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1 VYTAUTAS MAGNUS UNIVERSITY FACULTY OF NATURAL SCIENCES DEPARTMENT OF BIOLOGY NAME SURNAME AKASH CHANDRAHAS SHAMBHARKAR COMPARATIVE ANALYSIS OF BIOLOGICALLY ACTIVE COMPOUNDS IN EVERYDAY USED SPICES ALLIUM SATIVUM L., ELETTARIA CARDAMOM L., MYRISTICA FRAGRANS HOUTT, CINNAMOMUM ZEYLANICUM NEES. Master thesis Molecular Biology and Biotechnology Study program, Study code 621C71001 Biomedical sciences (Molecular Biology) Supervisor: Prof. Habil. Dr. Audrius Maruska... Defence: Prof. Habil. Dr. Saulius Mickevičius. KAUNAS, 2018

2 CONTENTS ABBRIVATION...4 SUMMARY...5 SANTRAUKA...7 LIST OF FIGURES...8 LIST OF TABLES INTRODUCTION LITERATURE ANALYSIS Ancient medical system Role of spices in traditional medical system Major chemical groups present in spices Phenolic compounds Flavonoid compounds Free radical scavenging activity Essential oils UV-spectrophotometer Gas chromatography High performance liquid chromatography MATERIALS AND METHODS Materials Chemicals and solvents Sample collection

3 3.1.3 Instruments Methods Preparation of samples Preparation of extracts Prepartion of suspension (QUENCHER) Determination phenolic content in extracts Determination of flavonoid content in extracts Determination of radical scavenging activity in extracts Determination of phenolic content in suspension (QUENCHER) Determination of flavonoid content in suspension (QUENCHER) Determination of radical scavenging activity content in suspension (QUENCHER) Gas chromatography mass spectrometry analysis Gas chromatography flame ionization detection Determination of polyphenolic by high performance liquid chromatography Statistic analysis RESULTS AND DISCUSSION Phenolic content in extracts sample Flavonoid content in extract sample Radical scavenging activity in extract sample Phenolic content in suspension sample (QUENCHER) Flavonoid content in suspension sample (QUENCHER) Radical scavenging activity in suspension sample (QUENCHER) Identification and quantification of essential oil by gas chromatography maas spectrometry

4 4.8 Identification and quantification of essential oil by gas chromatography flame ionization detection Polyphenolic determination by high performance liquid chromatography Gas chromatography CONCLUSIONS REFERENCES SUPPLEMENT

5 ABBRIVATIONS GC-MS GC-FID HPLC TFA DPPH Gas chromatography mass spectrometry Gas charomatography flame ionization detector High performance liquid chromatography Trifluoroacetic acid 2,2-diphenyl-1-picrylhydrazyl QUENCHER Quick easy new cheap reproducible (Suspension based sample prepartion) KTM Korean traditional medicine 5

6 SUMMARY Author of diploma paper: Akash Chandrahas Shambharkar Full title of diploma paper: Comparative analysis of biologically active compounds in everyday used spices Allium sativum, Elettaria cardamom, Myristica fragrans, Cinnamomum zeylanicum. Diploma paper advisor: Prof. Habil. Dr. Audrius Maruska Presented at: Vytautas Magnus University, Faculty of Natural Sciences, Kaunas, 2018 Number of pages: 72 Number of tables: 14 Number of pictures: 35 6

7 SUMMARY The study was conducted to identify and determine the concentration of biologically active compounds in daily used spices. The spices contain various category of chemical compound which has a health benefits against the various types of chronic disease like cancer, Alzheimer, blood pressure, hyperlipidemia and most of the infection diseases. Comparative study of biological active components of different family spices namely garlic, cardamom, nutmeg and cinnamon were done. It collected from local market source and investigated with the help of different analytical techniques i.e. UVspectrophotometry, gas chromatograph, liquid chromatography etc. The obtained result investigation of these spices have shown that great source of biologically active compounds includes polyphenol, flavonoids, radical scavenger and essential oils, which is useful for medicinal purpose as well as in dietary. 7

8 SANTRAUKA Tyrimas buvo atliktas siekiant nustatyti biologiškai aktyvias medžiagas kasdien naudojamuose prieskoniuose. Prieskoniai turi įvairių cheminių junginių, naudingų sveikatai, veikiančių prieš įvairių tipų lėtine ligas, vėžinius susirgimus Alzheimerio ligą, kraujospūdį, hiperlipidemiją ir infekcines ligas.. Darbe atlikta lyginamoji analizė biologiškai aktyvių komponentų skirtinguose prieskoniuose t.y. česnake (Allium sativum, L), kardamone (tikrasis kardamonas), muskato riešute (kvapusis muskatmedis houtt), cinamone (Cinnamomum zeylanicum Nees). Žaliava isigyta iš vietinio prekybos tinklo ir tirta įvairiais analizės metodais, t.y. UV-spektrometrija, dujų chromatografija, skysčių chromatografija ir kt. Gauti rezultatai parodė, kad prieskoniai yra puikus biologiškai aktyvių junginių šaltinis, kuris apima polifenolius, flavonoidus, radikalus surišančius junginius ir eterinius aliejus, kurie yra naudingi medicinos tikslais, taip pat kaip maisto sudedamosios dalys. 8

9 LIST OF FIGURES 1. Classification of phenolic compounds 2. Structure of flavonoid 3. Schematic diagram of general principal of UV-spectrophotometry 4. Schematic diagram of working principal of gas chromatography 5. General GC-MS procedure 6. General GC-FID procedure 7. Schematic diagram of working principal of HPLC 8. Used spices in research study 9. General procedure of sample prepartion for extracts 10. General procedure of sample prepartion for suspension (QUENCHER) 11. Graphical presentation of TPC in extracts 12. Graphical presentation of flavonoid in extracts 13. Graphical presentation of RSA in extracts 14. Graphical presentation of TPC in suspension 15. Graphical presentation of flavonoid in suspension 16. Graphical presentation of RSA in suspension 17. GC-MS chromatogram of garlic 18. Graphical presentation of garlic 19. Graphical presentation of cinnamon 20. Graphical presentation of nutmeg 21. Graphical presentation of cardamom 22. Chromatogram of nutmeg 23. Chromatogram of garlic 24. Chromatogram of cardamom 25. Chromatogram of cinnamon 26. Calibration curve of rutin 27. Calibration curve of rutin 28. Calibration curve of rutin 29. Calibration curve of rutin 30. Calibration curve of rutin 31. Calibration curve of rutin 9

10 32. Calibration curve of rutin 33. GC-MS chromatogram of cinnamon 34. GC-MS chromatogram of cardamom 35. GC-MS chromatogram of nutmeg 10

