results were expressed in mg GAE/g of the sample.
5.2.8 Colorimetric determination of total limonoid glucoside and limonoid aglycones
The total limonoid glucoside content was determined according to the method of Breksa and Ibarra (2007) with slight modifications. The colorimetric quantification was based on the formation of red to orange colored derivatives resulting from the treatment of the standard, limonin glucoside, or the neem extract with 4- dimethylamino benzaldehyde (DMAB) in the presence of perchloric and acetic acids.
5 mL of neem tea (control) or SPE-treated or AMB-treated tea was passed through a C18 SPE column. The limonoids trapped in the column were eluted with 3 mL of methanol and evaporated under a steady flow of nitrogen gas. The sample was reconstituted in 3 mL of 30% acetonitrile.
The total limonoid aglycone was also determined by the method of Breksa and Ibarra (Breksa and Ibarra 2007). The aglycones were extracted by chloroform. Neem tea was mixed with
117 chloroform in a 1:1 ratio and vortexed. Once the phases separated, the upper phase was discarded and the lower phase
(chloroform) was collected and evaporated to dryness under a steady flow of nitrogen gas. The sample was re-constituted in
2.5 mL acetonitrile and the colorimetric assay was performed exactly according to the procedure of limonin glucoside determination. The standard curve developed with the limonin aglycone as the standard (0-20 μg/mL).
Briefly, 2.1 mL of limonin glucoside standard or neem/ SPE/
AMB treated samples was mixed with 1.65 mL of DMAB indicator.
To this, 1.65 mL of stock acid solution is added. The test tubes were incubated for 30 minutes at room temperature and the color developed was measured at 503 nm. Total limonoid glucoside in the sample was determined in terms of limonin glucoside equivalents. This was done using a calibration curve generated from limonin glucoside made up in 30% acetonitrile (0- 100 μg/mL).
5.2.9 HPLC Analysis for the estimation of flavonols
HPLC analysis was carried out on an Agilent 1100 series liquid chromatographic system with a diode-array detector set at 370 nm. A Kinetex 250 X 4.6 mm, 5 μ C 18, 100 Å (Phenomenex Inc.,
Torrance, CA, USA) column was used for the separation of the flavonol aglycones. A gradient elution system comprising of two mobile phases, namely mobile phase A, 0.1 % trifluoroacetic acid in water and mobile phase B, 0.1% trifluoroacetic acid in acetonitrile, was used. The gradient
118 used was 80% A and 20% B at 0 min which was gradually reduced to 60% A at 20 mins and held for another 5 mins. A 5 min post time was added for the mobile phase concentrations to come back to initial levels. The flow rate was 1 mL/min and the column temperature was set at 40 °C. The identification of each compound was done by matching retention times with standards and quantification was based on the external standard curve method.
5.2.10 Analysis of volatiles by headspace-solid phase microextraction (HS-SPME)
A DVB/CAR/PDMS (Divinylbenzene/Carboxen/Polydimethylsiloxane) fiber with a 50/30 μm coating thickness (Supelco Inc.,
Bellefonte, PA) was used for this investigation. For SPME analysis, 5 mL of the tea/de-bittered tea sample was placed in a 10-mL vial, which was capped with a PTFE/Silicone septum
(Supelco Inc., Bellefonte, PA). 500 mg of sodium chloride was added and the sample was magnetically stirred to aid the volatilization process (Reto and others 2007). After equilibration at 70 °C for 3 hours, the fiber was exposed to the headspace to absorb/adsorb the volatiles for 1 hour.
Subsequently, the fiber was desorbed in the GC injection port for 5 mins before its next use.
5.2.11 GC-MS Analysis
A Varian GC 3400CX (Varian, Walnut Creek, CA, USA) equipped with a 1078 programmable injector connected to a Varian Saturn
2000 Mass spectrometer with an ion trap detector was used for
119 GC-MS analysis. Volatiles were separated by using a DB-5MS (J
& W Scientific, Folsom, CA, USA) UI, 30 m * 0.25 mm with 0.25
μm film thickness fused silica capillary column. Helium carrier gas flow rate was set at 1mL/min and injector, transfer line and ion trap temperatures were 250°, 250°, 150
°C, respectively. Desorption was done in the splitless mode and the post desorption split flow was 100 mL/min. The column temperature program used was: 35 °C held for 5 min followed by a linear temperature program from 35 to 250 °C at 3°C/min.
Identification of chemical compounds was established using mass spectra comparison with the NIST 1992 and Wiley 5 libraries, retention indices of standards, and literature values.
5.2.12 Color Properties
The color properties of neem tea, SPE-treated and Amberlite- treated tea, namely L*, 풶*, b * were measured by Minolta colorimeter CR-410 (Konica Minolta Sensing Inc., Osaka, Japan)
(Sigge and others 2001). Chroma and hue angle were calculated by the following formulae-
∗ ∗2 ∗2 퐶ℎ푟표푚푎 (푐푎푏) = 푎 + 푏
푏∗ 퐻푢푒 푎푛𝑔푙푒 (ℎ ) = 푡푎푛−1 푎푏 푎∗
The color difference between samples (ΔE*ab) is calculated from
L*, a*, b* by the formula-
∗ * 2 * 2 * 2 ∆퐸푎푏 =(ΔL ) + (Δ풶 ) + (Δb )
120 where ΔL, Δ풶*, Δb* are differences between neem tea and the de- bittered neem teas (SPE and Amberlite treated).
5.2.13 Statistical Analysis
Three different batches of Neem tea-cut leaves were purchased.
One sample from each batch was analyzed in duplicate to give triplicate analysis with two subsamples. Calculations of mean, standard deviation, analysis of variance (ANOVA) with Tukey’s post hoc test to determine significant differences, were performed in Minitab 16 statistical software (Minitab Inc.,
PA, USA). While comparing means using ANOVA, a randomized block design was used where the batch of the sample was blocked.
Principal Component Analysis (PCA) was performed in Minitab
16, on neem tea and the two de-bittered samples to visualize their clustering tendency based on their volatile profile and identify volatiles (variables) influencing their variability.
The data matrix consisted of 47 variables x 9 samples= 423 data points. Eigenvalues were calculated using a co-relation matrix among 47 volatile compounds as input, and the score and loading plots were generated using sample type and volatile constituents, respectively.
