Optimization of Supercritical Carbondioxide Extraction Parameters of Essential Oils from Various Plants

OPTIMIZATION OF SUPERCRITICAL CARBONDIOXIDE EXTRACTION PARAMETERS OF ESSENTIAL OILS FROM VARIOUS PLANTS

THESIS

SUBMITTED FOR THE AWARD OF THE DEGREE OF

DOCTOR OF PHILOSOPHY

CHEMISTRY

AARTI SINGH

DEPARTMENT OF CHEMISTRY

ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2016

CANDIDATE’S DECLARATION

I, Aarti Singh, Department of Chemistry, certify that the work embodied in this Ph.D thesis is my own bonafide work carried out by me under the supervision of Prof. Anees Ahmad at Aligarh Muslim University, Aligarh. The matter embodied in this Ph.D thesis has not been submitted for the award of any other degree. I declare that I have faithfully acknowledged, given credit to and referred to the research workers wherever their works have been cited in the text and the body of the thesis. I further certify that I have not willfully lifted up some other’s work, para, text, data, result, etc. reported in the journals, books, magazines, reports, dissertations, theses, etc., or available at web-sites and included them in this Ph.D. thesis and cited as my own work.

Date:

(Signature of the candidate) AARTI SINGH (Name of the candidate)

Certificate from the Supervisor

This is to certify that the above statement made by the candidate is correct to the best of my knowledge.

Signature of the Supervisor:

Name & Designation: Prof. Anees Ahmad

Department: Chemistry

(Signature of the Chairman of the Department with seal)

COURSE/COMPREHENSIVE EXAMINATION/PRE-SUBMISSION SEMINAR COMPLETION CERTIFICATE

This is to certify that Ms. Aarti Singh, Department of Chemistry has satisfactorily completed the course work/comprehensive examination and pre-submission seminar requirement which is part of her Ph.D. programme.

Signature of the Supervisor Signature of the Chairman

(Prof. Anees Ahmad) Department of Chemistry

Date:

Title of the Thesis: Optimization of Supercritical Carbondioxide extraction

parameters of essential oils from various plants.

Candidate’s Name: Aarti Singh

The undersigned hereby assigns to the Aligarh Muslim University, Aligarh,

Ph. D. degree.

(Signature of the Candidate)

AARTI SINGH Acknowledgement

First and foremost, I express my deep gratitude to the Almighty God for giving me the courage and strength to complete this work.

I express my sincere thanks to my supervisor Prof. Anees Ahmad for his guidance, support, advice and encouragements throughout my research work. I admire his invaluable comments and keen insight in research.

I would like to express my deepest gratitude to Chairman, Department of Chemistry, A.M.U. Aligarh, for support, motivation and providing me research facilities. I am also thankful to UGC-BSR, New Delhi, India, for financial assistance.

I also express my heartfelt gratitude to Dr. Abdul Latif for Identification and authentication of samples, Prof. Aquil Ahmed for providing me training on the MINITAB 14 statistical software package, Abbas F. M. Alkarkhi for clearing queries related to MINITAB 14 software, Dr. Sayeed Ahmad for my review article on carotenoids. I owe a debt of gratitude to you all for your kindness, active help and encouragements.

I owe a great debt of gratitude to my parents Dr. B. Lal and Asha Singh, without your love, support and prayer it would not have come true! Special mention goes to my brother Mr. Surya Singh for his affectionate encouragement and help. I would also like to thank Dr. Deepak Singh for his care, patience, motivation and kind help throughout my research work. I do remember the prayers, support and wishes from my friends and lab mates Dr. Rani Bushra, Mr. Seraj Anwar Ansari, Ms. Fauzia Khan, Ms. Nishat Khan, Ms. Yashfeen Khan and Ms. Nehal Zehra. ‘Thank you’ is just not enough to say, I appreciate your kind help throughout the journey.

Last but not least, I express my thanks to the staff from the Department of Chemistry, A.M.U., for their kind help and assistance during my research work. I am really grateful to you all.

(Aarti Singh)

Abstract

The thesis comprises of seven chapters. First chapter deals with the general introduction of the subject matter of the thesis which describes about essential oils, sources of essential oil, chemical constituents of essential oils, Methods of extracting essential Oils (Conventional methods and non-conventional methods), uses of essential oils and aims and objectives of the work.

Essential oils have a complex composition, containing from a few dozen to several hundred constituents, especially hydrocarbons and oxygenated compounds. The main group is composed of terpenes and terpenoids and the other aromatic and aliphatic constituents, all characterized by low molecular weight. Trace components are important, since they give the oil a characteristic and natural odour. They can be obtained from different parts of plants including flowers, buds, seeds, leaves, twigs, bark, herbs, fruits and roots. Oils are used in the embalming process, in medicine and in purification rituals. There are also over 200 references to aromatics, incense and ointments in the Old and New Testaments. Research has confirmed centuries of practical use of Essential Oils, and we now know that the 'fragrant pharmacy' contains compounds with an extremely broad range of biochemical effects. There are about three hundred essential oils in general use today by professional practitioners. Continual bombardment of viral, bacterial, parasitic and fungal contamination occurs in our body. Essential oils are of great benefit to help protect our bodies and homes from this onslaught of pathogens. Immune system needs support and these essential oils can give the required endorsement.

The ability of plants to accumulate essential oils is quite high in both Gymnosperms and Angiosperms, although the most commercially important essential oil plant sources are related to the Angiosperms. Most of the aromatic plants and essential oil commodities in terms of world trade belong to the families of Lamiaceae, Umbelliferae and Compositae.

An Essential Oil contains more than 200 chemical components, but some are many times more complex. Essential oils consist of chemical compounds which have hydrogen, carbon and oxygen as their building blocks. They can be essentially classified into two groups:

Volatile fraction: Essential oil constituting of 90-95% of the oil in weight, containing the monoterpene and sesquiterpene hydrocarbons, as well as their oxygenated derivatives along with aliphatic aldehydes, alcohols and esters.

Non-volatile residue: This comprises 1-10% of the oil, containing hydrocarbons, fatty acids, sterols, carotenoids, waxes and flavonoids.

Methods of extracting essential oils are classified as:

 Conventional methods

 Maceration  Cold Pressing  Enfleurage  Steam distillation and solvent extraction

 Non-conventional methods  Microwave assisted extraction  Ultrasonic assisted extraction  Supercritical fluid extraction

A supercritical fluid (SCF) is any compound at a temperature and pressure above the critical point. Above the critical temperature (Tc) of a compound, the pure, gaseous component cannot be liquefied regardless of the pressure applied. The critical pressure (Pc) is the vapor pressure of the gas at critical temperature. In the supercritical environment only one phase exists. The fluid, as it is termed, is neither a gas nor a liquid. This phase retains solvent power approximating liquids as well as the transport properties common to gases.

Applications of essential oils

 Food industry  Pharmaceutical industry  Cosmetic industry  Veterinary product industry

 Industrial deodorants  Tobacco industry  Biocides and insecticides  Antioxidants

Chapter two describes the critical review of the existing literature on the extraction of essential oils from various parts of the plants using conventional and non-convention extraction techniques.

Chapter three describes materials and methods which includes collection of plant material, identification and authentication of plant material, methods for extraction of essential oil (Hydro-distillation, Solvent Extraction, Ultrasonic Assisted

Extraction and Supercritical Carbon-dioxide (SC-CO2)). It also includes optimization

of SC-CO2 extraction parameters via Full Factorial Design (FFD), physiochemical characterization of essential oil, analytical Studies (SEM, HPTLC, GC-MS and FTIR) and antioxidant activity of essential oils. Use of natural antioxidants, as food additives for inactivating free radicals receiving a lot of attention nowadays, not only for their scavenging properties, but also because they are natural, non-synthetic products, and their appreciation by consumers is very favourable.

Chapter four describes optimization of total essential oil yield of

Cinnamomum zeylanicum N. by using SC-CO2 extraction. Essential oil was extracted via Hydro-distillation (HD), Solvent extraction (SE), Ultrasonic assisted extraction

(UAE) and Supercritical Carbon-dioxide (SC-CO2) extraction. Among all these methods SC-CO2 gave the highest percentage yield (3.8 %) at optimum conditions of o pressure (200 bar), temperature (40 C) and flow rate of CO2 (4 g/min) by FFD. The analysis shows that pressure and flow rate have shown to have direct effect on yield as p<0.05, While temperature, combination of pressure-flow rate, temperature-flow rate, pressure-temperature and pressure-temperature-flow rate combinations have no significance on oil yield as p>0.05. Furthermore evidences from GC-MS as well as

FTIR analysis showed that SC-CO2 is best technique for extraction of essential oils. Essential oil obtained from bark of C. zeylanicum has shown to possess significant antioxidant activity which was investigated by DPPH and Superoxide anion scavenging methods and was found to be 70.55 and 32.34 µg mL-1, respectively.

Chapter five describes Optimization of Supercritical Carbon dioxide extraction of essential oil from Cinnamomum tamala N. along with its chemical composition and antioxidant activity. In this work, essential oil from leaves of C. tamala was extracted via HD, SE, US and SC-CO2, out of all these methods SC-CO2 gave the highest percentage yield (3.64%) using FFD with optimum conditions of pressure (200 bar), o temperature (55 C) and flow rate of CO2 (15 g/min).The analysis shows that pressure, flow rate and interaction of pressure-flow rate have significant effect on oil yield while temperature, combination of pressure-temperature, temperature-flow rate and

pressure-temperatute-flow rate have no significant effect on yield as p>0.05. SC-CO2 is a green, environmentally friendly and sustainable extraction technique. Forty five compounds were identified by GC/MS. Essential oil obtained from leaves of C. tamala has shown to possess significant antioxidant activity which was investigated by DPPH and Superoxide anion scavenging methods and was found to be 89.4 and 32.24 µg mL-1, respectively.

Chapter six describes SC-CO2 extraction of essential oil from leaves of Eucalyptus globulus L., Analysis and their Application. Essential oil was extracted via

HD, SE, UAE and SC-CO2 extraction. Among all these methods SC-CO2 gave the highest percentage yield (3.6%) at optimum conditions of pressure (350 bar), o temperature (80 C) and flow rate of CO2 (12 g/min) as determined by FFD. Pressure, flow rate and combination of pressure-flow rate have shown to have direct effect on yield as p<0.05, hence pressure, flow rate and combination of pressure-flow rate are significant factors for influencing yield. While temperature, combination of pressure- temperature, temperature-flow rate and pressure-temperatute-flow rate have no significant effect on yield as p>0.05.

High pressure results in increased oil yield as the solvent power of SC-CO2 to dissolve essential oil in the powdered plant material increases to an extent. Increase in the temperature to a certain limit increases the solubility of the essential oil.

Increasing the CO2 flow rate results in better oil yield due to enhanced interaction

between CO2 and essential oil. Physicochemical characterization of essential oil extracted by HD, SE, UAE and SC-CO2 shows that the aldehyde, phenolic and

anthocyanin contents are highest in SC-CO2 extracted oil as all the phytoconstituents are retained in the extracted oil without any loss or addition of any adulterant and also

free from any toxic solvents as no organic solvents are used in the SC-CO2 extraction

of essential oil. Furthermore evidences from HPTLC, GC-MS as well as FTIR analysis showed that SC-CO2 is best technique for extraction of essential oils. Essential oil obtained from leaves of E. globulus has shown to possess significant antioxidant activity which was investigated by DPPH and Superoxide anion scavenging methods and was found to be 47.61 and 26.05 µg mL-1, respectively.