11 LIST OF TABLES 1. common use of spices 2. Ancient medicinal system 3. Spices incorporated with ancient medicinal system 4. Major chemical group present in spices 5. Chemicals used in research study 6. Investigated spices sample 7. List of equipments used in study 8. Rutin absorbance in TPC for extracts sample 9. Rutin absorbance in flavonoid for extracts sample 10. Rutin absorbance in RSA for extracts sample 11. Rutin absorbance in TPC for suspension sample 12. Rutin absorbance in flavonoid for suspension sample 13. Rutin absorbance in RSA for suspension sample 14. Rutin absorbance for HPLC 11

12 1. Introduction Spices and herbs have played a vital role in the modern civilization of mankind and in the history of a particular nation. The delightful flavor and pungency of spices make them indispensable in the preparation of palatable dietary. It has been used from very ancient time in the culinary process. In addition, they are reputed to possess several medicinal and pharmacological properties and hence find a position in the preparation of a various number of medicines. Basically, spices are the dried substance or sometimes it s a fresh part of specific plants. Spices either whole use or in the form of extract. It has been used frequently throughout history in the Ayurvedic system (ancient Indian medicine system), Unani system and Traditional Chinese medicine, for its good medicinal benefit. In ancient time, spices blend with other herbal material depending upon the desired medicinal properties. Throughout the history spices and its trade has been always contributed to the global economy. The current global production of spices estimated 8.5 million tons and this figure is very important as a primary source for the many developing countries. In the past centuries, the spice trade was globally important and it helped to make the connection between different continents in the world. The spice trade economically important and it was associated Asia, Middle East, Europe, America, and Africa. The spices production and consumption rate in India is highest, usually Indian and sub-continent people, they frequently used spices in their cuisine. It increases the flavor, color, and texture of the food and sometimes it used for the preservation purpose. India is a leading edge in the production of spices, 75 % of the spices produced in only India, along with it few percentages of India s economy depends upon the spices. According to the surveys, India is the global biggest producer of the spices and highly capable of exporting their spices into the world market. In comparison with the different continent, there are some common spices like Nutmeg, Garlic, Onion, Paper, Ginger, Cardamom, Cinnamon, Turmeric etc. The spices have been used in our day to day life in dietary for coloring, flavoring and increasing the food texture, along with the taste it has some potential health benefits. The spices contain lots of bioactive compounds, which is used in various disease and disorders like cardiovascular disease, cancer, diabetes, blood pressure, atherosclerosis, and hyperlipidemia. It highly praised by several authors as well as it is used as herbicidal and preservative and this bioactive compound present different concentration from a different family of spices. Generally, spices contain Volatile and Non-Volatile oils which include Alkaloids, Glycosides, Phenolic, Vitamins, Resin and Antioxidants which is necessary for the human body for the normal growth and maintains. The concentration of bioactive compounds is directly 12

13 proportional to the biological and pharmacological activity. High concentration leads to more beneficial and more desire pharmacological action. These spices having the very rich source of chemical compounds with great biological activity and often used in pharmaceutical, cosmetic, food and perfumery industries There are so many herbs use as medicinal plants even spices have play vital role in the treatment of various disease. The Garlic contain various essential compounds like, zinc, potassium, calcium, magnesium, vitamin (A, B1, C). It contains 33 types of sulfur compound and 17 amino acids(el- Bayoumy et al. 2006). The Garlic has ability to lower level of cholesterol as well as decrease lipid peroxidation that leads to inhibit plaque formation in artery and heart. This suppress low density lipoprotein and developed resistance against the LDL oxidation (Thomson et al. 2006). The mechanism of Garlic to stimulate the immune factor that is, T-cell and natural killer cell and it is show the synergistic effect combine with others anticancer drug(lamm and Riggs 2001). Other clinical uses including treatment in cardiovascular disease, natural immunity booster, diabetes, antibacterial (antifungal and antiviral)(thomson et al. 2006)(Jonkers et al. 1999). Nutmeg which have several clinical applications. The nutmeg contains myristicin which is active chemical ingredient and it is use as psychotropic agent(williams and West 1968). Table- 1. Common uses of spices Sr. no Name of spices Uses 1 Garlic Garlic contains a compound i,e ; Allicin, which has a potent medicinal properties like antioxidant and use against the cancer(thomson et al. 2006). It is closely related to the onion and it grows in many parts of the world. This is popular ingredient in cooking due to its smell and delicious taste. 2 Cinnamon Cinnamon is world recognized spice in food stuff and it has some good medicinal properties like anti-inflammatory, antioxidant, protect heart health etc(miller et al. 2002). 3 Nutmeg It is full of essential nutrient, vitamin, mineral. It is use in various disease and disorder insomnia, skin problem, teeth pain, joint pain, headache etc. (Williams and West 1968). 13

14 4 Cardamom Basically, cardamom is use as flavoring agent in food stuff for its good flavor and taste. Along with the taste, it has some medicinal benefits. This is a counteract digestive problems and also use in nausea, acidity loss of appetite(khan, Khan, and Gilani 2011). Apart from the known parameter of this spices, the spices were extensively investigated for the health benefit, which was resulted in more than 2000 publication over the last two decades which is proved that, this main area of research and many of research trying to investigate more about the spices. It is a significant challenge to the analytical chemist to identify and measure concentration the biological and chemical active compounds by using an instrumental analysis technique. MAIN GOAL OF THE WORK: Comparative analysis of composition and biological active compounds in everyday used spices Allium sativum (Garlic), Elettaria cardamomum (Cardamom), Myristica fragrans houtt (Nutmeg), Cinnamomum zeylanicum nees (Cinnamon) TASKS OF THE WORK: 1) Perform the spectrophotometry to quantitate different non-volatile compounds: a) Total phenolic content, b) Flavonoids, c) Radical scavenging activity, 2) Evaluate quantitative and qualitative analysis of essential oils in spices. 3) To perform qualitative and quantitative composition of phenolic compounds using HPLC. 4) To compare the results of different spices. 14

15 2 Literature review 2.1 Ancient medicinal system In the beginning of millennium AD there were principal medical system Ayurvedic, Unani and Traditional Chinese medicine, all they were deal with the herbs and animal. Throughout the history of civilization, humans have been totally dependent on the plant and animal for their survival, in the case of food, shelter and cloths, later they realized that some herbs and spices have potential to cure the disease. At the beginning they used some oils and plants extracts to treat the disease. In oldest medicine system there were few medicine systems which was often used, like Ayurvedic Medicine system, Traditional Chinese Medicine system and Unani. Ayurvedic system originated in India in 4000 BC, it derived from Sanskrit word Ayur (life) Veda (science), the Ayurveda means science of life(siraj Mays,2011). The Ayurveda speeded in China through the Tibet and they called it Traditional Chinese medicine. They have been started using around 2000 BC and still it exists(liu et al. 2014). In Arabic world, there was another Ancient medicine that is Unani, it evolved with beginning of Arab civilization, it spread Middle East, Turkey and Greece.(Halberstein 2005)(Koçyiğit 2005). In modern world still various universities carried research on ancient medicinal system. Table- 2. Ancient medicinal system Source: (Yuan et al. 2016). Name Origin Year Recent status Ayurveda India 4000 BC Indian Government has an official body to ensure Ayurvedic educational efforts, quality and practice Traditional Chinese medicine (TMC) China 2000 BC TMC still exists everyday health care and user of TMC are increasing day by day 15