5.3 Results and Discussion
5.3.1 Effect of DP on flavonols, total polyphenols and limonoids
Figs. 1-3 show us the mean levels of flavonol (quercetin), total polyphenols and limonoids, respectively. In addition to
121 quercetin, two other flavonols, tentatively identified as
myricetin and kaempferol, were extracted. However, myricetin
and kaempferol were extracted in amounts that were below the
quantitation limit of the method. The SPE DP completely
eliminates quercetin while Amberlite XAD-16 treated tea
retained quercetin to such a degree that there were no
significant differences to the control neem tea (Fig 1). The
reduction in flavonoids was also observed in previous
literature (Ribeiro and others 2002; Lee and Kim 2003), where
flavonoids where adsorbed effectively by polyadsorbent resins.
The ability of Amberlite XAD-16 to retain quercetin might be
important aspect of maintaining neem’s functionality.
Fig. 1. Effect of DP on quercetin in neem tea (n= 3 SD)
4.00 a 3.50 a 3.00
2.50
2.00
Quercetin (mg/g)Quercetin 1.50
1.00
0.50
0.00 Neem tea SPE AMB
Means that do not share the same letter are significantly different (p < 0.05)
122 Fig. 2. Effect of DP on the total phenolic content in neem tea (n=3 SD)
14.00 a
12.00 b c 10.00
8.00
6.00 4.00
Totalpolyphenols (mg/GAE) 2.00
0.00 Neem tea SPE AMB
Means that do not share the same letter are significantly different (p < 0.05)
The SPE DP reduced the total polyphenols by 91.17% while this
decrease was much lesser due to the AMB DP at 19.01%. The DP
caused a significant reduction (p<0.05) in both limonoid
aglycones and limonoid glucosides content. The amount of total
limonoid aglycones and limonoid glucoside was found to be 0.54
mg/g LE and 1.75 mg/g LGE (Fig. 3). These values are much
higher than in Washington Navel oranges and Rio star
grapefruit where LE were found to be 0.002 ± 0.00 and 0.01 ±
0.00 mg/g , and the limonoid glucoside content was found to be
0.14 ± 0.00 and 0.21± 0.01 mg/g LGE, respectively (Breksa and
Ibarra 2007).
123 Fig.3. Effect of de-bittering procedure on the limonoid content of neem tea (n= 3 SD)
2500.00 2500.00 a
2000.00 g/g) 2000.00
)
μ μg/g 1500.00 1500.00
1000.00 a 1000.00 b b b
500.00 b 500.00 Limoninequivalent (
0.00 0.00 Limonin glucoside Limonin glucoside equivalent ( Neem tea SPE AMB Neem tea SPE AMB
Means that do not share the same letter are significantly different (p < 0.05)
Both the SPE and Amberlite treatment resulted in a decrease of
limonoid aglycone, which is in agreement with the results of
Shaw and Buslig (Shaw and Buslig 1986); Johnson and Chandler
(Johnson and Chandler 1982) and Wilson et al. (Wilson III and
others 1989) who used XAD-4, XAD-7 and XAD- 16 for achieving
de-bittering, respectively. The loss of limonoid glucoside was
67% and 79% for SPE and Amberlite XAD-16 treated neem tea,
respectively, while the treatments reduced the limonoid
aglycones equally by 62%. This loss may have a negative impact
on the health benefits of de-bittered neem tea (Miller and
others 1989).
5.3.2 Effect of DP on anti-oxidant activity of neem tea
We observe a significant decrease (p<0.05) in DPPH activity and
FRAP assay in the two de-bittered teas (Fig. 4). This is commensurate with the significant (p<0.05) reduction in polyphenols and the limonoids. Although limonoids, as
124 determined by DPPH activity, are weaker anti-oxidants compared to flavonoids due to fewer number of hydroxyl groups (Yu and others 2005), its high concentration in neem (Champagne and others 1992) makes these compounds a potent contributor towards the overall anti-oxidant capacity. Thus, the removal of limonoids might have a more pronounced effect on the DPPH anti- oxidant capacity than in the FRAP assay.
5.3.3 Effect of DP on color properties
The de-bittering procedures affected the color properties of neem tea. The values of L*, a* and b* of neem and the two de- bittered teas are presented in Table 1. The lightness of the sample is not affect by the treatment.
Fig. 4. Effect of de-bittering procedure on
A) FRAP
25 a
20 oxidant
- b
15 c
10 Capacity(mg/g)
5 Vitamin Equivalent C Anti
0 Neem tea SPE AMB
Means that do not share the same letter are significantly different (p < 0.05) (n=3 S.D.)
125 B) DPPH anti-oxidant activities
25
20
15
a 10 b c
5 Gallic Gallic AcidEquivalent (mg/g)
0 Neem tea SPE AMB
Means that do not share the same letter are significantly different (p < 0.05) (n=3 S.D.)
The greenness of neem is reduced by the SPE treatment while it remains unaffected for Amberlite XAD-16 treated tea. However, the yellowness and chroma of neem tea is reduced significantly
(p < 0.05) by the two de-bittering procedures.
Table 1. The difference in color parameters of neem and de- bittered neem tea (n=3 S.D.)
Samples Neem tea SPE tea AMB XAD-16 tea
L* 55.62a 55.66a 57.03a
a* -0.71b 0.14a -0.46b
b* 9.60a 4.69b 5.85b
* a b b C ab 9.62 4.70 5.86
b a b hab -1.50 1.54 -1.49
Δ E*ab - 5.06 ± 0.85 4.04 ± 1.37
Means that do not share the same letter are significantly different (p < 0.05) L*= Lightness a*, b*= Chromacity co-ordinates C*ab= Chroma hab= Hue Δ E*ab= Total color difference
126 The hue angle remains unaffected. The total color difference (Δ
E*ab) between neem tea and SPE treated tea was 5.06 ± 0.85
CIELAB units while that between neem tea and Amberlite XAD-16 treated tea was 4.04 ± 1.37 CIELAB units. The mean value of Δ
E*ab was greater than the visual discrimination threshold (Δ E*ab
> 3) indicating that the color changes are visually appreciable
(Melgosa and others 2001). The color changes in the de-bittered samples can be explained by the decrease in the flavonol and the total polyphenolic content, which are known to contribute to color (Bao and others 2005).