Chapter seven describes optimization of SC-CO2 extraction, chemical composition and antioxidant activity of essential oil from Trachyspermum ammi L.

Essential oil from seeds of T. ammi was extracted via HD, SE, UAE and SC-CO2

Extraction. Out of all these methods SC-CO2 gave the highest percentage yield (2.64%) as compared to other extraction techniques. FFD was used to determine the

optimum conditions for SC-CO2 extraction of essential oil were pressure (225 bar), o temperature (32.5 C) and flow rate of CO2 (10 g/min). The analysis shows that pressure, temperature and flow rate effect yield of oil and combination of temperature-flow rate also shows significant influence on yield as p<0.05, while pressure-flow rate, pressure-temperature and pressure-temperature-flow rate combinations have no effect on oil yield as p>0.05. Furthermore evidences from

HPTLC, GC-MS as well as FTIR analysis showed that SC-CO2 technique is best

technique for extraction of essential oils. SC-CO2 is a green, environmentally friendly and sustainable extraction technique. Essential oil obtained from seeds of T. ammi has shown to possess significant antioxidant activity which was investigated by DPPH and Superoxide anion scavenging methods and was found to be 36.41 and 20.55 µg mL-1, respectively.

CONTENTS

Page No.

Chapter 1: General Introduction 1 - 22

Chapter 2: Literature Review 23 - 35

Chapter 3: Materials and Methods 36 - 53

Chapter 4: Optimization of total essential oil yield of Cinnamomum zeylanicum N. by using supercritical Carbon-dioxide extraction 54 – 77

Chapter 5: Optimization of Supercritical Carbon dioxide extraction of essential oil from Cinnamomum tamala N. along with its chemical composition and antioxidant activity 78 – 97

Chapter 6: Supercritical Carbon-dioxide extraction of essential oil from leaves of Eucalyptus globulus L., Analysis and their Application 98 - 124

Chapter 7: Optimization of Supercritical CO2 extraction, chemical composition and antioxidant activity of essential oil from Trachyspermum ammi L. 125 - 150

Appendix

Reprints of published papers

CHAPTER 1

GENERAL INTRODUCTION

INTRODUCTION

Essential oils represent a small fraction of a plant’s composition but confer the characteristic for which aromatic plants are used in the pharmaceutical, food and fragrance industries. Essential oils have a complex composition, containing from a few dozen to several hundred constituents, especially hydrocarbons and oxygenated compounds. Both hydrocarbons and oxygenated compounds are responsible for the characteristic odors and flavors [1]. The proportion of individual compounds in the oil composition is different from trace levels to over 90% (limonene in orange oil). The aroma’s oil is the result of the combination of the aromas of all components. The components include two groups of distinct biosynthetical origin. The main group is composed of terpenes and terpenoids and the other of aromatic and aliphatic constituents, all characterized by low molecular weight. Trace components are important, since they give the oil a characteristic and natural odor. Thus, it is important that the natural proportion of the components is maintained during extraction of the essential oils from plants by any procedure [2]. Since the middle ages, essential oils have been widely used for bactericidal [3, 4], virucidal [5], fungicidal [6], acaricidal [7], insecticidal [8, 9], medicinal and cosmetic applications [10], especially nowadays in pharmaceutical, sanitary, cosmetic, agricultural and food industries [50-61]. Because of the mode of extraction, mostly by distillation from aromatic plants, they contain a variety of volatile molecules such as terpenes and terpenoids, phenol-derived aromatic components and aliphatic components. In vitro physicochemical assays characterize most of them as antioxidants. However, recent work shows that in eukaryotic cells, essential oils can act as pro-oxidants affecting inner cell membranes and organelles such as mitochondria. Depending on type and concentration, they exhibit cytotoxic effects on living cells, but are usually non- genotoxic. In some cases, changes in intracellular redox potential and mitochondrial dysfunction induced by essential oils can be associated with their capacity to exert anti-genotoxic effects. These findings suggest that, at least in part, the encountered beneficial effects of essential oils are due to pro-oxidant effects on the cellular level [10].

The term ‘essential oil’ was used for the ﬁrst time in the 16th century by Paracelsus von Hohenheim, who named the effective component of a drug, ‘Quinta essential’ [11]. An essential oil is a concentrated, hydrophobic liquid containing

volatile aroma compounds from plants, produced as secondary metabolites in plants. Essential oils are also known as volatile, ethereal oils or aetherolea, or simply as the "oil of" the plant from which they were extracted, such as oil of clove. Oil is "essential" in the sense that it carries a distinctive scent, or essence, of the plant. Essential oils are frequently referred to as the “life force” of plants. The total essential oil content of plants is generally very low and rarely exceeds 1% [12]. They can be obtained from different parts of plants including flowers, buds, seeds, leaves, twigs, bark, herbs, fruits and roots. Oils are used in the embalming process, in medicine and in purification rituals. There are also over 200 references to aromatics, incense and ointments in the Old and New Testaments. Research has confirmed centuries of practical use of Essential Oils, and we now know that the 'fragrant pharmacy' contains compounds with an extremely broad range of biochemical effects. There are about three hundred essential oils in general use today by professional practitioners. Continual bombardment of viral, bacterial, parasitic and fungal contamination occurs in our body. Essential oils are of great benefit to help protect our bodies and homes from this onslaught of pathogens. Immune system needs support and these essential oils can give the required endorsement. Sources of essential oil The occurrence of essential oils is restricted to over 2000 plant species from about 60 different families, however only about 100 species are the basis for the economically important production of essential oils in the world [13]. The ability of plants to accumulate essential oils is quite high in both Gymnosperms and Angiosperms, although the most commercially important essential oil plant sources are related to the Angiosperms. Most of the aromatic plants and essential oil commodities in terms of world trade belong to the families of Lamiaceae, Umbelliferae and Compositae. Essential oils are isolated from various parts of the plant, such as leaves (basil, patchouli, cedar), fruits (mandarin, fennel), bark (cinnamon), root (ginger), grass(citronella), gum (myrrh and balsam oils), berries (pimenta), seed (caraway), flowers (rose and jasmine), twigs (clove stem), wood (amyris), heartwood (cedar), and saw dust (cedar oil) [14, 15]. Chemical Constituents of Essential oils An Essential Oil contains more than 200 chemical components, but some are many times more complex. Essential oils consist of chemical compounds which have

hydrogen, carbon and oxygen as their building blocks. They can be essentially classified into two groups:

Non-volatile residue: This comprises 1-10% of the oil, containing hydrocarbons, fatty acids, sterols, carotenoids, waxes and flavonoids.

The constituents can be again subdivided into 2 groups, such as the hydrocarbons which are made up of mostly terpenes and the oxygenated compounds which are mainly alcohols, aldehydes, esters, ketones, phenols and oxides. Some of the common components are listed below along with their properties. Classes of essential oil compounds Alcohols Properties: Anti-septic, anti-viral, bactericidal and germicidal Alcohols are the compounds which contains hydroxyl groups. Alcohols exist naturally, either as a free compound or combined with terpenes or ester. When terpenes are attached to an oxygen atom and hydrogen atom, the result is an alcohol. When the terpene is monoterpene, the resulting alcohol is called a monoterpenol. Alcohols have a very low or totally absent toxic reaction in the body or on the skin. Example:

• Geraniol in geranium and palmarosa

• Citronellol found in rose, lemon and eucalyptus. Aldehydes Properties: Anti-fungal, anti-inflammatory, anti-septic, anti-viral, bactericidal, disinfectant, sedative. Medicinally, essential oils containing aldehydes are helpful in treating inflammation, Candida and viral infections. Example:

• Citral in lemon.

• Citronellal in lemongrass, lemon balm and citrus eucalyptus.

Acids Properties: anti-inflammatory. Organic acids in their free state are generally found in very small quantities within essential oils. Plant acids act as components or buffer systems to control acidity. Example:

• Cinnamic and benzoic acid in benzoin.

• Citric and lactic. Esters Esters are formed through the reaction of alcohols with acids. Essential oils containing esters are used for their soothing, balancing effects. Because of the presence of alcohol, they are effective antimicrobial agents. Medicinally, esters are characterized as antifungal and sedative, with a balancing action on the nervous system. Example:

• Linalyl acetate in bergamot and lavender.

• Geranyl formate in geranium. Ketones Properties: Anti-catarrhal, cell proliferant, expectorant, vulnery. Ketones often are found in plants that are used for upper respiratory complaints. They assist the flow of mucus and ease congestion. Essential oils containing ketones are beneficial for promoting wound healing and encouraging the formation of scar tissue. Ketones are usually very toxic. The most toxic ketone is Thujone found in mugwort, sage, tansy, thuja and wormwood oils. Other toxic ketones found in essential oils are pulegone in pennyroyal and pinocamphone in hyssops. Some non-toxic ketones are jasmone in jasmine oil, fenchone in fennel oil, carvone in spearmint and dill oil and menthone in peppermint oil. Example:

• Fenchone in fennel, carvone in spearmint and dill, menthone in peppermint.

Phenol Properties: stimulating, warming, tonifying, immune stimulating, keratolytic. Structurally similar to alcohol volatile oil in that they have an OH side group but in case of phenols the basic molecule contains a benzene ring. Example:

• Thymol in Thymus vulgaris

• Eugenol in Eugenia caryophyllata Lactones Properties: Anti-inflammatory, antiphlogistic, expectorant, febrifuge. Lactones are known to be particularly effective for their anti-inflammatory action, possibly by their role in the reduction of prostaglandin synthesis and expectorant actions. Lactones have an even stronger expectorant action than ketones. Hydrocarbon Building blocks of Essential Oil are hydrogen and carbon. Basic Hydrocarbon found in plants is isoprene having the following structure.

CH3

H2C

H2C (Isoprene) Terpenes These components generally have names ending with “ene”. For example limonene, pinene, piperene, camphene etc. These components act as an antibacterial, antiviral, anti-inflammatory, antiseptic and bactericidal. Terpenes are further categorized as monoterpene, sesquiterpene and diterpenes. When two of the isoprene units are joined head to tail, the result is a monoterpene, when three are joined, it’s a sesquiterpene and similarly four linked isoprene units are diterpenes.

Monoterpene (C10H16) Properties: Analgesic, Bactericidal, Expectorant, and Stimulant. Monoterpenes are naturally occurring compounds, the majority being

unsaturated hydrocarbons (C10). But some of their oxygenated derivatives such as alcohols, Ketones and carboxylic acids are known as monoterpenoids.

CH3 CH3

OH CH H3C 2 H3C CH3 (Limonene) (Menthol)

Two isoprene units are present in these branched-chain C10 hydrocarbons and are widely distributed in nature with more than 400 naturally occurring monoterpenes. Moreover, besides being linear derivatives (Geraniol, Citronellol), the monoterpenes can be cyclic molecules (Menthol-Monocyclic, Camphor-bicyclic, Pinenes (α and β- Pine)) genera as well. Thujone (a monoterpene) is the toxic agent found in Artemisia absinthium (wormwood) from which the liquor absinthe is prepared. Borneol and camphor are two common monoterpenes. Borneol derived from pine oil is used as a disinfectant and deodorant. Camphor is used as a counterirritant, anesthetic, expectorant and antipruritic, among many other uses.