16 Unani Greco Arabic 2500 BC Unani acknowledge by the WHO and still it is used in practice Kampo Japan 6 th century It used along with the modern medicine Traditional Korean Medicine (TKM) Korea 19 th century TKM formula made by the certified practitioner and Korean government are fighting for the TMK survival 2.2 Role of spices in traditional medicine Several statements indicated that spices represent the oldest and widespread form of medication. In the recent era various medicine directly derived from the plant and animal sources (Eigner and Scholz 1999). Despite of synthetic medicine many tribal people still believed in natural medicine, as a natural medicine they used various varieties of herbs, spices, animal organs etc. (Halberstein 2005). In Ayurveda turmeric was used antiseptic to treat small cuts and wounds (Eigner and Scholz 1999), ginger was used in Chinese medicine to treat cough and joint pain (Cai et al. 2004; Wojdyło, Oszmiański, and Czemerys 2007). In traditional medicine, spices were the foremost ingredient, from the ancient era spices has been used against the various disease and disorder like Piper Nigrum sued as Antibacterial on topical wound (Reddy et al. 2004). There are various traditional medicinal plants and spices still exists in modern world for their potential benefits and most of the researcher are investigating bioactive compounds from spices to find out potential uses of for human health (Reddy et al. 2004)(Basile et al. 2009). 16

17 Table- 3. Spices incorporated with Ancient medicinal system. Name of Spices Medicinal system Ginseng Ginkgo Wolfberry Ginger Star anise Nutmeg Cinnamon Saffron Basil Black paper Cumin Clove Turmeric Coriander Cardamom Mint leaves Fennel Sesame oil Garlic Traditional Chinese Medicine Traditional Chinese Medicine Traditional Chinese Medicine Traditional Chinese Medicine/ Ayurveda/TKM Traditional Chinese Medicine Traditional Chinese Medicine/ Ayurveda Traditional Chinese Medicine/ Ayurveda/Unani Ayurveda/Unani Ayurveda Ayurveda/Unani Ayurveda Ayurveda/Unani Ayurveda/Unani Ayurveda Ayurveda Ayurveda Ayurveda TKM TKM/ Ayurveda Source- (Corson and Crews 2007)(Embuscado 2015)( Ayurveda -A Glance Ayurveda - A Glance 2011) 2.3 Major chemical groups in spices The spices have been playing one of the most important dietary and medicinal role in human s life for a century (Bozin et al. 2008). They are very rich source of important biologically active compounds which is used as the purpose of medicine to cure various disease (Reddy et al. 2004; Basile et al. 2009; Online et al. 2006). There are various common chemical groups which contain in the spices and this chemical 17

18 groups responsible for the pharmacological action (Kumar and Navaratnam 2013) (Shan et al. 2007). In most of the spices contain the Alkaloids, Glycosides, Flavonoids, Tannins, Phenolics, Resins, vitamins, minerals, amino acids. It also contains various Volatile oils and Non-Volatile oils in different concentrations (Gokhale, Mr SB. Pharmacognosy 2008) (Zachariah 2008). Table- 4. Major chemical group present in spices. Sr. no Spice Name Major Chemical group 1 Garlic Phenolic/ Flavonoids/ Saponin/ Proteins/ volatile oils 2 Nutmeg Alkaloids/ Essentials oil/ Polyphenol 3 Cinnamon Polyphenols/Tannins/Alkaloids/Sterol/Saponins/ Volatile oils 4 Cardamom Alkaloids/ Sterol/ Terpenoids/ Volatile oils These are the common chemical groups which is often present in spices. However, alkaloids are the potent psychoactive substance which was derived from the natural sources. The several known substances belong to alkaloids, such as caffeine, cocaine, nicotine, theobromine (Gokhale, Mr SB. Pharmacognosy 2008). All the spices possessed the pharmacology activity. Nowadays, most of the researcher are focusing on the natural products and trying to investigate more about the spices, because it has been using traditional medicine systems. 18

19 2.3.1 Phenolic compounds Phenolic compounds are the most important antioxidants in diet and it present in large quantity. The main source of polyphenol are fruits, vegetable, cereals, chocolate, legumes, spices as well as beverages such as juice, tea, coffee and wines (Manach et al. 2005). The spices play one of the important role as a source of phenolic compounds(mantas Stankevičius et al. 2011; Proestos et al. 2006).The intake of polyphenols in diet should be high 1gm/day. The phenolic compound divided into various category such as polyphenol, phenolic acid, tannins, flavonoids etc. Figure 1. classification of phenolic compound Source- (Gokhale, Mr SB. Pharmacognosy 2008) They all phenolic groups has a importance in human health and it has capability to prevent the chronic disease(santhosha, Jamuna, and Prabhavathi 2013; Bozin et al. 2008). Flavonoids are a group of polyphenolic compounds isolated from a wide range of plants(gopalakrishnan 1992; de Araújo et al. 2014). Their antioxidant properties and effects in diseases prevention were not truly been studied until 19

20 1995 (Manach et al. 2005). These phenolic compounds useful against the chronic disease such as cardiovascular disease, inflammation, oxidative stress. Overall, it reduces the risk factor of human health Flavonoids Flavonoids are the one of the most important class of phenolic compound. Flavonoids have their basic structure: the flavan nucleus (Figure 2.), which consists of 15 carbon atoms arranged in three rings (C6- C3-C6), which are labeled A, B, and C. Flavonoids may be further divided into several subclasses, i.e., flavones (and isoflavones), flavanones, flavonols, flavanols (also called catechins), and anthocyanidins (Lotito and Frei 2006). Studies found that consumption of flavonoid-rich foods, in particular fruits and vegetables, is associated with a lower incidence of heart disease, stroke, cancer, and other chronic diseases (Lotito and Frei 2006). It has been suggested that the health benefits of dietary flavonoids are due to their antioxidant properties(de Araújo et al. 2014). Many foods in human diets such as vegetables, fruits, tea and wine, contribute to the total daily intake of flavonoids in human. Figure 2. structure of Flavonoid The flavonoids sorted due to the adjusting atom present on the heterocyclic ring. This heterocyclic ring possessed the affinity towards the various chemical groups and shows the specific behavior of activity. The commonly known flavonoid compounds such as quercetin, kaempferol, fisetin, luteolin, apigenin, pachypodol, myricetin etc. 20