5.3.4 Effect of DP on the volatile profile of neem tea
Both the SPE and Amberlite XAD-16 treated led to the loss of major sesquiterpenes such as dihydroactinidiolide, spathulenol and caryophyllene oxide in neem tea (Table 2). These compounds are often found in essential oils possessing anti-inflammatory, anti-bacterial and anti-oxidant properties (Shimizu and others
1990); (Goff and Klee 2006; Wu and others 2014). Thus, their loss could affect the bioactive potential of neem tea.
Surprisingly, we observed the generation of some volatile compounds because of the treatment such as the generation of 2- heptanone, 2-ethyl hexanol in the SPE and AMB XAD-16 treated teas. The compounds 4- ethoxy benzoic acid and 3,5 ditert- butyl-4-hydroxy benzaldehyde were exclusively found in SPE and
AMB tea, respectively. The PCA score plot (Fig. 5) is able to separate neem tea from the treated samples.
127 Fig.5. Principal Component Analysis (PCA)- Score Plot. This shows the clustering tendency of neem tea (control) and the two de-bittered (treated) samples. Since they lie in different quadrants, it can be established that the de-bittering procedures impact the volatile profile (n=3)
5 SPE tea
4
3 SPE tea
2 SPE tea
1 NeemNeem teateaNeem tea 0
-1 F2 (15.65 (15.65 %)F2 AMB tea -2
-3
-4 AMB tea AMB tea -5 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 F1 (53.52 %)
The three samples are distinguished easily since they are clustered in three separate quadrants. The neem tea is separated from SPE tea on the first principal component (F1) while it separates from AMB tea on both the first and second principal component (F2). The loading plot (Fig. 6) reveals that neem tea is separated from the two de-bittered teas by sesquiterpenes, ketones and acids while the volatiles such as heptanal, benzeneacetaldehyde, 2 ethyl hexanol, dl –limonene load strongly on to F2 and distinguish SPE tea from AMB XAD-16 treated tea.
128 Fig.6. Loading plot of the variables reveals the specific volatiles (variables) that help discriminate between neem tea (control) and the two de-bittered samples (treatments) (n=3)
Variables (axes F1 and F2: 69.17 %)
1 heptanal 4 ethoxy benzoic3,5 di-tert acid-butyl ethyl-4- benzoic acid ethyl ester 2,4 dihydroxybenzaldehyde tertester butyl phenol 0.75 benzeneacetaldehyde
2 ethyl hexanol 0.5 dl limonene 2 nonanone 7R,8R-8-Hydroxy-4- 0.25 octanal isopropylidene3-isopropyl-7- -6,7- hexanal methylbicyclo[5.3.1]undecdimethyltricyclo[4.4.0.0(2,8hexahydro-8a-methyl- -,1,8 spathulenol (2H,5H)1unknown-Eudesmaene)]decaneeugenolbetacaryophyllene-unknownNaphthalenedionebetaDihydroactinolideunknownJuniperbeta ionone -ST-4,11unknown 9,10betaalphacyclocitral unknowniononesafranal - ST-diencamphor diolselineneepoxideST ionone oxide oxide- 2ST -STol methoxy benzene sulcatone 0 benzaldehyde
Farnesyl acetaldehyde F2 (15.65 (15.65 %)F2 -0.25 1 methyl 1 H pyrrole propanoicgeranyl acetone acid,2 methyl, 3 hydroxy 2,4,4 trimethylpentyl ester p cymenene -0.5 p benzoquinone methyl salicylate 2 heptanone
3,5 di-tert-butyl-4- linalool -0.75 hydroxybenzaldehyde
-1 -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 F1 (53.52 %)
129 Table 1. Volatile compounds in NT and the two de-bittered samples identified on a DB-5 MS column
Compounds Neem tea SPE AMB
1-methyl 1 H pyrrole + + +
Hexanal + + +
1,1,3 trimethyl cyclopentane + - -
2-heptanone - + +
Heptanal + + -
Methoxy benzene + + +
Benzaldehyde + + +
1 30
Sulcatone + + +
Octanal + + +
dl-limonene + + +
2-ethyl hexanol - + +
Benzeneacetaldehyde + + +
Linalool + + +
Methyl salicylate + + +
Safranal + - -
Compounds Neem tea SPE AMB
Eugenol + - -
Propanoic acid,2 methyl, 3 hydroxy 2,4,4 trimethylpentyl ester + - +
Alpha ionone + - -
Geranyl acetone + + +
p-benzoquinone + + +
Beta ionone + - - 1
31 Beta ionone epoxide + - -
4-ethoxy benzoic acid ethyl ester - + -
Unknown ST + - -
Unknown ST + - -
Hexahydro-8a-methyl-,1,8 (2H,5H)-Naphthalenedione + - -
Dihydroactinolide + - -
Spathulenol + - -
Caryophyllene oxide + - -
Unknown ST + - -
Unknown ST + - -
Compounds Neem tea SPE AMB
Beta selinene + - -
Unknown ST + - -
Unknown ST oxide + - -
Juniper camphor /globulol + - -
1 32
9.beta.-Acetoxy-3.beta.-hydroxy-3,5.alpha.,8-trimethyltricyclo[6.3.1.0] + - - dodec-8-ene
3-isopropyl-6,7-dimethyltricyclo[4.4.0.0(2,8)]decane-9,10-diol + - -
Eudesma-4,11-dien-2-ol + - -
3,5 ditert-butyl-4-hydroxy benzaldehyde +
7R,8R-8-hydroxy-4-isopropylidene-7-methylbicyclo[5.3.1]undec-1-ene + - -
Farnesyl acetaldehyde + + +
Di-tert-butyl-1-oxaspiro[4.5]deca-6,9-diene-2,8-dione + + +
6.4 Conclusions
Both treatments, SPE and XAD-16 resin, were able to reduce the bitterness in neem tea as demonstrated by the reduction of total limonoid aglycones. However, the Amberlite XAD-16 resin treatment was more effective in preserving the loss in bio- active compounds and anti-oxidant capacity than the SPE treatment. The encouraging results obtained should lead to further research with other styrene/divinylbenzene polymer resins such as Amberlite XAD-2, XAD-4, XAD-7 HP, XAD-16HP and
Dowex-L285 to see if they are able to preserve the bio-active polyphenols and anti-oxidant adequately while removing the bitterness completely.