Example: • Camphene and pinene in cypress oil. • Camphene, pinene and thujene in black pepper.

Sesquiterpene

Properties: Anti-inflammatory, anti-septic, analgesic, anti-allergic.

Sesquiterpenes are biogenetically derived from farnesyl pyrophosphate and in structure may be linear, monocyclic or bicyclic. They constitute a very large group of secondary metabolites, some having been shown to be stress compounds formed as a result of disease or injury.

Sesquiterpene Lactones

Over 500 compounds of this group are known; they are particularly characteristics of the compositae but do occur sporadically in other families. They not only have proved to be of interest from chemical and chemotaxonomic point of view, but also possess many antitumor, anti-leukemia, cytotoxic and antimicrobial activities. Chemically the compounds can be classified according to their carboxylic skeletons thus, guaianolides, pseudoguaianolides, eudesmanolides, eremophilanolides, xanthanolides, etc. can be derived from the germacranolides.

Structural features of all these compounds which appears to be associated with much of the biological activity, is the α, β -unsaturated- γ- lactones Example: Farnesene in chamomile and lavender. β-caryophyllene in basil and black pepper.

Diterpenes

Properties: Anti-fungal, expectorant, hormonal balancers, hypotensive.

Diterpenes are made up of four isoprene units. This molecule is too heavy to allow for evaporation with steam in the distillation process, so is rarely found in distilled essential oils. Diterpenes occur in all plant families and consist of compounds having a C20 skeleton. There are about 2500 known diterpenes that belong to 20 major structural types. Plant hormones gibberellins and phytol occurring as a side chain on chlorophyll are diterpenic derivatives. The biosynthesis occurs in plastids and interestingly mixture of monoterpenes and diterpenes are the major constituents of plant resins. In a similar manner to monoterpenes, diterpenes arise from metabolism of geranyl geranyl pyrophosphate (GGPP). Diterpenes have limited therapeutical importance and are used in certain sedatives (coughs) as well as in antispasmodics and anxiolytics.

Example:

Sclareol in clary sage is an example of a diterpene alcohol

Methods of extracting essential Oils

Oils contained within plant cells are liberated through heat and pressure from various parts of the plant matter; for example, the leaves, flowers, fruits, grass, roots, wood, barks, gums and blossoms. The extraction of essential oils from plant material can be achieved by various methods and can be classified into two categories.

Conventional methods Maceration Maceration actually creates more of an “infused oil" rather than an "essential oil". The plant matter is soaked in vegetable oil, heated and strained at which point it can be used for massage. Cold Pressing Cold pressing is used to extract the essential oils from citrus rinds such as orange, lemon, grapefruit and bergamot. This method involves the simple pressing of the rind at about 120 degrees Fahrenheit to extract the oil. The rinds are separated from the fruits are ground or chopped and are then pressed. The result is a watery mixture of essential oil and liquid which will separate, given time. Little, if any, alteration from the oil's original state occurs - these citrus oils retain their bright, fresh, uplifting aromas like that of smelling a wonderfully ripe fruit. It is important to note that oils extracted using this method have a relatively short shelf life, so make or purchase only what you will be using within the next six months. Enfleurage It is an intensive and traditional way of extracting oil from flowers. The process involves layering fat over the flower petals. After the fat has absorbed the essential oils, alcohol is used to separate and extract the oils from the fat. The alcohol is then evaporated and the essential oil is collected. Steam distillation and solvent extraction The steam distillation is a traditional technique for essential oils [16-20]. This is a very simple process but suffers many drawbacks: Thermal degradation, hydrolysis and solubilization of some compounds in water that alter the flavour and fragrance profile of many essential oils extracted by this technique. Chyau [21] studied on the essential oil of Glossogyne tenuifolia using a simultaneous steam distillation and solvent-extraction (SDE) apparatus for the first time. However hydro- and steam- distillation have several disadvantages, such as incomplete extraction of essential oils from plant materials, high operating temperatures with the consequent breakdown of thermally labile components, promotion of hydration reactions of chemical constituents and require a post extraction process to remove water. In case of solvent extraction, a hydrocarbon solvent is added to the plant material to help dissolve the

8 essential oil. When the solution is filtered and concentrated by distillation, a substance containing resin (resinoid), or a combination of wax and essential oil (known as concrete) remains. From the concentrate, pure alcohol is used to extract the oil. When the alcohol evaporates, the oil is left behind. Solvent extraction overcomes the drawbacks of distillation, but has the major disadvantage of solvent residue in the extracts [22]. Ideally, extraction procedures should be environmentally friendly and should not create additional pollution. Steam extraction and solvent extraction do not meet these criteria because they generate large volumes of contaminated, hazardous solvents and emit toxic fumes. Recently clean techniques, such as SFE, microwave and ultrasound, for extracting essential oils from complex matrices have been developed where they can be used routinely. Non-conventional methods Microwave assisted extraction Microwave radiation interacts with dipoles of polar and polarizable materials. The coupled forces of electric and magnetic components change direction rapidly (2450 MHz). In microwave assisted extraction (MAE), the heat of the microwave irradiation is directly transferred to the solid without absorption by the microwave- transparent solvent, the intense heating causes instantaneous heating of the residual microwave-absorbing moisture in the solid, the heated moisture evaporates, creating a high vapor pressure, the vapor pressure generated by the moisture, breaks the cell and the breakage of cell walls releases the oil trapped within it [23]. The use of microwaves for isolating essential oils has recently been reported [24, 25]. Microwave technology has allowed the development of rapid, safe and cheap methods for extracting essential oil and does not require samples devoid of water [26-28]. Recently, extraction equipment that combines microwave energy with small volumes of solvent has appeared, resulting in the procedure known as microwave-assisted extraction [29]. Results from various types of biological samples obtained by this method are qualitatively and quantitatively compared to the steam distillation method [30-34]. However, it still uses the organic solvent, not a green method. Ultrasonic assisted extraction The procedure involves the use of ultrasound with frequencies ranging from 20 - 2000 kHz; this increases the permeability of cell walls and produces cavitations.

The extraction mechanism involves two types of physical phenomena, diffusion through the cell walls and washing out the cell contents once the walls are broken, which are affected by ultrasonic irradiation. Ultrasound Assisted Extraction (UAE) has been widely used for the extraction of nutritional material, such as lipids [22], proteins [35], flavoring [36, 37], essential oils [38] and bioactive compounds e.g., flavonoids [39], carotenoids [40, 41] and polysaccharides [42-45]. Compared with traditional solvent extraction methods, ultrasound extraction can improve extraction efficiency and extraction rate, reduce extraction temperature and increase the selection ranges of the solvents [46]. Also the ultrasonic does not affect the composition of the almond oil but the ultrasonic cavitating energy can cause structure breakage of the almond powder and greatly reduce the extraction time [47]. In view of its growing use for isolating organic compounds and its significant advantages, the future introduction and dissemination of ultrasound equipment seem to be assured for more essential oils extraction. Super critical fluid extraction A supercritical fluid (SCF) is any compound at a temperature and pressure above the critical point. Above the critical temperature (Tc) of a compound, the pure, gaseous component cannot be liquefied regardless of the pressure applied. The critical pressure (Pc) is the vapor pressure of the gas at critical temperature. In the supercritical environment only one phase exists. The fluid, as it is termed, is neither a gas nor a liquid. This phase retains solvent power approximating liquids as well as the transport properties common to gases.

Fig. 1. Supercritical fluid phase diagram

Principle of supercritical fluid extraction Supercritical fluid is the region where maximum solvent capacity and the largest variations in solvents can be achieved with small changes in temperature and pressure. It offers very attractive extraction characteristic, owing to its favorable diffusivity, viscosity, surface tension and other physical properties. Its diffusivity is one to two orders of magnitude higher than those of liquids. The diffuseness facilitates rapid mass transfer and faster completion of extraction than conventional liquid solvent. The low viscosity and surface tension enable it to easily penetrate the botanical materials from which the active component is extracted. The gas like characteristics of supercritical fluid provides ideal condition for extraction of solutes giving high degree of recovery in short period of time. Supercritical Solvents The most desirable supercritical fluid solvent for extraction of natural

compounds is carbon dioxide (CO2). It is inert, inexpensive, easily available, odorless, tasteless, environmentally friendly and generally regarded as safe solvent. In

the processing with CO2 there is no solvent residue in the extract, as it becomes gas in the ambient condition. Its near ambient critical temperature, 31.1 °C, makes it ideally suitable for thermolabile natural products extraction. Due to its low latent heat of vaporization, low energy input is required for the extraction separation system. CO2

produces the most natural smelling extracts, since the hydrolysis does not occur in the process. Advantages over traditional methods

• Due to their low viscosity and relatively high diffusivity, supercritical fluids have better transport properties than liquids. • Can diffuse easily through solid materials and can therefore give faster extraction yields. • Possibility of modifying the density of the fluid by changing its pressure and temperature. • Since density is directly related to solubility by altering the extraction pressure, the solvent strength of the fluid can be modified. • Use of solvents generally recognized as safe. • The higher efficiency of the extraction process (in terms of increasing yields and lower extraction times).

• Possibility of direct coupling with analytical chromatographic techniques such as gas chromatography (GC) or supercritical fluid chromatography.

Uses of essential oils Food industry They are used to season or condiment meats, dried and cured meats, soups, ice-cream, cheese. The most commonly used essential oils are cilantrum, orange and mint. They are also used in the elaboration of alcoholic and soft drinks, especially the latter. Essences of orange, lemon, mint and fennel are used in the making of sweets and chocolates. Pharmaceutical industry They are used in toothpastes (mint and fennel essences), analgesics and decongestant inhalers (eucalyptus). Eucalyptol is also widely used in dentistry. They are used in many medicines to neutralize unpleasant tastes (essence of orange or mint). Cosmetic industry This industry uses essential oils to make cosmetics, soaps, scents, perfumes, and make-up. Geranium, lavender, roses and patchouli essences as common examples. Veterinary product industry This industry uses the essential oil of the Chenopodium ambrosoides, which is highly prized for its ascaridol (worm-killer) content. Limonene and menthol are also used to make insecticides. Industrial deodorants At present, the use of essences to disguise the unpleasant smell of industrial products like rubber, plastic and paint is being developed. Paint manufacturers use limonene as a biodegradable solvent. Toys are also scented. In the textile industry they are used to mask unpleasant smells before and after dyeing. In paper manufacture, products such as notebooks, toilet paper and face wipes are scented. Tobacco industry Requires menthol for mentholated cigarettes.

Biocides and insecticides There are certain substances such as thyme, cloves, salvia, mint, oregano, pine with bactericidal properties. Others are insecticides:

• Against ants: Mentha spicata (spearmint), Tanacetum pennyroyal. • Against aphids: garlic, other Allium, coriander, aniseed, basil. • Against fleas: lavender, mints, lemongrass, etc. • Against flies: rue, citronella, mint, etc. • Against lice: Mentha spicata, basil, rue, etc. • Against moths: mints, Hisopo, rosemary, dill, etc. • Against coleoptera: Tanacetum, cumin, wormwood, thyme, etc. • Against cockroaches: mint, wormwood, eucalyptus, laurel, etc. • Against nematodes: Tagetes, Salvia, Calendula, Asparagus, etc.