21 2.3.3 Free radical scavenging activity Free radical formed during the metabolic processes and it has ability to trigger the peroxidation of membrane lipid and leads to the accumulation of lipid peroxide which is associated to aging and other various disease. Antioxidant are the compounds that obstruct or restrain the oxidation of the other molecule by obstructing the commence or propagation of oxidizing chain reaction(de Araújo et al. 2014). The key role of antioxidant is to induce the mechanism of protection against the damage caused by reactive oxygen and degenerative disease(coskun et al. 2005). Free radicals and some causative species attack on lipid, protein DNA and urge their oxidation which resulted into oxidative damage(coskun et al. 2005) and oxidative damage related with an elevated risk of chronic disease, such as cardiovascular morbidity and neoplasm (Ninfali et al. 2005; Valko et al. 2006). Antioxidant has a high potential to promote the protection mechanism and it can protect human body from oxidative damage by adsorbing and neutralizing free radical, quenching singlet and triple oxygen, or deactivating peroxide by their redox properties(bouayed and Bohn 2010).The main source of polyphenol are fruits, vegetable, cereals, chocolate, legumes, spices as well as beverages such as juice, tea, coffee and wines (Manach et al. 2005).The most important groups are polyphenolics, such as flavonoids including flavonols, isoflavones, and anthocyans, also some pigments like chlorophyllin, phytosterines and allysulfides were found which possessed antioxidant activities. Vitamins are the prototype example of radical scavenger which in vitamin A, C, E and folic acid. It acquires the health benefits and protect the human health from the various risk factor which is harmful to human health Essential oil Plant derived antimicrobial agents are becoming increasingly popular as food, cosmetic and pharmaceutical preservatives. Essential oils isolated from widely used spices and their plant sources contain compounds known for their antimicrobial activity (Dorman and Deans 2000). Oils containing specific phenols are generally the most effective for antimicrobial application, including those from clove, oregano, and thyme (Dorman and Deans 2000) (Holley and Patel 2005). Many of these oils are most effective against Grampositive organisms, though some are more antagonistic towards Gramnegative species (Holley and Patel 2005). Additional derivatives of these oils may be non-phenolic in nature but still exhibit profound antimicrobial activity, such as allyl isothiocyanate, which may be used against Gram-negative bacteria and fungi (KYUNG and FLEMING 1997). 21

22 2.4 UV-Spectrophotometer UV-spectrometry is one of commonly used instrument for determine the concentration from the sample solution. This is very convenient and common method (Beers and Sizer n.d.). UV-Vis spectroscopy is used to determine the absorbance of the sample at the different wavelength. The absorbance A, of the sample is the factor of the extinction coefficient at the specific wavelength ελ the concentration of the substance in the sample C, and path length of the light B. Absorbance is a measure of the light that does not pass through the sample and corresponds to the base ten log of incident light, ten divided by light registered by the detector. This known as the Lambert-Beers law. A = log10 Io I = ελ CB Equation 1. Where, A = log10 Io I expressed as absorbance, ελ denoted the wavelength, C indicate the concentration of the solution and B path length of the light. Figure- 3. Schematic diagram of basic principle of UV-Spectrophotometry Source- (Gurdeep, R. and Anand, K.S., 1998.) 22

23 2.5 Gas chromatography A gas chromatography (GC) is an analytical instrument that measure the content of various components in a sample material. This technique associates with the quantitative and qualitative analysis. This technique mostly used in analytical chemistry for separating and analyzing the sample that can be vaporized but without decomposition of the chemical components. GC is able to analyze organic and inorganic compound but it has notable limitation which is the analyze compound must be in volatile in nature, because principle is based on volatility of compounds.(hussain and Maqbool 2014) The principle of gas chromatography is similar with the Column chromatography, High performance liquid chromatography and Thin layer chromatography(shackley et al. 1998). The basic principle behind this technique is that, the sample solution injected into the instrument which enter a gas stream and move to the separating tube which named column. In this method, mobile phase is important, it transports the solution and for this it used a carrier gas, usually inert gas such as Helium, Nitrogen and most of the instruments carries analysis with the Hydrogen for the better separation. The number of components separated inside the column. The detector measures the quantity of compounds which does exit the column. The GC has significant compatibility with the various detector such as, Flame ionization detector Thermal conductivity detector Mass spectrometer Infrared detector Electron capture detector Catalytic combustion Photo ionization detector 23

24 Figure- 4. Schematic diagram of working principle of GC Source- (Gurdeep, R. and Anand, K.S., 1998.) crushed material place in glass bottle analysis process in instrument detection by detector (MS) chromatogram result Figure- 5. General GC-MS procedure 24

25 methanolic extracts (75%) place in glass bottle analysis process in instrument detection by detector (FID) chromatogram result Figure- 6. General GC-FID procedure These all most common detector are used in GC technique. The selection of detector is depending upon the selected method and compound to be detected. To measure a sample with unknown concentration, a standard sample with known concentration is injected into the injector. The standard sample peak appearance time and area are compare to test sample for determining the concentration (Mirocha and Pathre 1973; Hussain and Maqbool 2014). In my research study, mainly I had focused on flame ionization detector and mass spectrometer detector. 2.6 High performance liquid chromatography HPLC, it is an analytical technique which is used to separate, identify and quantify of compounds from the mixture. The basic principle behind the HPLC is that, the separation occurs in between mobile phase and stationery phase, where the mobile phase refer as liquid and stationery phase is column(khoddami, Wilkes, and Roberts 2013). It depends on pump to pass high pressurize liquid to the containing sample mixture via column filled with solid absorbent material. 25

26 Figure-7. Schematic diagram of working principle of HPLC Source- (Gurdeep, R. and Anand, K.S., 1998.) The analysis performance is done by according to sample with the different flow rate. Most of the HPLC instrument also have the oven which associated with the column which allows adjusting temperature while performing the separation process. It has ability to perform analysis in various ways such as 26

27 Partition chromatography Normal phase chromatography Displacement chromatography Reverse phase chromatography Aqueous normal phase chromatography Size exclusion chromatography Bio-affinity chromatography The HPLC instrument dedicated to analysis of organic molecule, biomolecule, polymer, ions etc. It has great compatibility with very common detector such as, UV-Vis absorbance detector Charged aerosol detector HPLC has been continuously developing great advancement, in recent advancement it has able to analyses the Glycobase material (Campbell et al. 2008). 27