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Chapter 6
Effect of tea matrix and type of milk on the recovery of flavonols, total phenolic content and anti-oxidant activity with an application towards Ready-to-Drink beverages (RTD’S)
6.1 Introduction
Tea is a traditional beverage that may provide numerous health benefits due to its high concentrations of antioxidants in the form of polyphenols (Chen and others
2015; Miura and others 2015). In many societies, teas are traditionally consumed with milk, and more recently ready-to-drink (RTD) beverages, including teas, have become popular.
An RTD is any packaged beverage that is sold in a prepared form, ready for consumption. The Ready-to-Drink (RTD) product segment has seen significant growth over the last few years with new entrants and innovation, mainly due to a new wave of consumers seeking convenient alternatives that fit their active lifestyles. Many tea-based RTD’s are sold in the market with added dairy components.
Due to the boom in this specific beverage market segment, research of milk protein-polyphenol interaction has assumed great significance. Both covalent and non-covalent bonding
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has been suggested as possible mechanisms of bonding between polyphenols and proteins. It was suggested that hydrophobic forces initiate interactions, which are consequently stabilized by hydrogen bonding (Prigent and others 2003).
Further evidence of hydrophobic interactions in protein- polyphenol interactions was provided through fluorometry and isothermal calorimetric analysis (Yuksel and others 2010).
Determining the nature and quantum of binding in-vitro can be helpful for understanding the dissociation and availability of the protein-flavonoid complex and subsequent absorption of flavonoids in-vivo. So far, there have been a multitude of studies on flavan-3-ols (catechin and its derivatives) and milk protein interactions and their effect on anti-oxidant capacity. But these studies have revealed contradictory results. Three types of results have been observed. Firstly, some authors (Kyle and others 2007; Richelle and others 2001;
Leenen and others 2000) observed no effect of milk or milk proteins on the anti-oxidant potential of the teas. Other authors (Stojadinovic and others 2013; Xiao and others 2011;
Sharma and others 2008; Arts and others 2002; Serafini and others 1996) observed a binding effect of milk with flavonoids that led to a reduction in anti-oxidant activity of the tea-milk mixture. Thirdly, it was also observed that while the addition of milk to green, Darjeeling and English breakfast teas lead to a decrease in the anti-oxidant activity (ABTS+ free radical scavenging), it had an enhanced
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chain breaking anti-oxidant effect (lipid peroxidation method) (Dubeau and others 2010).
While most of the research has focused on the binding and recovery of flavan-3-ols post milk addition, our study determined the effect of milk addition on flavonols.
Flavonols are an important group of compounds whose health effects have been well documented. They have been shown to have a protective effect on DNA damage in human lymphocytes (Duthie and others 1997), reduce the risk of oxidative metabolism in neurons (Oyama and others 1994), lower plasma glucose (Liu and others 2005; Park 1999), reduce the risk of coronary heart disease (Huxley and
Neil 2003), in addition to numerous other beneficial effects (Guardia and others 2001; Pavanato and others
2003; Noroozi and others 1998).
We used three different teas or infusions, specifically one medicinal infusion (Neem) and two conventional teas
(green and black) in our study. Neem (Azadirachta indica
A. Juss) is an evergreen tree belonging to the Meliaceae family, cultivated in various parts of the Indian sub- continent (Biswas and others 2002). It has been in use in
Indian folk medicine for centuries because of its therapeutic value (Mishra and others 1995; Kaushik and others 2012). Recently, our lab has shown that it is a rich source of flavonols with levels even higher than those in green and black tea [unpublished]. Given its
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myriad of purported health benefits, and a general consumer trend towards functional foods and nutraceuticals, Neem leaf powder is being marketed and consumed as tea.
Our aims in this study were two-fold. Firstly, we observed if there was a difference in the in-vitro anti-oxidant activity due to a decrease in polyphenols and other bioactives, when bovine milk is added to different tea matrices, i.e. neem, green and black tea. Secondly, plant based milks such as almond, coconut, soya and rice milk have great potential for application in Ready to Drink health beverages (RTD’s), given their lactose free nature. Being sourced from plants, they are a popular option among vegans. Keeping in mind this trend of increased consumption of these alternative, non-dairy sources of milk, we investigated the differences in effect of adding bovine, soya and almond milk on in-vitro phenolic content and anti-oxidant activity.
6.2 Materials and methods
6.2.1 Samples
Three different lots of Neem (Azadirachta indica) leaf powder were purchased from Neem Tree Farms Inc. (Brandon,
FL., USA), and three lots of Great Value Green tea and
Schnucks 100% Natural Orange Pekoe & Pekoe Cut Black tea were purchased from local Walmart and Schnucks super markets in Columbia, MO, respectively.
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Skimmed milk (Prairie Farms Dairy Inc., Carlinville, IL) and Silk (Light original) soymilk (White Wave Services
Inc., Broomfield, CO) were bought from the local Hyvee store and unflavored (original) almond milk (So Delicious
Dairy Free, Springfield, OR) was bought from the local
Lucky’s supermarket. Three samples of commercial ready to drink (RTD) green tea-bovine milk Taste Nirvana Real
Green Tea Latte (Nirvana Foods & Commerce International
Co., Ltd., Walnut, CA) were bought online.
6.2.2 Chemicals and standards
The flavonol standards myricetin, quercetin and kaempferol were bought from Cayman Chemical Co. (Ann
Arbor, MI). HPLC grade acetonitrile and water were purchased from Thermo Fisher Scientific Inc. (Waltham,
MA, USA) while ascorbic acid, trifluoroacetic acid and
DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent was purchased from Sigma Aldrich Co. (St. Louis, MO, USA).
6.2.3 Sample preparation
Teas were prepared by brewing 5 g of Neem leaf powder or green tea or black tea in 100 mL of de-ionized water for 2 hours at 100 °C. Each solution was then vacuum-filtered through a Fisher brand P8 filter paper. The filtered solutions were then made up to a volume of 100 mL in a volumetric flask using de-ionized water.
Fifteen (15) mL of milk, diluted 2:3 (v/v) with de-ionized water and maintained at approximately 4ºC, was added to 10 mL
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of tea (Neem, green or black tea) in plastic centrifuge tubes at room temperature. The samples were centrifuged at 11,000 x g for 30 mins and the supernatants were made up to 25 mL in volumetric flasks using de-ionized water.