Antioxidant

Reactive oxygen and nitrogen species, ROS/RNS are essential to energy supply, detoxification, chemical signaling and immune function. They are continuously produced in the human body and they are controlled by endogenous enzymes (superoxide dismutase, glutathione peroxidase, catalase). When there is an over-production of these species, an exposure to external oxidant substances or a failure in the defense mechanisms, damage to valuable biomolecules (DNA, lipids, proteins) may occur [48]. This damage has been associated within degenerative diseases like cancer, cataract, immune system weakness and brain problems. Free radicals can also induce nutrition and medicine deterioration. Fortunately, formation of free radicals is controlled by a variety of systems which is called "antioxidant". Antioxidants are defined as compounds which can reduce oxidation rate considerably. When availability to antioxidants depot in our body cells decrease or in the cases of oxidants attack (like smoking, air pollution, inflammation and ischemia) free radicals induce oxidative damages [49].

Antioxidant property of Phenolic compound

The term “phenolic” is used to define substances that possess one or more hydroxyl groups (OH) substituent bonded onto an aromatic ring. Compounds that have several or many phenolic hydroxyl substituents are often referred to as polyphenols. Due to its chemical structure phenolic compounds have the ability of

13 phenoxide ion delocalization. The phenoxide ion can lose a further electron to form the corresponding radical which can also delocalize. In reference to this property, phenolic compounds have radical scavenging and antioxidant activity. Phenolics are large and heterogeneous groups of secondary plant metabolites that are distributed throughout the plant kingdom. They have been implicated in a number of varied roles including UV protection, pigmentation, disease resistance and nodule production. They are well known as radical scavengers, metal chelators, reducing agents, hydrogen donors and singlet oxygen quenchers. Phenolics have a wide variety of structures. Flavonoids, tannins and phenolic acids are the main phenolic compounds.

Antioxidant activity of essential oils

Use of natural antioxidants, as food additives for inactivating free radicals receiving a lot of attention nowadays, not only for their scavenging properties, but also because they are natural, non-synthetic products and their appreciation by consumers is very favourable. Some known natural aroma components, such as 4- hydroxy-2, 5-dimethyl-3(2H)-furanone, maltol and 5-hydroxy methylfurfural have been reported to possess appreciable antioxidant activities. There is now evidence of beneficial influence of essential oils on lipid metabolism, ability to stimulate digestion and antioxidant properties. Eugenol (clove), linalool (coriander) and cuminaldehyde (cumin) were found to be effective antioxidants. These compounds inhibited lipid peroxidation by quenching oxygen free radicals and by enhancing the activity of endogenous antioxidant enzymes, superoxide dismutase, catalase, glutathione peroxidase and glutathione transferase. At the same time, there was no alteration in the fatty acid composition of membrane lipids and the levels of endogenous antioxidants, vitamin E, ascorbic acid and glutathione.

Objective of work

Essential oils (EOs) are valuable natural products that are popular nowadays in the world due to their effects on the health conditions of human beings and their role in preventing and curing diseases. In addition, essential oils have a broad range of applications in foods, perfumes, cosmetics and human nutrition. The composition of essential oil may vary considerably between plant species and varieties and within the same variety but with different climates, seasonal and geographic conditions and harvest periods. In addition, the composition of essential oil from different parts of the

same plant can also vary widely. Considering all the aforementioned differences in essential oil composition, it is clear that only a detailed knowledge of constitutes of an essential oil will lead to a proper application of its components. However, such a detailed knowledge can only be obtained by means of applying suitable extraction techniques and carefully performed chromatographic analysis. The essential oil from plants has usually been isolated by either steam distillation or solvent extraction. These techniques present some shortcomings, namely losses of volatile compounds, low extraction efficiency, long extraction time and degradation of unsaturated compounds and toxic solvent residue, which makes it mandatory to use the alternative techniques for the extraction of essential oils.

Phenolic compounds in plants are recognized as important compounds in conferring stability against oxidation. Natural antioxidant phenolics can be classified into a lipophilic group, tocopherols and a hydrophilic group, including simple phenolics, phenolic acids, anthocyanins, flavonoids and tannins. Aromatic plant extracts rich in, phenol rich essential oils are gaining interest because of their relatively safe and wide acceptance by consumers. Antioxidant properties of phenolic compounds also play a vital role in the stability of food products, as well as in the antioxidative defense mechanisms of biological systems. The aim of this work is to develop a method for the principal application of environmentally safe technology,

SC-CO2 extraction technique for extraction of essential oils, optimization of SC-CO2 parameters and compare the results with those obtained by conventional techniques, in order to introduce this advantageous solvent free alternative in the pharmaceutical field.

Aims and Objectives

The main objectives of the study are:

1. To compare the effectiveness of the conventional methods with SC-CO2 extraction.

2. Optimization of SC-CO2 extraction parameters for extraction of essential oils. 3. To perform the qualitative analysis of the extracted essential oil. 4. Identification of essential oils. 5. To perform the in vitro bioactivity.

PLAN OF WORK 1. Collection of plant material Table 1: List of plants selected for proposed work S. No. Common Name Botanical Name 1. Cinnamon (Bark) Cinnamomum zeylanicum N. 2. Cinnamon (Leaf) Cinnamomum tamala N. 3. Eucalyptus (Leaf) Eucalyptus globulus L. 4. Ajowan (seed) Tachyspermum ammi L. 2. Sample preparation (extraction of essential oils) • Hydro distillation • Solvent extraction • Ultrasonic assisted extraction

• SC-CO2 extraction

3. Optimization of SC-CO2 extraction parameters • Factorial design • Contour/Surface plots • Optimized conditions 4. Quality control parameters/Physicochemical properties

• Optical rotation • Specific gravity • Refractive index • Solubility in alcohol • Aldehyde content • Acid value • Ester value • Total Phenolic content • Total Anthocyanin content 5. Development of analytical methods • SEM • HPTLC • GC-MS • FTIR 6. In vitro bioactivity • Antioxidant activity

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22A CHAPTER 2

LITERATURE REVIEW 1. Cinnamomum zeylanicum N. Synonym – Kalmi dalchini Part used – Bark

Fig. 1. Cinnamomum zeylanicum (Dalchini)

Taxonomy

Kingdom Plantae Subkingdom Tracheobionta Superdivison Spermatophyta Division Magnoliophyta Class Magnoliopsida Subclass Magnolidae Order Laurales Family Lauraceae Genus Cinnamomum Species Cinnamomum zeylanicum

Phytochemistry

Cinnamon bark yields 0.4-0.8% oil, C. verum cinnamon bark oil contains cinnamic aldehyde (usually 60-80%) as its major component, other major constituents include sesquiterpenoids (4-5%) (e.g., α-humulene and β-caryophyllene that make up 3–4% of the total, limonene and others), eugenol, eugenol acetate, cinnamyl acetate, cinnamyl alcohol, methyl eugenol, benzaldehyde, cuminaldehyde, benzyl benzoate, monoterpenes (e.g., linalool, pinene, phellandrene, and cymene), carophyllene, safrole and others.

The antioxidant activities of various parts (barks, buds and leaves) of Cinnamomum cassia was investigated by extracting with ethanol and SFE. For the antioxidant activity comparison, IC50 values of the SFE and ethanol extracts in the DPPH scavenging assay were 0.562-10.090 mg/mL and 0.072-0.208 mg/mL, and the Trolox equivalent antioxidant capacity (TEAC) values were 133.039-335.779 mmole Trolox/g and 6.789-58.335 mmole Trolox/g, respectively. In addition, the total flavonoid contents were 0.031-1.916 g/100 g dry weight of materials (DW) and 2.030-3.348 g/100 g DW and the total phenolic contents were 0.151-2.018 g/100 g

DW and 6.313-9.534 g/100 g DW in the SC-CO2 and ethanol extracts, respectively. Based on the results, it was concluded that the ethanol extracts of Cinnamon barks have potential value as an antioxidant substitute and also SC-CO2 is a better technique to extract the natural antioxidant substances from C. cassia.

The genus Cinnamomum comprises of several hundreds of species which are distributed in Asia and Australia. Cinnamomum zeylanicum, the source of cinnamon bark and leaf oils, is an indigenous tree of Sri Lanka, although most oil now comes from cultivated areas. C. zeylanicum is an important spice and aromatic crop having wide applications in flavoring, perfumery, beverages and medicines. Volatile oils from different parts of cinnamon such as leaves, bark, fruits, root, flowers and buds have been isolated by hydro-distillation/steam distillation and SFE. The chemical compositions of the volatile oils have been identified by GC and GC-MS. More than 80 compounds were identified from different parts of cinnamon. The leaf oil has a major component called eugenol. Cinnamaldehyde and camphor have been reported to be the major components of volatile oils from stem bark and root bark, respectively. Trans-cinnamyl acetate was found to be the major compound in fruits, flowers and fruit stalks. These volatile oils were found to exhibit antioxidant, antimicrobial and antidiabetic activities. C. zeylanicum bark and fruits were found to contain proanthocyandins with doubly linked bis-flavan-3-ol units in the molecule. The present review provides a coherent presentation of scattered literature on the chemistry, biogenesis, and biological activities of cinnamon [5].

SC-CO2 extraction of the volatile oil from cinnamon bark was carried out under different conditions of temperature (30, 50 and 70 °C), pressure (16, 20 and 24 MPa) and dynamic extraction time (20, 40, 60 and 80 min), in order to evaluate their influence on the global yield and the composition of the volatile oil using a pilot-scale

supercritical fluid extraction (SFE) system. The chemical composition of each SFE sample was analyzed by gas chromatography- mass spectrometry (GC-MS) [6].

The volatile oil of the bark of Cinnamomum zeylanicum was extracted by

means of SC-CO2 extraction in different conditions of pressure and temperature. Its chemical composition was characterized by GC-MS analysis. Nineteen compounds, which in the supercritical extract represented >95% of the oil, were identified. (E)- Cinnamaldehyde (77.1%), (E)-β-caryophyllene (6.0%), α-terpinol (4.4%) and eugenol (3.0%) were found to be the major constituents [7].

Volatile oils from different parts of cinnamon such as leaves, bark, fruits, root bark, flowers and buds have been isolated by hydro distillation/steam distillation and SFE. The chemical compositions of the volatile oils have been identified by GC and GC-MS. More than 80 compounds were identified from different parts of cinnamon. The leaf oil has a major component called eugenol. Cinnamaldehyde and camphor have been reported to be the major components of volatile oils from stem bark and root bark, respectively. Trans-cinnamyl acetate was found to be the major compound in fruits, flowers and fruit stalks. These volatile oils were found to exhibit antioxidant, antimicrobial and antidiabetic activities. C. zeylanicum bark and fruits were found to contain proanthocyandins with doubly linked bisflavan-3-ol units in the molecule [8].