28 3 Material and methods 3.1 Material Chemical and solvents All the chemical and solvents were collected from Instrumental Analysis Open Access Centre FNS, Vytautas Magnus University, Kaunas, Lithuania. and all chemicals and solvents which was used in study were %. analytical grade. Chemicals used are listed in table no.5. Table- 5. Chemicals used in the study. Sr. No Chemicals Concentration 1 Sodium carbonate 4 % 2 Methyl alcohol 75 % 3 Rutin 0-1gm/ml 4 Folin Ciocalteu 6 Acetic acid 33 % 7 Hexamethylentetramine 5 % 8 Aluminium chloride 10 % 10 DPPH 100 mm 11 Acetonitrile 100 % 12 Micro- crystalline cellulose 100 % 13 TFA 0.05 % 14 Methyl alcohol 25 % 15 Acetonitrile 25 % 16 Distilled water 2 Times The used chemicals during the research study were precisely checked the all contents and information, includes purity, manufacturing date, expiry date, molarity etc. which was mentioned on the label of the chemical s container. 28

29 3.1.2 Sample collection The sample of spice were collected from the local supermarket Maxima Kaunas, Lithuania. Here we purchased fresh garlic bulb and other (cinnamon, nutmeg, Cardamom) were labeled. To avoid the oxidation and evaporation of volatile oil and deterioration, the spice materials were stored in umber glass airtight container away from the direct sunlight. Spice materials used in study listed below table no. 6. Table- 6. Investigated spice sample. Sr. No Spice Name Labeled 1 Garlic Fresh bulb (Lithuania) 2 Nutmeg Saldva (India) 3 Cinnamon Saldva (India) 4 Cardamom Saldva (India) There were very difficult to protect the spice materials from degradation, microbial contamination and from the direct sunlight during the analysis, for protection the materials, we used aluminium foil and plastic zip-locks bags. A- Garlic 29

30 B- Nutmeg C- Cardamom 30

31 D- Cinnamon Figure -8. Spices used for research study. Source- (Gokhale, Mr SB. Pharmacognosy 2008) 31

32 3.1.3 Instruments All the analysis was performed in Instrumental Analysis Open Access Centre FNS, Vytautas Magnus University, Kaunas, Lithuania. in faculty of Natural Sciences. The instruments used during study listed below. Table- 7. List of equipment used for analysis. Sr. Name of instruments Company No 1 Uv-Spectrophotometer (spectronic-1207) UV- Milton Roy (USA) 2 GC-MS (QP-2010) Shimadzu (Japan) 3 HPLC Shimadzu (Japan) 4 Mechanical stirrer VWR 5 Centrifuge (Labofuge) Cap-Heraeus Sepatch 6 Micro pipette Thermo Fisher 8 GC-FID (QP-2010) Shimadzu (Japan) The instruments which was used during the research study, were precisely calibrate according to the standard procedure and the protocols and along with the conditions were properly maintained before and after the analysis. 32

33 3.2 Methods Preparation of sample Preparation of extracts The extraction process was done by the very common and conventional method. The dried spices cardamom, nutmeg, cinnamon (0.5gm) and garlic (1gm) were crushed in small pieces of 2-6 mm using cylindrical crusher, and crushed material mixed with organic solvent (methanol 75%) and kept for 24 hours on mechanical stirrer, after the overnight continuous stirrer the concentrated solution was filtered by Whatman filter paper. collection of spices crushed into fine powder mixed with 75% methanol and kept 24hrs on mechanical stirrer filtered stored in glass container Figure- 9. General procedure for sample preparation 33

34 The extraction of spice materials is very significant step for the investigation of material. There was tried to achieve maximum extraction from the spice materials. The obtained extracts were stored in dark glass bottle in fridge for further processing Preparation of suspension (QUENCHER) This is one of the finest method of sample preparation for UV-spectrophotometric analysis. QUENCHER stands for Quick Easy New Cheap Reproducible which sense immediately as a new approach in the world of spectrophotometry. This method is suspension base sample preparation for the analysis on UV- Spectrophotometry analysis. QUENCHER is very significant step, in this method cellulose used as a solvent and this is type of solid-solid extraction. 100mg of crushed material mixed with 900mg of microcrystalline cellulose in the ratio 1;9 and for the further process there is 10mg material required form the dilution (Kopjar, Tadić, and Piližota 2015). This procedure overcome the problem related to losses inherent to commonly conventional processes of method of sample preparation which employ solvent extraction or hydrolysis(gökmen, Serpen, and Fogliano 2009). This QUENCHER procedure avoids these solvent and hydrolysis process of preparation and thus reagent directly act on the sample and possibly get more accurate result(kopjar, Tadić, and Piližota 2015). reaction mixture 1 Incubation and centrifuga tion measurement 1 crushed raw material microstalline cellulose mix compound ratio (1;9) reaction mixture 2 Incubation and centrifuga tion measurement 2 reaction mixture 3 Incubation and centrifuga tion measurement 3 Figure- 10. General principle of QUENCHER procedure 34

35 The major drawback with the conventional method is that proper choice of solvent for extraction but few phenolic compound has a favor of solubility with the particular type of solvent but with this method it can be easily minimize this error because reagent is directly associate with the soluble and non-soluble phenolic compounds(m Stankevičius et al. 2010) and it has possibility to achieve more precise analysis Determination total phenolic content in extracts The method of determination of total phenolic content by UV-Spectrophotometer was consider from various demonstrated methods(kaškonienė, Ruočkuvienė, et al. 2015). The total soluble phenolic compounds in the different spice (cinnamon, garlic, cardamom, nutmeg) extracts were determined with Folin-ciocalteu reagent using rutin as a standard with different concentration ranging from 0.05 to 1.00 mg/ml, 0.1 ml extract of sample spices (garlic, cardamom, nutmeg and cinnamon), mixed with the 3 ml stock solution of sodium carbonate (4%) and mixed. Subsequently, add 0.1 ml Folin reagent (2N) with inverting vial two times. After 30 min incubation in room temperature, absorbance measured at 760 nm verses blank. The blank solution prepared in same way without addition of extract of spices. If the absorbance exceeded more than 1.00 AU limit, the extracts should additionally dilute ten times with methanol (75%). The sample were prepared in triplicate for each analysis and the mean value of absorbance was obtained. The total phenolic content was calculated according to given equation no 2. TPC = a γ( V ) 100.Equation- 2 m Where TPC- total phenolic content, a- number of dilution, γ- concentration obtained from calibration curve, V- volume of methanol used for extraction, m- weight of dry spice material. 35