Parallel sets of tea-water preparations, as controls, were made in an identical manner by diluting teas with de-ionized water in order to assess the impact of milk on polyphenol and flavonol recovery.
6.2.4 Preparation of flavonol standards and flavonol extraction
Prior to extraction, tea–water controls and tea-milk treatment solutions were subjected to alcoholic hydrolysis
(Wang and Helliwell 2001) to form the aglycones of the flavonol glycosides. To eight (8) mL of hydrolyzed sample, 12 mL of absolute (100%) ethanol was added to set the ethanol concentration at 60%. For hydrolysis, 5 mL of 6N hydrochloric acid was added to this mixture. The samples were then refluxed using a condenser in a water bath set at 95° C for 2 hours. The hydrolyzed solution was cooled, filtered using Fisherbrand P8 filter paper and made up to 25 mL in a volumetric flask with 60% ethanol. After filtering again, using a 0.22 μm filter, 30 μL was injected into the HPLC.
Flavonol standards myricetin, quercetin and kaempferol were prepared by making stock solutions dissolved in dimethyl sulfoxide (DMSO). Working standard curves ranging from 0-
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0.008, 0-0.032 and 0-0.006 mg/mL were prepared for myricetin, quercetin and kaempferol, respectively.
6.2.5 HPLC analysis
Analysis of flavonols was performed using an Agilent 1100 series HPLC system equipped with a diode-array detector set at 370 nm. An Aqua 250 x 4.6 mm 5 μ C 18, 100 Å (Phenomenex
Inc., Torrance, CA) reverse-phase column was used for the separation of the flavonol aglycones. A gradient elution system comprising of two mobile phases, namely mobile phase
A, 0.1 % trifluoroacetic acid in water and mobile phase B,
0.1% trifluoroacetic acid in acetonitrile, was used. The gradient used was 80% A and 20% B at 0 min which was gradually reduced to 60% A over 27 minutes. A 3 min post time was added for the mobile phase concentrations to come back to initial levels. The flow rate was 1 mL/min and the column temperature was set at 40 °C.
Identification of each compound was accomplished by comparing its retention times with those of chemically pure standards and quantification was based using external standard curves.
6.2.6 Total phenolic content by Folin-Ciocalteau assay
Total phenolic content was determined by the Folin–
Ciocalteu assay as performed by Thaipong and others
(2006). To 50 µL of sample, 250 µL of Folin’s reagent
(1:3 dilution) and 750 µL of 7.5% sodium carbonate along with 3 mL of de-ionized water was added. The solution was vortexed, incubated for 2 hours and absorbance was
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measured at 725 nm using a Varian Cary® 50 UV-VIS spectrophotometer (Agilent Technologies Inc., Santa
Clara, CA). Gallic acid was used to prepare a standard curve with a concentration range of 0.1-0.7 mg/mL and the results were expressed in gallic acid equivalents (GAE).
6.2.7 DPPH Anti-oxidant activity
The method of Thaipong and others (2006) was followed with slight modifications. The stock solution was prepared by dissolving 24 mg DPPH in100mL of methanol, and it was then stored at -20 °C until used. The working solution was obtained by mixing 20 mL of stock solution with 80 mL methanol.
To 2 ml of de-ionized water, 200 μL of sample was added and was allowed to react with 2800 μL of the DPPH solution. The extent of de-colorization was measured at
515 nm after 90 minutes, using a Varian Cary® 50 UV-VIS spectrophotometer. The blank was prepared by using de- ionized water. Percentage (%) DPPH anti-oxidant activity was calculated as follows-
% DPPH activity= (Absorbance of control- Absorbance of sample)/ (Absorbance of control) * 100 (Yen and Duh 1994)
The percentage (%) anti-oxidant values were converted to vitamin C equivalents (VCEAC) by using a standard curve of ascorbic acid that was found to be linear between
0.0176-0.1408 mg/mL.
6.2.8 Statistical analysis
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Three different batches of Neem leaf powder, green and black tea were brewed and each batch was subjected to duplicate analysis.
In order to determine the in-vitro effect of tea matrix and milk type on in-vitro flavonol concentration, total polyphenolic content and % DPPH anti-oxidant activity, an
Analysis of Variance (ANOVA) was conducted according to the procedure of General Linear Model using Minitab 17 statistical software (Minitab Inc., Philadelphia, PA,
USA). Tukey’s post hoc test was used to determine the significant differences of the mean values among treatments (P < 0.05).
6.2.9 Analysis of commercial sample
Twenty-five (25) mL of Taste Nirvana green tea latte was pipetted into a centrifuge tube and subjected to centrifugation for 30 mins at 11,000 x g. Afterwards, the supernatant was made up to volume with de-ionized water in a
25 mL volumetric flask. Eight (8) mL of the sample was hydrolyzed using the exact same procedure as the tea-milk mixtures and control samples.
The pellet was dried in a vacuum oven at 80°C. The dried pellet was transferred onto a round bottom flask and was subjected to alcoholic hydrolysis by adding 20 mL of 60% ethanol and 5 mL of 6N hydrochloric acid.
Both the hydrolyzed solutions of the supernatant and the pellet were made upto volume in a 25 Ml volumetric flask
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and 30 µL was injected for HPLC analysis.
6.3 Results and Discussions
6.3.1 Effect of tea matrix on in-vitro flavonols, total phenolic content and DPPH anti-oxidant activity
The three flavonols of interest, namely myricetin, quercetin and kaempferol were easily separated on a C18 column by RP-HPLC (Figure 1). The amounts of each flavonol for the three tea types are shown in Table 1.
Table 1. Amount of flavonols-myricetin, quercetin and kaempferol in tea samples. Results are expressed in mg/gram of sample (n=3 ± standard deviation)
Samples Myricetin Quercetin Kaempferol
Neem tea 0.83 ± 0.24 4.24 ± 0.52 0.69 ± 0.12
Green tea 0.69 ± 0.05 1.72 ± 0.05 0.66 ± 0.02
Black tea - 1.68 ± 0.18 0.71 ± 0.10
Although myricetin was detectable in black tea, levels were below quantitation limits and hence, not considered for any calculations.