Solvent-assisted supercritical fluid extraction is superior to Soxhlet extraction, solvent extraction at reflux and simultaneous steam distillation solvent extraction for the isolation of semi volatile compounds from the cinnamons of commerce. Optimized extraction conditions are described using carbon-dioxide at 300 bar and 70°C to extract 0.5 g of powdered cinnamon mixed with 4.5 g of anhydrous sodium sulfate to which 1.0 ml of acetonitrile was added at the start of the sequential 30 min static and 30 min dynamic extraction. The semi volatile compounds were collected by solvent trapping in ethyl acetate and analyzed directly by series coupled-column gas chromatography after filtration and partial solvent evaporation. Twenty-one compounds identified by mass spectrometry were quantified in twenty-four samples of cinnamon and cassia purchased in Sri Lanka, Korea, United Kingdom, and the United States of America. True cinnamon is easily distinguished from cassia by the presence of eugenol, absence of δ-cadinene, much lower amounts of coumarin and larger amounts of benzyl benzoate. Principal component analysis using the relative composition of the twelve major semivolatile compounds provides a clear distinction

25 between true cinnamon and cassia as well as useful interspecies differences which can be used for further classification [9].

2. Cinnamomum tamala N. Synonym – Tejpat Part used – Leaves

Fig. 2. Cinnamomum tamala (Tejpat)

Taxonomy

Kingdom Plantae Subkingdom Tracheobionta Superdivison Spermatophyta Divison Magnoliophyta Class Magnoliopsida Subclass Magnolidae Order Laurales Family Lauraceae Genus Cinnamomum Species Cinnamomum tamala

Phytochemistry

Cinnamon leaf oil contains high concentrations of eugenol (Ceylon type 80- 88%; Seychelles type 87-96%), it also contains many of the major constituents present in cinnamon bark oil (e.g., benzyl benzoate (6%), cinnamaldehyde, cinnamyl acetate, eugenol acetate, benzaldehyde, linalool, α-terpinene, and others) as well as

26 other minor compounds including α-humulene, β-caryophyllene, α-ylangene, methyl cinnamate and cinnamyl acetate.

Variations in constituents of leaf essential oils are characterized by enantio- GC-FID, capillary GC-FID and GC-MS. The oil samples were analyzed for two consecutive years. (E)-Cinnamaldehyde which was the principal component, was higher in the first year oil samples but lower in the second year samples. Similarly, (Z)-cinnamaldehyde was 5.8-7.1% in the first year oils and 1-1.1% in the second year oils. Linalool content varied (6.4-8.5% and 5.7-16.2%, respectively) in first and second year oil samples, while (E)-cinnamyl acetate was higher (10.0-22.7%) in oil samples of the second year collection. Using a 10% heptakis(2,3-di-O- methoxymethyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin as the chiral stationary phase, optically pure (3S)-(+)-linalool was found in both the oil samples leading to complete enantiomeric excess for (3S)-(+)-enantiomer [10].

The leaf terpenoid compositions of nine Lauraceae species, viz., Neolitsea pallens, Lindera pulcherrima, Dodecadenia grandiflora, Persea duthiei, Persea odoratissima, Persea gamblei, Phoebe lanceolata, Cinnamomum tamala and Cinnamomum camphora collected from the Himalayan region (India) were examined by GC, GC-MS and NMR analyses in order to determine the similarities and differences among their volatile constituents. Furano-sesquiterpenoids were the principal constituents of N. pallens, L. pulcherrima, and D. grandiflora. (E)- Nerolidol, limonene, β-pinene, and α-pinene were the major constituents of P. duthiei; α-pinene, sabinene and β-caryophyllene were predominant in P. odoratissima, while the oils of P. gamblei and P. lanceolata possessed β- caryophyllene as common major constituent. C. camphora and C. tamala were marked by the presence of camphor and cinnamaldehyde, respectively. Cluster analysis of the oil composition was carried out in order to discern the differences and similarities within nine species of six genera of Lauraceae [11].

The leaves of Cinnamomum tamala L. (Lauraceae) were collected from seven plants occurring in seven different areas of Manipur and analyzed for their essential oil and eugenol contents using GC and GC/MS. The yield of the oil was found to vary from 1.5-5.6% (w/w), on a dry weight basis. A total of 79 compounds were identified in the oils. Eugenol was found as a major compound in the leaf oils and its concentration varied from 35.1-94.3% followed by α-phellandrene (1.0-25.4%) [12].

Eugenol is used as a flavor in the food industry, has a variety of biological activity and can serve as a biomarker. Because eugenol is present in the leaves of Cinnamomum tamala Nees and Eberm., which are used as a spice, a sensitive and reliable quantitative high performance thin-layer chromatographic method has been established for quantification of the compound in the leaves of the plant. A methanol extracts of the powder of dried leaves of Cinnamomum tamala Nees and Eberm. was applied to silica gel 60 F254 TLC plates and these were developed with toluene- ethylacetate-formic acid, 90:10:01 (v/v/v), as mobile phase. Detection and quantitation was performed by densitometry at λ = 280 nm. The accuracy of the method was checked by conducting recovery studies for two different levels of eugenol, the average recovery was found to be 98.39% [13].

A hydro-distilled oil was obtained from the leaves of Cinnamomum tamala Nees and Eberm., grown in India, was analysed by capillary GC and GC-MS. The leaf volatile oil contained 40 constituents, of which high proportions were monoterpenes (65.6%). The predominant monoterpenes were trans-sabinene hydrate (29.8%), (Z)-β- ocimene (17.9%), myrcene (4.6%), α-pinene (3.1%) and β-sabinene (2.3%). Among 21 sesquiterpenes (32.9%), the major ones were germacrene A (11.3%) and α- gurjunene (4.7%) [14].

Water distilled essential oil of Cinnamomum tamala (Ham) Nees and Eberm (Lauraceae) leaves was analysed by GC_MS. Sixty three compounds representing 94.3% of the oil were identified. β-Caryophyllene (25.3%), Linalool (13.4%) and caryophyllene oxide (10.3%) were the main constituents of the oil obtained in 0.03% yield [15].

3. Eucalyptus globulus L. Synonym - Eucalyptus, Dinkum oil Part used- Fresh and Dry leaf

Fig. 3. Eucalyptus globulus

Taxonomy

Kingdom Plantae Subkingdom Tracheiobionta Superdivision Spermatophyta Division Magnoliophyta Class Magnoliopsida Order Myrtales Family Myrtaceae Genus Eucalyptus Species Eucalyptus globulus

Phytochemistry

Eucalyptus leaves contain 0.5-3.5% volatile oil, tannins, polyphenolic acids (Gallic, caffeic, ferulic, gentisic, protocatechuic acids, etc.), flavonoids (quercetin, quercitrin, rutin, hyperoside, eucalyptin etc.), wax and others. Eucalyptus oil contains usually 70-85% of eucalyptol (1,8-cineole); other constituents present are mostly monoterpene hydrocarbons (α-pinene, δ-limonene, p-cymene, β-pinene, α- phellandrene, camphene, γ-terpinene, etc., with the first three in major amounts), with lesser amounts of sesquiterpenes (e.g., aromadendrene, allo-aromadendrene, globulol,

epiglobulol, ledol and viridiflorol), aldehydes (e.g, myrtenal), ketones (e.g, carvone and pinocarvone) and others.

The SFE of E. globulus bark was carried out with carbon-dioxide at 100, 140 and 200 bar at 40, 50 and 60 °C, and the extracts were analyzed by GC–MS. The results were compared with those obtained by soxhlet with dichloromethane and the influence of the operating conditions upon the global yield and the individual yields of the triterpenic acids was discussed [16].

The solubility of 1, 8-cineole, α-pinene, limonene, their mixtures and the

extract of eucalyptus leaf oils in SC-CO2 were determined using the dynamic method at 80, 100, 150 and 250 bar and 40 and 60 °C for each pressure. The results showed an increase in the solubility of all oils with an increase in pressure and a decrease with temperature. The mixtures of both pure blends as well as the eucalyptus oil extracts exhibited lower solubility than the pure single oil components in the same conditions. The oil of E. radiata exhibited a better separation of 1, 8-cineole at 80 bar and 60 °C [17]. The volatile oil compositions of Eucalyptus camaldulensis var. brevirostris leaves obtained by hydro-distillation (HD) and SFE were analysed qualitatively and quantitatively by GLC-MS. Ninety different components were separated and most of them were identified. In both extracts the main constituents were found to be b- phellandrene (8.94 and 4.09%), p-cymene (24.01 and 10.61%), cryptone (12.71 and 9.82%) and spathulenol (14.43 and 13.14%). The yield of the monoterpene hydrocarbons in HD oil (0.288 g/100 g fresh leaves) was slightly higher compared

with that in the SFE extract (0.242 g/100 g fresh leaves). The SC-CO2 extract possessed higher concentrations of the sesquiterpenes, light oxygenated compounds and heavy oxygenated compounds than the HD oil. The relationship between the antioxidant activity and chemical composition of the extracted oils was investigated. The concentration of both compounds, but especially that of p-cymen-7-ol (2.25%), is higher in the SC-CO2 extract. This corresponds with the higher antioxidative activity

of the SC-CO2 compared with the HD extract. p-cymen-7-ol, a compound newly identified in leaves of Eucalyptus species, exhibited superior antioxidant activity in comparison with that of butylated hydroxyanisole [18].

4. Trachyspermum ammi L. Synonym- Ajwain, Bishop seed Part used- Dried ripe seeds

Fig. 4. Trachyspermum ammi (Ajowan)

Taxonomy

Kingdom Plantae Subkingdom Tracheobionta Superdivison Spermatophyta Division Magnoliophyta Class Magnoliopsida Subclass Rosidae Order Apiales Family Apiaceae Genus Trachyspermum Species Trachyspermum ammi

Phytochemistry Major chemical constituents responsible for physicochemical and therapeutic action of the herb are 2 to 4 % brownish essential oil with thymol as the major constituent (35 to 60 %), p-cymene about 50.0 to 55.0 % and terpinene about 30.0 to 35.0 %, fat (18.0 to 21.0 %), proteins (12.0 to 18.0 %), carbohydrates (20.0 to 25.0 %). The non-thymol fraction (thymene) contains para-cymene, γ-terpenine, α- and β- pinenes, dipentene, α-terpinene and carvacrol. Minute amounts of camphene,

myrcene, and α-3-carene have also been found in the plant. Alcoholic extracts contain a highly hygroscopic saponin. The principal oil constituents of T. ammi are carvone (46%), limonene (38%) and dillapiole (9%).

A volatile concentrate of caraway was isolated by SC-CO2 extraction coupled to a fractional separation technique. GC-MS analysis of the various fractions obtained at different extraction and fractionation conditions allowed the identification of the operating conditions to isolate the volatile concentrate. A good extraction performance was obtained operating at 90 bar and 50 °C. The optimum fractionation was achieved by operating at 90 bars and -12 °C in the first separator and at 10 bars and 17 °C in the second. The influence of the extraction time on the composition was evaluated. A comparison with the hydro-distilled was also proposed [1].