36 3.2.3 Determination of flavonoid content in extracts The total flavonoids content of spice extracts (cinnamon, garlic, cardamom, nutmeg) were determined using aluminiun chloride assay with slight modification described in Kaškonienė et al., (2015), according to this method, Briefly prepared the stock solution of 60 ml methanol (100%), 3 ml acetic acid (33%), 12 ml hexametyleneteramine (5%), 9 ml aluminium chloride (10%), 60 ml of bi-distilled water and incubate in refrigerator at temperature ml spice extract mixed with 1.92 ml of stock solution and incubated for 30 min at temperature 2. After the incubating time, the absorbance had taken at 407 nm versus blank. The calibration curved constructed using rutin, it is ranging from 0.05 to 1.00 mg/ml. If the value of absorbance exceeded more than 1.00 AU limit, the extracts were additionally diluted ten times with methanol (75%). The cinnamon extract was additionally diluted ten times with methanol (75%). The samples were triplicate for the better result. The total amount of flavonoid was calculated as per equation no 3. TFC = a γ( V ) 100.Equation- 3 m Where TFC- total phenolic content, a- number of dilution, γ- concentration obtained from calibration curve, V- volume of methanol used for extraction, m- weight of dry spice material Determination of radical scavenging activity in extracts The radical scavenging activity of spices extract (cinnamon, garlic, cardamom, nutmeg) were determined by the using 1,1-dyphenyl-2- picrylhydrazyl (DPPH) assay with modification according to extract sample need (Kaškonienė, Ruočkuvienė, et al. 2015; Ninfali et al. 2005). The 10 mg DPPH dissolved in 125 ml Acetonitrile and then add 125 ml methanol. After the completion of solution, stored in dark for 30 min and then check absorbance, it should be in between ( ), 0.07 ml extract mixed with 3 ml of stock solution of DPPH and incubated in dark for 15 min and took absorbance at 507 nm. The rutin range 0.05 to 1.00 mg/ml were used for the standard calibration curve. All the spice extracts sample were diluted ten times with the methanol (75%) and measurements were performed in triplicate and result was the average of three values. The DPPH radical scavenging activity (%) was calculated according to below equation no

37 RSA (%) = A control -A sample / A control 100(%).. Equation- 4 Where, RSA is a radical scavenging activity, A control is absorbance of blank, and A sample is stand for absorbance of sample Determination of total phenolic content in suspension (QUENCHER) The determination total phenolic content was done by QUENCHER approach with the UV- Spectrophotometry method which previously described in Kopjar, Tadić and Piližota, (2015) Palombini et al., (2016) with the slightly modification. 10mg of sample were taken from the dilution previously weigh crushed spices and microcrystalline cellulose and mixed with 0.08 ml methanol (75%), 6 ml sodium carbonate (4%) and 0.2 ml Folin reagent. In the next step, this mixture stirred on mechanical stirrer for 60 minutes, later there was a centrifugation step for 5 minutes at 3000 rpm for settling down of solid particle and withdraw 2 ml solution from the top and measured the absorbance at 760nm verses blank (4% sodium carbonate). The standard calibration curve constructed using rutin with different concentration 0.05 to 1.00 gm/ml. The sample were triplicate for each analysis and mean value of absorbance was obtained. The total phenolic content was calculated using according to follow equation no 5. TPC = a γ( V ) 100.Equation- 5 m Where TPC- total phenolic content, a- number of dilution, γ- concentration obtained from calibration curve, V- volume of methanol used for extraction, m- weight of dry spice material. 37

38 3.2.6 Determination of flavonoid in suspension (QUENCHER) The flavonoid content was determined flavonoid assay with some modification in actual method which was previously described research articles (Kopjar, Tadić, and Piližota 2015; Gökmen, Serpen, and Fogliano 2009; Serpen, Gökmen, and Fogliano 2012). In this method, 15mg of blended materials were mixed 0.12 ml methanol (75%) along with 5.75 ml stock solution of flavonoid reagent and leaved this solution mixture on mechanical stirrer for the 60 minutes. After the continuous stirring this mixture loaded on centrifugation for 5 minutes at 3000 rpm, for settling the solid particle. In the final step, 2 ml mixture withdrawal from the top without shaking and measured absorbance at 407nm against blank (stock solution of flavonoid). The rutin solution used as standard with ranges 0.05 to 1.00 mg/ml. The standard calibration curve plotted using rutin absorbance. The cinnamon sample was diluted ten times with methanol (75%) because absorbance of cinnamon was more than 1.00 AU. The total flavonoid concentration was determined by using equation no 6. TFC = a γ( V ) 100.Equation- 6 m Where TFC- total phenolic content, a- number of dilution, γ- concentration obtained from calibration curve, V- volume of methanol used for extraction, m- weight of dry spice material Determination of radical scavenging activity (QUENCHER) The radical scavenging activity of spices (cinnamon, garlic, cardamom, nutmeg) were determined by the QUENCHER procedure associated with(kopjar, Tadić, and Piližota 2015) using 1,1-dyphenyl-2- picrylhydrazyl (DPPH) assay with modification according to sample need (Kaškonienė, Ruočkuvienė, et al. 2015; Ninfali et al. 2005). The 10 mg DPPH dissolved in 125 ml Acetonitrile and then add 125 ml methanol. After the completion of solution, stored in dark for 30 min and then check absorbance, it should be in between ( ), 5 mg previously prepared dilution of spices powder and microcrystalline cellulose mixed with 0.24 ml methanol (75%) and 18 ml of DPPH reagent and incubated in dark for 15 min without shaking and measured absorbance at 515nm of 2ml solution from top. The rutin range 0.05 to 1.00 mg/ml were used for the standard calibration curve. All the spice extracts sample were diluted ten times with the methanol (75%) and measurements were performed in triplicate and result was the 38

39 average of three values. The DPPH radical scavenging activity (%) was calculated according to equation no 7. RSA (%) = A control -A sample / A control 100(%).. Equation- 7 Where, RSA is a radical scavenging activity, A control is absorbance of blank, and A sample is stand for absorbance of sample Gas chromatography mass spectrometry analysis The quantitative and qualitative analysis of spice (Garlic, Cinnamon, Nutmeg, Cardamom) materials were performed using gas chromatography instrument with mass spectrometry detector, the adopted method used for GC-MS analysis which was signified in number of research articles(kaškonienė, Stankevičius, et al. 2015a; Ligor et al. 2014). This GC-MS method is very sensitive and highly effective, it has ability to detect the compound even amount of sample is very low. The GC-MS system 2010 model with mass spectrometric detector GCMS-QP2010 (SHIMADZU, Tokyo, Japan). Mass spectrometer was operated in the electron impact ionization mode (70eV). The compounds were separated using RTX- 5MS column (30 m, 0.25 i.d, 0.25 μm film thickness) (Restec, Bellefonte, PA, USA). The carrier, Helium was used (99.99% AGA Lithuania) with adjusted flow rate 1.2 ml/min(kaškonienė, Stankevičius, et al. 2015a). For the sample injection, split mode was used at ratio of 1:10 and injector temperature was maintained 240 C. The oven temperature was controlled such as, initial temperature was maintained 30 C and increased to 200 C at time 5 C/min, and again hiked to 280 C at 20 C/min and it was held for 2 minutes. The ion root and interface were adjusted at 220 and 260 C respectively(lee et al. 2005; Viuda-Martos et al. 2007). In every analysis of specific compounds, sample preparation is vital step and its depending upon the method selection. In this analysis study, the sample materials were used in powder form which was crushed in cylindrical crusher. The 10 mg amount of powder sample were kept in small glass container and placed into GC-MS instrument, due to the risen temperature of oven the volatile components of test materials were evaporate and mixed with the inert carrier gas and transport to the 39