We observed that the amount of tea flavonols recovered from the supernatant was diminished by the addition of milk significantly (p<0.05). The mean decrease in myricetin was similar in green tea and neem tea, with values of 33.3 % and
30.3%, respectively (Figure 2A, 2B and 2C). A similar pattern was seen with both quercetin and kaempferol levels, reducing significantly (p<0.05) within each tea type. But since, this
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decrease was significant (p<0.05) across all three types of tea, we did not see a matrix effect on flavonol levels.
Our results were deduced from the absence of significant interaction (p>0.05) between the factors- ‘tea’ and ‘sample type’, for any of the three flavonols, indicating that the reduction in individual flavonols-myricetin, quercetin and kaempferol, is not significantly affected by the matrix they are present in.
Figure 1. HPLC chromatograms of control (above) and treatment (below) samples
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Figure 2A. Comparative reduction in myricetin due to bovine milk addition in different tea matrices (n=3 ± S.D.)
1.2 a 1 b 0.8 a
0.6
0.4
0.2 Flavonolcontent (mg/g) 0 Green tea Neem tea
Tea-water control Tea-milk treatment
Means that do not share a common letter are significantly different (p<0.05)
Figure 2B. Comparative reduction in quercetin due to bovine milk addition in different tea matrices (n=3 ± S.D.)
5 4.5 4 b 3.5 3 2.5 c c 2 d 1.5 1 0.5
Flavonolcontent (mg/g) 0 Green tea Neem tea Black tea
Tea-water control Tea-milk treatment
Means that do not share a common letter are significantly different (p<0.05)
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Figure 2C. Comparative reduction in kaempferol due to bovine milk addition in different tea matrices (n=3 ± standard deviation)
0.9 a a 0.8 a 0.7 b b 0.6 b 0.5 0.4 0.3 0.2
0.1 Flavonolcontent (mg/g) 0 Green tea Neem tea Black tea
Tea-water control Tea-milk treatment
Means that do not share a common letter are significantly different (p<0.05)
This decrease in flavonols within each tea type, can be attributed to protein-flavonoid interactions (Xiao and others 2011; Serafini and others 2009; Sharma and others
2008; Arts and others 2002). However, it is unknown which protein fraction (alpha, beta or kappa casein) contributes most to the binding effect and hence, is an area of further research. Arts et al. (2002) and Ye et al. (2013) showed that this binding or masking effect is not only dependent on the protein-flavonoid pair but also on the matrix, a phenomenon not seen with flavonols, as evidenced in this study.
The initial phenolic content in the three teas is shown in Table 2.
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Table 2. The total phenolic content of various tea samples as determined by the Folin’s assay. The results are expressed in mg GAE/gram of the sample (n=3 ± standard deviation)
Samples Total phenolic content
Neem tea 28.73 ± 0.81
Green tea 114.48 ± 2.27
Black tea 56.30 ± 6.59
The total phenolic content showed significant (p<0.05) reduction in the supernatant of the green and black tea- milk mixtures of 26.2 and 29.4 %, respectively. However, we did not see any such decrease in neem tea, indicating a matrix effect (Fig.3). According to a study by Ye and others (2013), the binding affinities of tea catechins with milk protein fractions are affected by the coexisting phenolics in the compound system. This explains the matrix effect with regards to the total phenolic content but fails do so in the case of individuals flavonols. One of the major bio-active compounds in neem are a group of triterpenoids called limonoids(Parida and others 2002).
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Fig.3. Reduction in total phenolic content on addition of bovine milk to different tea matrices (n=3 ± standard deviation)
140 a 120
100 b
80 c 60 e e d 40
20
0 mg/gGallic equivalent acid Green tea Neem tea Black tea
Tea-water control Tea-milk treatment
Means that do not share a common letter are significantly different (p < 0.05)
The pattern of decrease in DPPH anti-oxidant activity in the three teas is commensurate with the reduction in phenolics, as total phenolic content and anti-oxidant activity are highly co-related (Zhao and others 2014; Bhoyar and others 2011). The DPPH anti-oxidant activity of neem, green and black tea samples are provided in
Table 3. The DPPH activity decreased significantly in both green and black tea by 22.4 and 23.3 %, respectively
(Fig.4). But, this reduction (16.5%) was not found to be significant in neem tea. This observation may be due to the lack of binding of milk proteins with limonoids, a group of triterpenoids present in high quantities in neem
(Parida and others 2002). Although limonoids are weaker
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anti-oxidants compared to flavonoids due to fewer number of hydroxyl groups (Yu and others 2005), its high concentration in neem (Champagne and others 1992) makes these compounds a potent contributor towards the overall anti-oxidant capacity. The minimal loss of phenolics and hence, anti-oxidant activity in neem tea is perhaps due to the low levels of phenolics in neem that leads to a low phenolic/protein ratio. The low phenolic/protein ratio, as suggested by Prigent and others
(2003), affects the protein-phenolic interaction .
Table 3. The DPPH anti-oxidant activity of various tea samples. The results are expressed in mg of vitamin C equivalent/ gram of sample (n=3 ± standard deviation)
Samples Anti-oxidant activity
Neem tea 17.90 ± 0.47
Green tea 165.40 ± 2.24
Black tea 63.90 ± 4.83
Our results also indicate no quantitative difference in the decrease of phenolic content and DPPH values in the supernatant of green and black tea. This is in contradiction to the results of Dubeau and others (2010) in which the authors suggest that larger polyphenols such as thearubigins and theaflavins in black tea, have a greater binding affinity for milk proteins as compared to smaller polyphenols.
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Figure 4. Reduction in DPPH anti-oxidant activity on addition of bovine milk to different tea matrices (n=3 ± standard deviation)
180 a 160 - b 140
120
100
80 c d 60
oxidantactivity 40 e e 20
mg/gVitamin C Equivalentanti 0 Green tea Neem tea Black tea
Tea-water control Tea-milk treatment
Means that do not share a common letter are significantly different (p < 0.05)
6.3.2 Effect of different types of milk on flavonol binding, total phenolic content DPPH anti-oxidant activity
Three types of milk, bovine, soya and almond milk were used in our analysis and their nutritional differences have been tabulated in Table 4.