Essential oil of Carum copticum cultivated in Iran was obtained by hydro distillation and SC-CO2 extraction methods. The oils were analyzed by capillary gas chromatography, using flame ionization and mass spectrometric detection. The compounds were identified according to their retention indices and mass spectra (EI, 70 eV). The effects of different parameters such as pressure, temperature, modifier volume and extraction time, on the supercritical fluid extraction of C. copticum oil were investigated. The results showed that under pressure of 30.4 MPa, temperature 35 °C, methanol 0% and dynamic extraction time of 30 min, the method was most selective for the extraction of thymol. Eight compounds were identified in the hydrodistilled oil. The major components of C. copticum were thymol (49.0%), c- terpinene (30.8%), p-cymene (15.7), b-pinene (2.1%), myrcene (0.8%) and Limonene (0.7%). However, by using supercritical carbon-dioxide under optimum conditions, only three components constituted more than 99% of the oil. The extraction yield, based on hydro distillation was 2.8% (v/w). Extraction yield based on the SFE varied in the range of 1.0–5.8% (w/w) under different conditions. The results show that in Iranian C. copticum oil, thymol is a major component [2].

The process conditions during the extraction of carvone and limonene from

caraway seed (Carum carvi L.) with SC-CO2 have been optimized with respect to

pressure, temperature and CO2 flow and extraction time in order to selectively obtain the essential oil. Using Soxhlet extraction, limonene, carvone and fatty oils in both the raw material and the residual matrix material were extracted. From GC analysis of both extracts and the isolated compounds using SFE yields for limonene, carvone and

32 total oil (C16, C18) were determined. The highest yield of carvone and limonene was obtained at an extraction pressure 125 bar and a temperature 32 °C. CO2 flow rate and extraction time both affected the yield of carvone and limonene under these conditions [3].

Caraway seeds were extracted with liquid and SC-CO2 at 23-40 °C and 90- 100 bar. The extracts were analyzed for carvone and limonene and were compared with essential oil obtained by steam distillation. Slight fractionation was observed during the extraction. The effect of process parameters such as temperature, pressure and solvent flow rate and direction, on essential oil recovery was described using a simple model with mass transfer between a liquid mixture of essential and fatty oils and the solvent, and with diffusion resistances both in the liquid mixture and in the walls of caraway cells [4].

References:

1. M. Cabizza, G. Cherch, B. Marongiu and S. Porcedda, Isolation of a volatile concentrate of caraway seed. J. Essent. Oil Res., 2001; 13: 371-375. 2. M. Khajeh, Y. Yamini, F. Sefidkon and N. Bahramifar, Comparison of essential oil composition of Carum copticum obtained by supercritical carbon dioxide extraction and hydrodistillation methods. Food Chem., 2004; 86: 587- 591. 3. T. Baysal and D.A.J. Starmans, Supercritical carbon dioxide extraction of carvone and limonene from caraway seed. J. Supercrit. Fluids., 1999; 14: 225- 234. 4. H. Sovová, R. Komers, J. Kuc̆ era and J. Jez̆ , Supercritical carbon dioxide extraction of caraway essential oil. Chem. Eng. Sci., 1994; 49: 2499-2505. 5. G.K. Jayaprakasha and J.M. Rao, Chemistry, Biogenesis, and Biological Activities of Cinnamomum zeylanicum. Food Sci Nutr., 2011; 57: 547-562. 6. H. Baseri, M.N. Lotfollahi and A. Haghighi, Effects of some experimental parameters on yield and composition of supercritical carbon dioxide extracts of cinnamon bark. J. Food Process. Eng., 2011; 34: 293-303. 7. B. Marongiu, A. Piras, S. Porcedda, E. Tuveri, E. Sanjust, M. Meli, F. Sollai,

P. Zucca and A. Rescigno, Supercritical CO2 extract of Cinnamomum zeylanicum: Chemical characterization and antityrosinase activity. J. Agric. Food Chem., 2007; 55: 10022-10027. 8. G.K. Jayaprakasha, M.O. Kameyama, H. Ono, M. Yoshida and J. Rao, Phenolic constituents in the fruits of Cinnamomum zeylanicum and their antioxidant activity. J. Agr Food Chem., 2006; 54: 1672-1679. 9. K.G. Miller, C.F. Poole and T.N.P. Chichila, Solvent-assisted supercritical fluid extraction for the isolation of semi volatile flavor compounds from the cinnamons of commerce and their separation by series-coupled column gas chromatography. J. Sep. Sci., 1995; 18: 461-471. 10. C.S. Chanotiya and A. Yadav, Enantioenriched (3S)-(+)-Linalool in the Leaf Oil of Cinnamomum tamala Nees et Eberm. from Kumaon. J. Essent. Oil Res., 2010; 22: 593-596.

11. S.C. Joshi, R.C. Padalia, D.S. Bisht and C.S. Mathela, Terpenoid Diversity in the Leaf Essential Oils of Himalayan Lauraceae Species. Chem Biodivers., 2009; 6: 1364-1373. 12. V.S. Rana, C.B. Devi, M. Verdeguer and M. Blázquez, Variation of Terpenoids Constituents in Natural Population of Cinnamomum tamala (L.) Leaves. J. Essent. Oil Res., 2009; 21: 531-534. 13. V. Dighe, A. Gursale, R. Sane, S. Menon and S. Raje, Quantification of eugenol in Cinnamomum tamala nees and eberm. leaf powder by highperformance thin-layer.Chromatography. J. Planar. Chromatogr., 2005; 18: 305-307. 14. R.M. Showkat, A. Mohammaed and R. Kapoor, Chemical composition of essential oil of Cinnamomum tamala Nees and Eberm. Leaves. Flavour Frag. J., 2004; 19: 112-114. 15. A. Ahmed, M.I. Choudhary, A. Farooq and E. Demirci, Essential oil constituents of the spice Cinnamomum tamala (Ham.) Nees & Eberm. Flavour Frag. J., 2000; 15: 277-289. 16. M.R. Melo, E.L.G. Oliveira, A.J.D. Silvestre and C.M. Silva, Supercritical fluid extraction of triterpenic acids from Eucalyptus globulus bark. J. Supercrit. Fluids., 2012; 70: 137-145. 17. J.C. Francisco and B. Sivik, Solubility of three monoterpenes, their mixtures and eucalyptus leaf oils in dense carbon dioxide. J. Supercrit. Fluids., 2002; 23: 11-19. 18. H. Fadel, M. Marx, A.E. Sawy and A.E. Ghorab, Effect of extraction techniques on the chemical composition and antioxidant activity of Eucalyptus camaldulensis var. brevirostris leaf oils. Z Lebensm Unters Forsch A., 1999; 208: 212-216.

CHAPTER 3

MATERIALS AND METHODS

MATERIAL AND METHODS

1. Collection of plant material

Drug samples were collected from market and campus of AMU, Aligarh.

2. Identification and authentication of plant material

Before carrying out the research work, the collected plant materials were authenticated in Department of Ilmul Advia by Dr. Abdul Latif (Associate Professor and Chairman, Aligarh Muslim University, Aligarh). Voucher specimens of same material were deposited in the museum of the same department.

3. Extraction of essential oil

3.1. Extraction of essential oil from different plant samples using Hydro- distillation method

Procedure

The hydro-distillation was accomplished in Clevenger-type apparatus. The dried plant materials were grounded by domestic grinder to obtain coarse powder and were then sieved (50 mesh) and used for extraction. About 150 g grounded powder and 500 mL distilled water was mixed in round bottomed flask (1 L). The system was boiled using a 2 L heating mantle for 5 h to yield essential oil. The oil is less dense than water, hence floats on water which is then collected. The extract was centrifuged at 10,000 rpm for 10 min in order to separate small water droplets present in the essential oil. The oil was kept at 4 oC until further use and percentage yield was calculated [1].

3.2. Extraction of essential oil from different plant samples using Solvent Extraction

Procedure

The dried plant materials were grounded by domestic grinder to obtain coarse powder and were then sieved (50 mesh) and used for extraction. About 150 g coarse powder was extracted using solvent hexane (100 mL) was kept in conical flask in 1:2 ratio for 24 hrs. The extract was then filtered and concentrated under reduced pressure in rotary vacuum evaporator at 40 °C. The oil thus obtained was kept at 4 oC for further quantification. The percentage yield was calculated.

3.3. Extraction of essential oil from different plant samples using Ultrasonic Assisted Extraction

Procedure

The dried plant materials were grounded by domestic grinder to obtain coarse powder and were then sieved (50 mesh) and used for extraction. The 150 g of coarse powder were taken in a 250 mL conical flask along with 100 mL hexane. The flask was covered and then placed in ultrasound water bath apparatus for 30 min. Ultrasonic extractions were performed with an ultrasound water bath (frequency 33 kHz). The temperature of the water bath was held constant at 25 °C. The extract was filtered and concentrated under reduced pressure in rotary vacuum evaporator. The oil thus obtained was kept at 4 °C for further quantification. The percentage yield was calculated [2].

3.4. Extraction of essential oil from plant samples using SC-CO2 Extraction

Procedure

A SFE-1000M1- 2- C50 system (Pittsburgh, PA) was used for all extractions. The extraction vessel was 200 mL stainless steel. Grounded plant materials (150 g)

were loaded in the extractor. The CO2 was allowed to pass through the seed matrix at desired pressure, temperature and flow rate. After the completion of extraction, the extracted oil was collected from the collecting vessel, percentage yield was calculated

and stored for further analysis [3]. SC-CO2 extraction parameters were decided by design of experiment, extraction of essential oils from various drugs was carried out and optimum conditions to get maximum yield were obtained.

4. Optimization of SC-CO2 extraction parameters

4.1. Design of Experiment (DOE)

Factorial design

Factorial design was used to examine the relationship among the experimental factors. An experiment using factorial design allows one to examine simultaneously the effects of multiple independent variables at their different levels and their degree

of interaction. Moreover, factorial designs provide information on whether the factors act on the experimental units independently of one another or not, indicating the presence or absence of interaction between the factors. Factorial designs allow one to study the change in response produced by changing the level of the factor which is called the main effect and the difference in response between the levels of one factor and all levels of another factor which is called the interaction [4, 5].

A two level factorial design is called the special case of factorial design. This type of design is used to find p-value when the variables are at 2 levels. Factors are denoted by A, B, C...., and treatment conditions are by a, b, c..... The levels are denoted as +1 and -1 for high level and low level, respectively. A complete set of this design is required 2p factorial design. Statistical model for a 2p design would include p main effects, two interactions, three factor interactions and one p-factor 푃 푃 interaction [4]. 퐶2 퐶3 In this study, optimization was done by using factorial design with three factors A, B and C were denoted for Pressure, Temperature and Flow rate respectively, while output response is % yield.

4.2. Contour/ 3D Surface plots

A contour plot is used to explore the potential relationship between three variables. Contour plots display the 3-dimensional relationship in two dimensions, with x- and y-factors (predictors) plotted on the x- and y-scales and response values represented by contours. A contour plot is like a topographical map in which x-, y-, and z-values are plotted instead of longitude, latitude and elevation.

3D surface plots are graphs that can be used to explore the potential relationship between three variables. The predictor variables are displayed on the x- and y-scales and the response (z) variable is represented by a smooth surface (3D surface plot).

Characteristic patterns that occur in contour plots and 3D surface plots

Simple maximum pattern

The following 3D surface and contour plots represent a response surface with a simple maximum. As the colour gets darker, the response increases.