40 column. In the column, volatile compound splits and detector detect the components and recorded on the chromatogram and analysis time was 30 minutes. The identification of compounds was done according to their mass spectra and matching their spectra to data in NIST v1.7 library. Positive identification was considered when matches more identical of mass spectra were achieved Gas chromatography flame ionization detection The quantitative and qualitative analysis was done using GC-FID method, according to the described method(mantas Stankevičius et al. 2015)(Ferreira et al. 2007). In this method, flame ionization detector was used for the detection of chemical compounds along with the determination of concentration. The flame ionization detector works only for organic and hydrocarbon containing compounds because, it has ability to carbon to form cation. FID has a low detection limit. In this method methanolic extracts (75%) of spices namely garlic, cinnamon, cardamom and nutmeg were used. The identification of compounds was done according to their mass spectra and matching their spectra to data in NIST v1.7 library Carbon containing compound burnt on flame and form into cation, goes to excite state and recorder record the excite state of compound(jirovetz et al. 2002). The working principle is same as the GC-MS but method of sample preparation was different. The GC-FID method conditions were perfectly maintained. The injector and detector temperature condition were adjusted at 350 C. The temperature acclivity was set 60 C and maintained for 1 min, further temperature was hiked at 10 C/min up to120 C, at 5 C/min it was adjusted to up to 180 C, on the 10 th minute it was inclined up to 330 C and maintained for rest 7 minute. Hydrogen was used as carrier gas and flow rate was controlled 1.4 ml/min. The analysis retention time was 35 minutes(ferreira et al. 2007). The injected sample extracts amount was 1 μl. The injection was carried out in split mode 1:10 and three injection of sample extracts were performed and selected a best average of result. 40

41 Determination of polyphenols by HPLC The determination of phenolic compounds from the spice extracts (Garlic, Cardamom, Cinnamon, Nutmeg) were performed using HPLC system with UV-vis detector. The methanolic (75%) extracts were used. The HPLC (Shimadzu, Japan) system consist of LC-20AD, a model CTO10ASvp column and data station for data acquisition and analysis. The column is heart of HPLC system, it has specification such as KROMASIL 100 C10 dp=5 µm, ( mm) was used. The used column thermostated at 25 C. The 20 µl sample extracts were injected. The method conditions were perfectly maintained, solution A mixture TFA and two times distilled water (99:1 v/v), solution B was mixture of gradient grade methanol and TFA (99:1 v/v) and solution c was water (50%), methanol (25%), acetonitrile (25%). The flow rate was adjusted to 1.0 ml/min. Following gradient was used for extraction 1% of solution B at 0 min, 99 % of solution B at 40 min, 99% solution B at 45 min, in next it had declined 48 min and 58 min the solution B was 1 %. The wavelength 254 nm was used for determination of phenolic compound. The analysis time was 60 min(kaškonienė, Stankevičius, et al. 2015b). The identification of each compound was performed out comparing the retention time and UV-Vis spectra peaks. The analysis was performed thrice and got selected best result Statistic analysis All experimental measurements were carried out in triplicate and were expressed as average of three analyses and standard deviation were calculated using MS-excel with number of absorbance and its average, calculated deviation according to the standard deviation formula in excel. 41

42 4 Result and discussion 4.1 Phenolic content in extracts The determination of total phenolic content was done by sodium carbonate assay. The standard calibration curve constructed by using rutin. The standard calibration curve is linear trend line. The obtained result showed that cinnamon contained highest amount of phenolics 23.72% and nutmeg contained 21.49%, cardamom 10.37%. Garlic possessed the lowest amount phenolic compounds 7.91%. The cinnamon and nutmeg are the rich source of phenolic compounds as compare to other two spice material. 30 TPC Cardamom Garlic Nutmeg Cinnamon Total conc in % Figure 11. Graphical presentation of TPC 42

43 4.2 Flavonoid content in extracts The concentration of flavonoids in various extracts of spices were determined using aluminum chloride. The content of flavonoids was expressed in terms of rutin equivalents. The concentration was determined by using linear curve. Flavonoid is one of the most important calls of phenolic compound. The obtained data expressed that all the spices contained flavonoid compound with different concentration. Nutmeg and cinnamon possessed the % and % of flavonoids but in the case of garlic and cardamom it had very low quantity 1.43% and 5.58 respectively. 25 flavonoid content Cardamom Garlic Nutmeg Cinnamon flavonoid in % Figure 12. Graphical presentation of flavonoid content 43

44 4.3 Radical scavenger in extracts The antioxidant activity of different extracts of spices were determined using methanol solution of DPPH reagent. DPPH is a very stable free radical. The effect of an antioxidant on DPPH radical scavenging is due to their hydrogen donating ability or radical scavenging activity. When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form diphenylpicrylhydrazine with the loss of its violet color. The calibration curve equation was linear. The collected denoted that cinnamon and nutmeg possessed the highest amount of radical scavenging activity as compare to garlic and cardamom. Cinnamon contain % of free radical, this is highest concentration possessed of RSA and Garlic has %, this is smallest amount among the all spices. 60 RSA Cardamom garlic Nutmeg Cinnamon RSA in % Figure 13. Graphical presentation of radical scavenger 44

45 4.4 Phenolic content in suspension (QUENCHER) The obtained result showed that cardamom and cinnamon contain the maximum amount of phenolic content as compare to garlic and nutmeg. The concentration was determined by using linear equation. Cardamom and cinnamon possessed the 42% and 41% respectively. Garlic hold the lower amount that is 11% and nutmeg owned the 20 % of phenolic content. 60 TPC Cardamom Garlic Nutmeg Cinnamon TPC in % Figure 14. Graphical presentation of TPC (QUENCHER) 4.5 Flavonoid content in suspension (QUENCHER) The concentration of flavonoids in various extracts of spices were determined using aluminum chloride. The content of flavonoids was expressed in terms of rutin equivalents. The determination of flavonoid content by QUENCHER method, I had found that cinnamon carried 62 %, cardamom 40 %, nutmeg 18 % and garlic 4%. Cinnamon carried sharp amount of flavonoid as compare to other spice material 45