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Table 4. Nutritional differences between skimmed milk, soya milk and almond milk. All amounts are per serving (240 mL)
Nutrient Skimmed Soya milk Almond milk milk
Total fat 0 g 1.5 g 2 g a) Saturated fat 0 g 0 g 0 g b) Polyunsaturated - 1 g - fat c)Monounsaturated - 0.5 g - fat
Cholesterol 5 mg 0 mg 0 mg
Sodium 120 mg 135 mg 95 mg
Potassium - 340 mg 35 mg
Total carbohydrates 11 g 5 g 8 g a) Dietary fiber 0 g 1 g 0 g b) Sugars 11 g 3 g 8 g
Protein 8 g 6 g 5 g
We observe that myricetin, quercetin and kaempferol reduce significantly (p<0.05) in bovine and almond milk but not in soya milk (Fig. 5A, 5B and 5C). Hence, in the two-way ANOVA analysis, the two factors- ‘milk type’ and
‘sample type’ show significant interaction (p < 0.05) for quercetin and kaempferol but not for myricetin (p >0.05 or p=0.087). This indicates that the type of milk added affects the decrease of quercetin and kaempferol in the tea-milk supernatant.
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Figure 5A. Comparative reduction in myricetin due to the addition of different types of milk to green tea = (n=3 ± S.D.) 0.9 a a 0.8 a ab 0.7 bc 0.6 c 0.5 0.4 0.3 0.2
0.1 Flavonolcontent (mg/g) 0 Bovine milk Soya milk Almond milk
Tea- water control Tea- milk treatment
Means that do not share a common letter are significantly different (p<0.05)
Figure 5B. Comparative reduction in quercetin due to the addition of different types of milk to green tea = (n=3 ± S.D)
2 a a a 1.8 a 1.6 b 1.4 b 1.2 1 0.8 0.6
0.4 Flavonol Flavonol (mg/g)content 0.2 0 Bovine milk Soya milk Almond milk
Tea- water control Tea- milk treatment
Means that do not share a common letter are significantly different (p<0.05)
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Figure 5C. Comparative reduction in kaempferol due to the addition of different types of milk to green tea = (n=3 ± S.D) 0.8 a a a 0.7 a bc 0.6 c 0.5
0.4
0.3
0.2
0.1 Flavonolcontent (mg/g) 0 Bovine milk Soya milk Almond milk
Tea- water control Tea- milk treatment
Means that do not share a common letter are significantly different (p<0.05)
The recovery of the total phenolic content in the tea-milk mixture supernatant is dependent on the type of milk added. While bovine and almond milk show significant (p < 0.05) decrease in total phenolics, this was not seen for soya milk, which is in consonance with the behavior of individual flavonols- quercetin and kaempferol. Also, the decrease seen for bovine milk was quantitatively more than almond milk (Fig.6).
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Fig.6. Reduction in total phenolic content on addition of different types of milk to green tea (n=3 ± standard deviation) 140 a 120 a a a
100 b c 80
60
40
20
mg/g mg/g Gallic acidequivalent 0 Bovine milk Soya milk Almond milk
Tea-water control Tea-milk treatment
Means that do not share a common letter are significantly different (p<0.05)
A similar phenomenon was seen for DPPH anti-oxidant activity, where the decrease was the least and statistically insignificant (p>0.05) for soya milk
(Fig. 7). Addition of bovine and almond milk led to significant decreases (p< 0.05) although there were no significant differences (p>0.05) in the quantum of decrease.
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Figure 7. Reduction in DPPH anti-oxidant activity after the addition of different types of milk to green tea (n=3 ± S.D.)
180 a a a a 160 b 140 b 120 100 80 60
40 oxidantactivity
- 20 0
anti Bovine milk Soya milk Almond milk mg/gVitamin C Equivalent Tea-water control Tea-milk treatment
Means that do not share a common letter are significantly different (p<0.05)
Our results are partly in consonance with those of Ryan and
Sutherland (2011) , who observed higher antioxidant values
(FRAP) or no change after the addition of soya milk to black tea, as compared to semi-skimmed bovine milk.
According to previous literature (Ryan and Petit 2010;
Ferruzzi and Green 2006), increase in protein content led to higher protein-phenolic interaction. Our study contradicts these observations, because despite having the highest protein content of 8 g, the recovery of phenolics after the addition of bovine milk is comparable to that of almond milk with 5 g of protein. Soya milk with 6 g of protein, shows higher recovery than both bovine and almond milk defying previous observations on protein-phenolic interactions.
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This unusual variation in our observations can be explained by three reasons. Firstly, the presence of higher protein content may not ensure more precipitation and thus, lower recovery. An important aspect to consider would be the casein/whey protein (insoluble/soluble protein) ratio. As observed by Ye and others (2013), the polyphenols bind differently to the casein and the whey protein fraction.
Therefore, an analysis into the soluble and insoluble fractions of the different milk proteins could provide us with a cogent explanation.
Another factor that could contribute to our observation, is the different chemistry of the proteins involved.
Although the composition of soya milk is complex, conglycinin and glycinin represent the majority of soy proteins (Riblett and others 2001) ,while there are soy proteins that are rich in the sulfur amino acid- methionine (Young 1991). Polyphenols bind to the proline rich regions of conglycinin and glycinin
(Maruyama and others 2004) with subsequent complexation
(Luck and others 1994). However, our results suggest that the binding of polyphenols to soya proteins is not similar quantitatively to bovine milk proteins.
The almond milk used in our study was fortified with unknown proportions of pea and rice protein. There is not enough literature about rice and pea protein
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interaction with polyphenols which can help explain our results and therefore, needs to be researched.
Thirdly, there can be extraneous factors such as the presence of fat in soya and almond milk that could affect the protein-polyphenol interactions.
Serafini and others (2009) propounded that longer peptide chains, the formation of which is apparently associated with increasing fat content, could result in increased polyphenol complexation perhaps similar to the interactions described between β-lactoglobulins and phenolic acids (Stojadinovic and others 2013). The role of fat content on the anti-oxidant effect is unclear.
While Langley-Evans (2000) has shown that high-fat milk digested in vitro with black tea had a more pronounced negative impact on antioxidant capacity than low fat milk,
Ryan and Petit (2010) contradicted them by observing that the lower fat content of skimmed milk had a more drastic reduction in anti-oxidant activity due to its lower content of milk polyphenols.