Simple maximum 3D surface plot Simple maximum contour plot

Minimax pattern

The following 3D surface and contour plots represent a minimax response surface. As the color gets darker, the response increases. Note the relationship between the shape of the surface and the shape of the contours. From the stationary point (saddle point) near the center of the design, increasing or decreasing both factors at the same time leads to a decrease in the response. But from the stationary point (saddle point), increasing either factor while decreasing the other leads to an increase in the response.

Minimax 3D surface plot Minimax contour plot

Stationary ridge pattern

The following 3D surface and contour plots represent a stationary ridge surface. As the color gets darker, the response increases. A stationary ridge is shaped like an arch. In these graphs, there are many possible factor settings that maximize the response.

Stationary ridge 3D surface plot Stationary ridge contour plot

Rising ridge pattern

The following 3D surface and contour plots represent a rising ridge surface. As the color gets darker, the response increases. The response increases as you decrease time and increase temperature at the same time.

Rising ridge 3D surface plot Rising ridge contour plot

4.3. Optimized conditions

Response optimization helps to identify the combination of variable settings that jointly optimize a single response or a set of responses. This is useful when there is a need to evaluate the impact of multiple variables on a response.

In Minitab, Response Optimizer is used to search for optimal responses based on the requirements defined for each response:

• Minimize the response (smaller is better, for example, cost) • Target the response (target is best, for example, part dimension) • Maximize the response (higher is better, for example, flavour, yield)

5. Quality control analysis of volatile oils

5.1. Physicochemical Characterization of Essential Oil 5.1.1. Organoleptic properties

The essential oil obtained by extraction process was characterized for the physical properties like nature, color and odor. The volatile oil was placed in transparent bottle over white background and color and clarity was observed, the characteristic odor was determined by sniffing and to determine its characteristic feel to touch, it was rubbed between fingers.

5.1.2 Determination of specific gravity

Procedure

The actual weight or the tare of a vial was determined accurately. The vial was filled with water and weighed. The procedure was repeated using the volatile oil in place of water. The specific gravity of the oil is expressed as the ratio of the weight of the volume of oil to that of an equal volume of pure water when both are determined at 25 °C. The specific gravity [6] was determined using the formula:

Specific gravity = (1)

Weight of essential oil Weight of an equal volume of distilled water weight of essential oil = weight of bottle filled with oil – weight of empty bottle

5.1.3. Determination of refractive index

Procedure

The Abb's refractometer was used for the determination of refractive index. The test plate was attached to the refracting prism of the Abb’s refractometer. The light was focused on the test plate. The instrument was adjusted until the borderline of the critical angle coincides with the cross hairs in the telescope and the reading of the refractive index was taken. The refractometer was standardized with distilled water which has refractive index of nD29.5= 1.3315. Then, it was cleaned with acetone and dried with cotton. The test plate was removed, cleaned and 2‐3 drops of the volatile oil were placed on the prism and the prism was clamped together firmly. The light source was fixed so that the light is reflected through the prisms and the instrument adjusted until the borderline between the light and dark halves of the field of view

exactly coincides with the cross hairs of the telescope. The refractive index was then read. The refractive index is denoted by nD25 where n is the refractive index at 25°C taken with sodium light (D - line) [6]. 5.1.4. Determination of optical rotation Procedure The extent of optical activity of oil was determined by a polarimeter which measured the degree of rotation. 10 mL polarimeter tube was cleaned, dried and filled with 10% alcoholic solution of the volatile oil. Care was taken in filling the tube to avoid the entrance of air bubble which could disturb the rotation of light. It was then placed in the trough of the instrument between polarizer and analyzer. The analyzer was rotated until equal illumination of light of the two halves of the visual field is achieved. The zero point of the polarimeter was adjusted and determined. The direction of rotation was determined, if the analyzer was turned counter clock wise from the zero position to obtain the final reading, the rotation is dextro (+) if clock wise and levo (-) if anti clockwise. The zero correction is the average of the blank reading and is subtracted from the average observed rotation, if 2 figures are of the same sign or added if they are opposite in sign [6]. 5.1.5. Determination of solubility in alcohol Procedure Essential oil (0.5 mL) was placed in a 10 mL of glass-stoppered cylinder. It was then kept in a constant temperature device, maintained at a temperature of 20 ± 0.2 °C. A burette was filled with alcohol (95%), the alcohol was added by increments of 0.1 mL until solution is completely clear, shaken frequently and vigorously. The volume of alcohol added was recorded when a clear solution has been obtained [7]. 5.1.6. Determination of Acid value Reagents

• Phenolphthalein indicator • 0.1M Potassium hydroxide titrant - Potassium hydroxide (5.61 g) was placed in a 100 mL volumetric flask. Made up to the mark with water. • Ethanol-ether solution - Prepared a mixture of ethanol (95%) and diethyl ether (1:1, v/v).

Procedure

Essential oil (1.0 gm) was dissolved in 5 mL of a mixture of equal volumes of ethanol (95%) and ether, previously neutralized with 0.1M potassium hydroxide to phenolphthalein solution. As the sample does not dissolve in the cold solvent, flask was connected with the reflux condenser and warmed slowly, with frequent shaking, until the sample dissolves. 1 mL of phenolphthalein solution was added and titrated with 0.1M potassium hydroxide until the solution remains faintly pink after shaking for 30 sec. (I. P., 2007), the acid value was calculated from the expression [6].

Acid value = 5.61 n/w (2) where n = number of mL of 0.1M potassium hydroxide required, w = weight in gm of the substance

5.1.7. Determination of ester value (Saponification value) The acid value and the saponification value of the substance under examination were determined. The ester value was calculated from the expression:

Ester value = Saponification value – Acid value (3)

Reagents

• Phenolphthalein indicator • 0.5M Potassium hydroxide - Potassium hydroxide (2.8 gm) was placed in a 100 mL volumetric flask. Made up to the mark with ethanol. • 0.5M Hydrochloric acid titrant - Hydrochloric acid (1.8 mL) was placed in a 100 mL volumetric flask. Made up to the mark with water Procedure

Essential oil (1 gm) was added into a 250 mL Erlenmeyer flask fitted with a reflux condenser. After adding 6.25 mL of 0.5M ethanolic potassium hydroxide, a reflux condenser was connected and was then heated on boiling water bath for 30 minutes. The mixture was allowed to cool, 3 drops of phenolphthalein was then added and the excess alkali was titrated with 0.5M hydrochloric acid. A blank titration omitting the sample was performed. The saponification value was calculated from the expression [6].

Saponification value = 28.05 (b-a)/w (4)

where w = weight of the substance in gm, a = mL of KOH required to neutralize the substance, b = mL of KOH required for blank

5.1.8. Determination of aldehyde content Reagents • Ethanolic hydroxylamine hydrochloride solution (90%) - Hydroxylamine hydrochloride (7.0 gm) was dissolved in 90 mL of ethanol. Warmed gently to dissolve. • 0.5M Ethanolic Potassium hydroxide solution (60%) - Potassium hydroxide (2.8 gm) was placed in 100 mL of volumetric flask. Made up to the mark with ethanol. Procedure Essential oil (1.0 gm) was added to 5 mL of toluene and 15 mL of alcoholic hydroxylamine solution in glass-stopperd tube, shaken vigorously. The liberated hydrochloric acid was titrated immediately with 0.5M ethanolic potassium hydroxide using methyl orange as indicator until red color changes to yellow. Continued shaking and neutralizing until full yellow color of indicator is permanent in lower layer. The quantity of aldehyde is expressed in percentage [6]. Calculated by following formula:

( )×( . .)× % = ×( . ) (5) 퐵−푆 퐸푞 푤푡 100 표푓 푎푙푑푒ℎ푦푑푒 푐표푛푡푒푛푡 1000 푤푡 표푓 푠푎푚푝푙푒 where B = volume of acid required for blank, S = volume of acid required for sample, Eq. wt of KOH = 56 gm eq wt

5.1.9. Determination of total phenolic content

Reagents

10 % Folin Ciocalteu (F.C) reagent in distilled water, Na2CO3 (1M) in distilled water and Standard (Gallic acid) 1mg/ml solution in methanol was prepared. Different dilutions of standard gallic acid (20 μg/ml-100 μg/ml) were made in methanol.

Preparation of F.C. Reagent: The commercial F.C reagent was diluted (1:10) with distilled water on the day of use.

Preparation of aqueous Na2CO3: 1M Na2CO3 was prepared by dissolving 106 gm

of Na2CO3 in 1000 mL distilled water.

Blank solution: 0.5 ml methanol and 5 ml F.C. reagent was taken and 4 ml Na2CO3 solution was added to this.

Procedure

Different dilutions were prepared from 20 - 100 μg mL-1 of standard Gallic acid in distilled water. Each standard dilution (0.5 mL) was taken into a test tube. 5

mL of F.C. reagent (10%) and 4.0 mL of 1.0 M (Na2CO3) solution were added to the test tubes. The 0.5 mL of each sample extract was also taken into test tube and similarly reagents were added to it and kept for 15 min. Appearance of the blue color was noted. The absorbance was measured at 765 nm against blank solution containing

0.5 mL of methanol and 5.0 mL F.C. reagent along with 4.0 mL of Na2CO3 solution. After measuring the absorbance of standard dilutions, calibration curve was plotted and linear regression equation was numerated [8] as has been shown in Fig 1.

0.6

0.5

0.4

0.3 y = 0.0156x + 0.173 R² = 0.974 0.2 Absorbance 0.1

0 0 5 10 15 20 25 30 Concentration (µg/mL) Fig. 1. Calibration plot for total phenolic contents using Gallic acid as standard

5.1.10. Determination of total anthocyanin content

Reagents

• 0.025M Potassium chloride buffer (pH 1.0) - Potassium chloride (1.86 gm) was taken into a beaker and 980 mL of distilled water was added. The pH was measured and adjusted to 1.0 (±0.05) with hydrochloric acid. Transferred to 1 L flask and diluted to volume with distilled water.

• 0.4M Sodium acetate buffer (pH 4.5) - Sodium acetate (32.8 gm) was taken into a beaker and 960 mL of distilled water was added. The pH was measured and

adjusted to 4.5 (±0.05) with hydrochloric acid. Transferred to 1 L flask and diluted to volume with distilled water.

Preparation of Test Sample

The essential oil samples were diluted (1:4) with buffer solutions of pH 1.0 and pH 4.5.

Procedure

Absorbance of test portions diluted with pH 1.0 and pH 4.5 buffers at both 520 nm and 700 nm were determined. The diluted test portions were read against blank cell filled with distilled water. The absorbance was measured within 20-50 min of preparation [9]. Anthocyanin pigment concentration was calculated and expressed as cyanidin- 3- glucoside equivalents, as follows:

Absorbance (A) = (A520 nm – A700 nm) pH 1.0 – (A520 nm – A700 nm) pH 4.5 (6)

× × × % = (7) ɛ× 퐴 푀푊 퐷퐹 100 푤 where A = Absorbance,⁄푤 MW = molecular1 weight of anthocyanin (449.2), ε = cynidin- 3-glucoside molar-extinction coefficient (L × moL-1 × cm-1), DF = dilution factor

5.2. Analytical Studies

5.2.1. Scanning Electron Microscopy (SEM) The surface morphology of oily and non-oily biomass of plant material before

and after SC-CO2 extraction was examined by scanning electron microscopy (SEM) using Model JSM-6510LV, JEOL, (Japan). The SEM was operated at 10 kV, working distance of 11 and 12 mm, at magnification of 1000x and 2000x.