46 Flavonoid Cardamom Garlic Nutmeg Cinnamon Flavonoid in % Figure 15. Graphical presentation of flavonoid (QUENCHER) 4.6 Radical scavenger in suspension (QUENCHER) The effect of an antioxidant on DPPH radical scavenging is due to their hydrogen donating ability or radical scavenging activity. The linear calibration curve equation for the determination of concentration. When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form diphenylpicrylhydrazine with the loss of its violet color. The collected results shown that all the spices carried almost same percentage of RSA.66%, 64%, 65%, 68% respectively. The investigated results were lies on the same plane and it indicated that all the spices possessed the almost same percentage of radical scavenging activity. 46

47 RSA Cardamom Garlic Nutmeg Cinnamon RSA in % Figure 16. Graphical representation of RSA (QUENCHER) 4.7 Identification and quantification of essential oils by GC-MS The total chemically active compounds in spices like Cinnamon, Nutmeg, Cardamom, Garlic were determined using GC-MS. However around more than ten chemically active compounds were found in each spice material. They varied from the different concentration, some in very minute concentration and some of them are in highest concentration. In this spices cinnamaldehyde, linalool, terpinyl acetate, limonene, sulphide, disulphide, propyl cyanide, butanon, myristicin respectively. This chemically active compound has a vitalness to the body as well as various health benefits against various chronic disease and disorders like antibacterial, antifungal, anticholinergic agents, anti-inflammatory, herbicidal. 47

48 Figure 17. GC-MS chromatogram of garlic In garlic, 14 essential oils compounds were investigated which includes thiophene (3.88%), methoxymethyl (3.33%), tetrasulfide (13.73%), propyl cyanide (16.14%), allyl methyl trisulfide (1.23%), thiophene (14.91%), 1,2,3- thiadiazole,5-methyl (1.23%), thiazole (22.70%), menthalactone (14.66%) etc. In same with the garlic, cinnamon possessed the 10 compounds which comprises cinnamaldehyde (62.04%), this is dominant compound that investigated. Other compounds also found, terpinyl acetate (25.63%), limonene (2.99%), linalool (2.80%). The lowest concentration was (0.40 %) in bomeol. In cardamom there was 19 compounds found which 4 compounds were dominant limonene, terpineol, linalyl acetate, terpinyl acetate (12.76%, 10.02%, 15.00%, 39.80%). Nutmeg ruled among the all spices which was investigated. In nutmeg there was 21 essential oil compounds were present. The obtained result is that, 3 compounds were completely shown the dominance in all result, sabinene (15.03 %), ocimene (17.45 %), terpinene-4-ol (16.42 %). The other compounds were shown lowest concentration which include thujene (0.87%), phellandrene (0.91 %). The most significant compound found in nutmeg is myristicin (4.53 %), it has more pharmacological importance and it is prototype example of psychoactive substance. 48

49 4.8 Identification and quantification of essential oils by GC-FID The collected result of GC-FID analysis displayed that all the spices garlic, cinnamon, nutmeg, cardamom is the great source of essential oils and this, essential oils is a beneficial to the human health. Cardamom possessed the 18 compound of essential oils which is highest number among the all spice materials. Nutmeg contained 14 compounds, garlic carried 4 compounds and cinnamon possessed 6 compounds. These compounds have a vitalness towards the human life for the normal growth and maintenance. Most of the found compounds has pharmacological benefits, which has been using often in modern and traditional medicine. In garlic 4 compounds were present thiophene (14.91%) and this compound shown the dominance. 1,2,3- thiadiazole,5-methyl (1.23%), thiazole (22.70 %), Garlic Figure 18. Graphical presentation of garlic No1. Thiazole, no2. 1,2,3- thiadiazole,5-methyl, no4. Thiophene 49

50 Cinnamon Figure 19. Graphical presentation of cinnamon No1. Bomeol, no 2. Cinnamaldehyde, no 3. linalyl format, no 4. Limonene, no 5. terpinyl acetate. cinnamaldehyde (62.04%), this is dominant compound that investigated. Other compounds also found, terpinyl acetate (25.63%), limonene (2.99%), linalool (2.80%). The lowest concentration was (0.40 %) in bomeol, linalyl format (1.97%). Nutmeg Figure 20. Graphical presentation of nutmeg. No 10. Ocimene, no 6. terpinene-4-ol, no 7. Sabinene, no 14. myristicin 50

51 Nutmeg ruled among the all spices which was investigated. In nutmeg there was 14 essential oil compounds were present. The obtained result is that, 3 compounds were clearly indicates the higher concentration in all the investigated results, sabinene (15.03 %), ocimene (17.45 %), terpinene-4-ol (16.42 %). The other compounds were shown lowest concentration which include thujene (0.87%), phellandrene (0.91 %), safrole (6.51%), careen (0.88%). The most notable biological substance found in nutmeg is myristicin (4.53 %), it has more pharmacological importance and it is prototype example of psychoactive substance Cardamom Figure 21. Graphical presentation of cardamom. No 2. terpenyl acetate, no 16. Terpineol. In cardamom there was 18 volatile compounds were acquired and 4 major biological compounds were ruled in all obtained result. Terpine-4-ol (1.14%), terpineol (10.02%), terpenyl acetate (39.80%), limonene oxide (1.27%), selinene (1.71%), these all investigated compounds are biologically active and it owned the health benefits. It often noticed in preparation of modern medicine. 4.9 Polyphenolic determination by HPLC 51

52 The obtained result of HPLC analysis exhibit that all the spices such as garlic, cinnamon, nutmeg, cardamom is the rich source of phenolic compounds. All the chromatogram of respectively spices show confirmation of group of phenolic compounds which includes flavonoid, tannins, phenolic acid, coumarin, hydroxybenzoic acid, hydroxycinnamic acid. Figure 22. Chromatogram of nutmeg 52

53 Figure 23. Chromatogram of garlic Figure 24. Chromatogram of cardamom 53

54 Figure 25. Chromatogram of cinnamon In nutmeg investigation of nutmeg, there is 32. dominated compounds were found, in cinnamon 19. compounds, garlic 31. compounds and in cardamom there was 21. Phenolic compounds were present. From the above chromatogram it clearly represented, nutmeg and garlic owned the maximum possession of the polyphenolic as compare to the rest of other two spice materials. Cinnamon resulted the least compound of polyphenolics and cardamom showed the near to minimum possession of the polyphenolic. In contracts all the spice materials are the significant source of polyphenolic which has pharmacological importance and health beneficial. It often used in pharmaceutical industry for the preparation of modern medicine and external supplement of nutrients and xenobiotics. 54