The decrease in the anti-oxidant activity is consistent with the observations of Gallo and others (2013);
Arts and others (2002); Dubeau and others (2010) and
Sharma and others (2008), in which the authors suggested a masking effect of bovine milk protein components on polyphenolic components. The consonance between the phenolic recovery and anti-oxidant levels can be
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explained by the fact that the antioxidant ability of the polyphenol–milk protein complex does not depend on the sum of the antioxidant ability of polyphenol molecules bound but simply on the number of polyphenols unbound Xiao and others (2011).
6.3.3 Analysis of commercial samples
Unlike the tea- milk model mixtures, we did not observe any flavonols in the supernatant. Instead all the flavonols were isolated form the dried pellet.
The amount of myricetin, quercetin and kaempferol recovered from the pellet was 0.01 ± 0.00, 0.19 ± 0.04 and 0.13 ± 0.03 mg/280 mL of the sample.
This may be due to the presence of various other components such as fats, sugars and dietary fibers, all of which may lead to the binding of flavonols.
6.4 Conclusions
From a product development perspective for Ready to
Drink (RTD) beverages, striking the ideal balance between protein and flavonoid content is the key.
From our studies, we observe that the recovery of polyphenols and the preservation of anti-oxidant activity is the highest for neem tea and for soya milk among different tea and milk types, respectively. Therefore, neem tea-soya milk based beverages can play an important role in the growth of the RTD beverage industry.
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Future direction of research
Our study focused on neem leaf and bark and the elucidation of their bio-active potential. While we restricted our research to flavonols, other groups of health promoting compounds such as phenolic acids, flavones, flavan-3-ols and flavanones are probably present in neem. Obtaining qualitative and quantitative information about these compounds would give us a better idea of the bio-active profile of neem. Furthermore, this profiling can be extended to neem seeds, flowers and twigs which have also found use in ancient medical practice.
The neem leaf powder bought from Neem Tree Farms in
Florida is sourced from India and Mexico while the fresh
Neem leaves are grown in Florida. Since the suppliers are able to provide this information today, one can perform a study to see if a distinction can be drawn between neem samples from different countries based on their chemical constitution. The results from a source/origin based LC-
MS and GC-MS profiling of neem would be valuable to the cultivators of neem in these countries helping them in adopting practices that would enhance neem’s bioactive potential.
Counteracting the bitterness of neem is essential in making it more palatable and is a key aspect of expanding
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its commercial applications. The bitter principle in neem comprises of a group of compounds called limonoids.
Although neem contains over 3,000 different limonoids and their derivatives, attempts must be made in isolating as many limonoids as possible, by trying to remove them selectively to understand the effect of each limonoid on the bitterness and bio-activity of neem. A big step forward would be the ability to isolate the bitter compounds individually and remove them selectively in order to preserve or cause minimal loss to the overall bio-activity of neem.
The ideal bitterness reducing strategy is the one which is not only effective in reducing bitterness but is able to do so with minimal cost and least damage or loss to the bioactive nutrients and organoleptic properties of the food/beverage in question. Our study was able to successfully remove the bitterness from neem but had the disadvantage of causing nutritional and sensorial loss to neem tea. While the results for Amberlite XAD-16 were more encouraging than those for SPE, there was still some loss in polyphenols, anti-oxidant capacity, color and volatiles, which can perhaps be averted by exploring other strategies of de-bittering. These may include the use of other styrene/divinylbenzene polymer resins such as Amberlite XAD-7 HP, XAD-16HP and Dowex-L285; cyclodextrins; enzymes catalyzing the conversion of
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limonin from immobilized bacterial cells and altering cultivation practices to ensure lesser production of bitter limonoids.
Understanding the effect of de-bittering on sensorial properties of neem tea is an area which needs more understanding. While our study conclusively showed that the debittering procedure led to a significant decrease in bitterness, it did not provide us information on the effect on volatiles of neem tea. A descriptive sensory panel with extensive training is required for this purpose as an untrained panel cannot reveal any useful information.
The growing popularity of ready to drink beverages
(RTD’s) has demanded a better understanding of milk protein-flavonoid interactions which may ultimately affect the bio-availability of flavonoids in-vivo. Our study expands on the knowledge gained about the behavior of catechins (flavan-3-ols) to the interaction of flavonols with different milk proteins-bovine, soy and almond. With the increased usage of plant-based milks in
RTD’s, it will be knowledge-worthy to expand the study to various other plant-based milks available in the market place such as rice, flaxseed and coconut milk. While our study encompassed the applied aspects of tea-milk interaction, it would be worthwhile to extend the study to answer some of the basic questions involved. These
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include exploring the mechanisms of interaction between proteins in soya, almond, rice and flaxseed milk with different flavonoids in tea because our understanding of flavonoid-protein binding studies is so far limited to bovine milk proteins. One interesting observation from our study was that although the addition of bovine milk to neem tea did not affect the amount of polyphenols in the supernatant, we did see an overall decrease in the anti-oxidant capacity. We suspect that this might be due to the binding of casein with limonoids. This behavior is very poorly understood and requires further research.
The role of milk fat in affecting the anti-oxidant capacity of tea-milk systems is also controversial. A previous study had suggested that reducing the fat content of bovine milk leads to a significant reduction in the anti-oxidant values because of a decrease in fat soluble anti-oxidants, such as carotenoids, tocopherols and retinols. However, these results were contradicted by another author observing that the highest decrease in
FRAP anti-oxidant values were due to the addition of whole milk as compared to semi-skimmed or skimmed milk.
They suggest a direct interaction between tea catechins and milk fat similar to the interaction between tea catechins and lipoprotein fractions in human plasma. Yet others have suggested that milk fat leads to the formation of longer peptide chains, thereby increasing
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protein-polyphenol interactions. The effect of fat in plant based milks on polyphenols in different tea matrices and its effect on the in-vitro anti-oxidant activity is an area of interest for us.
Besides fat, the role of sugars and dietary fibers is a direction of research that can be pursued to gather more knowledge about RTD’s.
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VITA
Abhinandya Datta was born in Kolkata, India on 17th
January, 1987. He earned his Bachelor of Science degree in Biochemistry at Rama Krishna mission Vivekananda
College, University of Madras, India in 2008. He continued his graduate study in Biochemistry by pursuing a Master of Science degree in Medical Biochemistry at
Manipal University, Karnataka, India. He graduated from this course in 2011. In the fall of 2011, he joined the
Ph.D. program in the department of Food Science at the
University of Missouri, Columbia. He received his doctoral degree in 2014.
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