5.2.2. High performance thin layer chromatography (HPTLC) profiling

Sample preparation

The essential oil obtained by various extraction techniques were further diluted by adding 190 µL of hexane to 10 µL of oil.

Development of solvent system for separation of oil sample by TLC

The solvent system for the separation of oil samples was developed in different organic solvent. The summary of spots observed after using those solvents is given in Table 1.

Table 1. Solvent system tried for separation of phytoconstituents of essential oil S.no. Solvent system Ratio (v/v/v) Observation 1. Hexane : dichloromethane 95 : 5 No separation 2. Toulene : diisopropyl ether : ethyl acetate 80 : 10 : 10 No separation 3. Toulene : diisopropyl ether 95 : 5 No separation 4. Petroleum ether : ethylacetate : formic acid 95 : 5 : 1 No separation 5. Cyclohexane : ethylacetate 90 : 10 No separation 6. Chloroform : methanol : formic acid 90 : 2 : 1 Little separation 7. Chloroform : methanol : formic acid 90 : 3 : 1 Little separation 8. Chloroform : methanol : formic acid 90 : 1: 1 Little separation 9. Toulene : methanol : formic acid 90 : 7.5 : 1 Little separation 10. Toulene : methanol : formic acid 90 : 6 : 1 Little separation 11. Toulene : chloroform : formic acid 90 : 2 : 1 Little separation 12. Toulene : acetone 95 : 5 No separation 13. Toulene : ethyl acetate 97 : 5 Fair 14. Toulene : ethyl acetate 97 : 3 Fair 15. Toulene : ethyl acetate : formic acid 95 : 5: 1 Fair 16. Hexane : ethyl acetate : formic acid 90 : 1 : 1 Good separation 17. Hexane : ethyl acetate : formic acid 70 : 30 : 1 Good separation 18. Hexane : ethyl acetate : formic acid 70 : 20 : 1 Best separation

Application of the sample

The samples were applied in triplicate (4.0 µL each) and the width of the track was kept to 3.0 mm and distance between tracks was 13 mm on precoated silica gel

60 F254 plates 10 cm × 10 cm (E. Merck, 0.20 mm thickness) using Linomat V (HPTLC sample applicator). Chromatographic rectangular glass chamber (20 cm × 10 cm) was used in the experiment. To avoid insufficient chamber saturation and the desirable edge effect, a smooth sheet of filter paper approximately 15 × 40 cm size was placed in the chromatographic chamber in a “U” shape and allowed to be soaked in developing solvent. The chamber was allowed to saturate for 20 min before use. The solvent was allowed to run up to ¾th distance (80%) of the height of the TLC

plate. The plates were taken out and air- dried after marking the solvent front. The experiment is carried out at room temperature in diffused daylight.

Detection of spots

The densitometric scanning was performed on CMAG TLC scanner 3 in the absorbance mode at 254 and 366 nm (obtained from UV spectrum) using slit dimension 4 × 0.30 mm. The plate was then sprayed with anisaldehyde-sulphuric acid reagent and after heating at 110 °C for 5 min followed by air drying, scanning was done in the absorption mode at 580 nm using slit dimension 4 × 0.30 nm. The scanned TLC plates at 254, 366 and 580 nm with the chromatogram and comparative

Rf values with area percent was determined.

5.2.3. GC-MS analysis of essential oil obtained by different extraction techniques

Sample preparation

The essential oil obtained by different extraction techniques were further diluted by adding 1998 µl of hexane to 2 µl oil (Hydro-distilled oil) and 1990 µl of hexane to 10 µl oil (other extracted oils). The Operating conditions of GC: Capillary column with composition of 5% phenyl polymethyl siloxane, length (30 m), thickness (0.25 μm), diameter (0.25 mm), Carrier gas (He), Column flow rate (1mL/min), Carrier gas temperature (65 °C). Oven having injection temperature of 65 °C (3.0 min), The column temperature was raised at a rate of 10 oC/min from 160 oC to 302 oC. Splitless sample injection volume 2 µl. Detector type is MS with detector interface temperature of 250 °C.

By comparing with the National Institute of Standard and Technology (NIST) library, compounds were detected and identified. Comparative GC-MS chromatograms along with the name, retention time and peak area of essential oil constituents of different drugs were obtained [10].

5.2.4. Fourier transform infrared spectroscopy (FTIR) analysis of essential oil samples extracted by different extraction techniques

Procedure

FTIR spectrometer was used that was equipped with a deuterated triglycine Sulfate (DTGS) as a detector. IR spectra were recorded in the 400-4000 cm−1 (mid

infrared) range with a resolution of 4 cm−1. The room was kept at a controlled ambient temperature (25 °C) and relative humidity (30%). Dried potassium bromide powder (200 mg) was made into pieces of transparent blank potassium bromide pellets that were approximately 5 mm in diameter and approximately 1 mm in thickness. Volatile oils (2 µL) were coated on the potassium bromide pellets to form thin liquid films for infrared spectrometric analysis. The prepared samples were placed between zinc silicate (Zn2SiO4) round crystal windows (25 mm x 2 mm) and the transmission path length was fixed at 15 μm with a polytetrafluoroethylene (PTFE) spacer. The cell was then placed in the press lock demountable cell holder (Thermo Scientific) and the sample was scanned. For each spectrum, 45 scans were accumulated at resolution of 4 cm-1. Spectra were collected in triplicate for each sample and was averaged using the software supplied from the manufacturer of the spectrometer, OMNIC 7.3 (Thermo Electron Corp.). All spectra were smoothed using the “automatic smooth” function of the OMNIC 7.3 software, which uses the Savitsky-Golay algorithm (95-point moving second-degree polynomial). Subsequently the baseline was corrected using the “automatic baseline correct” function (polynomial). The spectra and peak table of different drugs were obtained [11].

6. In vitro bioactivity

6.1. Antioxidant activity

6.1.1. Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method

Reagents- DPPH (0.002% w/v) was prepared in 95% methanol, Standard (Ascorbic acid) 1mg/mL solution in methanol was prepared.

Procedure

The samples were mixed with 95% methanol to prepare the stock solution (1 mg/ml). 100 µL of 0.5 mM 2, 2-diphenyl-1-picryl hydrazyl radical (DPPH) in methanol was mixed with 100 µL of samples in 96 well plate at various concentrations (0.781, 1.56, 3.12, 6.25, 12.5, 25.0, 50.0 and 100.0 µg) in duplicate. The 96 well plates were allowed to stand at room temperature for 30 min in dark. The control was prepared as described above without sample or standards, whereas blank was prepared without DPPH containing sample and methanol. The changes in absorbance of all the samples and standards were measured at 540 nm in Elisa plate

reader (Bio Rad 680). Radical scavenging activity was calculated using the corrected ODs (COD) of control and samples as per the below mentioned formula:

= (8)

푐표푛푡푟표푙 푐표푛푡푟표푙 푠푎푚푝푙푒 퐶푂퐷 푂퐷 − 푂퐷

(%) = × (9) 퐶푂퐷푐표푛푡푟표푙−퐶푂퐷푠푎푚푝푙푒 푅푎푑푖푐푎푙 푠푐푎푣푒푛푔푖푛푔 퐶푂퐷푐표푛푡푟표푙 100

IC50, which is the concentration of sample required to scavenge 50% of free radicals was calculated [12].

6.1.2. Superoxide anionic scavenging method

Reagents

• 120 mM Phenazine methosulfate (PMS) - Phenazine methosulphate (306.34 mg) was dissolved in 1000 mL of volumetric flask. Made up to the mark with distilled water. • 300 mM Nitroblue tetrazolium (NBT) - Nitroblue tetrazolium (24.5 mg) was dissolved in 1000 mL of volumetric flask. Made up to the mark with distilled water. • 936 mM Nicotinamide adenine dinucleotide (NADH) - nicotinamide adenine dinucleotide (62.2 mg) was dissolved in 1000 mL of volumetric flask. Made up to the mark with distilled water. • 100 mM of Phosphate buffer (pH 7.4) - Monosodium phosphate (0.3117 gm) and disodium phosphate (2.074 gm) were dissolved in 100 mL of distilled water.

Procedure

0.3 ml of different concentrations (10 - 50 μg/mL) of the sample was taken. The superoxide anion was generated in 3 ml of phosphate buffer (100 mM, pH 7.4) containing 0.75 ml of NBT (300 μM) solution, 0.75 ml of NADH (936 μM) solution. The reaction was initiated by adding 0.75 ml of PMS (120 μM) to the mixture. After 5 min of incubation at room temperature, the absorbance at 560 nm (Schimadzu UV- Vis 1601) was measured in spectrophotometer. The superoxide anion scavenging activity was calculated according to the following equation:

% Inhibition = [(A0-A1) / A0 × 100] (10)

50 where A0 was the absorbance of the control (without extract) and A1 was the absorbance of the extract or standard.

IC50, which is the concentration of sample required to scavenge 50% of free radicals was calculated [13].

References:

1. G. Wenqiang, Li. Shufen, Y. Ruixiang, T. Shaokun and Q. Can, Comparison of essential oils of clove buds extracted with supercritical carbon dioxide and other three traditional extraction methods. J. Food Chem., 2007; 101: 1558- 1564. 2. R. Kowalskia and J. Wawrzykowskib, Effect of ultrasound-assisted maceration on the quality of oil from the leaves of thyme Thymus vulgaris L. Flavour Frag. J., 2009; 24: 69-74. 3. M. Khajeh, Y. Yamini, F. Sefidkon and N. Bahramifar, Comparison of essential oil composition of Carum copticum obtained by supercritical carbon dioxide extraction and hydrodistillation methods. Food Chem., 2004; 86: 587- 591. 4. G.P. Roger, Design and analysis of experiments. USA: Marcel Dekkar. 1985. 5. O.K. Robert, Design of experiments: statistical principles of research design and analysis. USA: Duxbury Press., 2000. 6. Indian Pharmacopeia., 2007; 1. 7. European Pharmacopeia., 2005; 5. 8. F. Pourmorad, S.J. Hosseinimehr and N. Shahabimajd, Antioxidant activity, phenol and flavonoid contents of some selected Iranian medicinal plants. Afr J Biotechnol., 2006; 5: 1142-1145. 9. R. Durst and E.R. Wrolstad, Determination of Total Monomeric Anthocyanin Pigment Content of Fruit Juices, Beverages, Natural Colorants, and Wines by the Ph Differential Method: Collaborative Study. J. AOAC Int., 2005; 88: 1269-1278. 10. E. Derwich, Z. Benziane and R. Taouil, GC/MS Analysis of Volatile Compounds of the Essential Oil of the Leaves of Mentha pulegium growing in Morocco. Chem Bull "POLITEHNICA" Univ (Timisoara)., 2010; 25: 103- 106. 11. Y.Q. Li, D.X. Kong and H. Wua, Analysis and evaluation of essential oil components of cinnamon barks using GC-MS and FTIR spectroscopy. Ind Crop Prod., 2013; 41: 269-278.

12. G. Sacchetti, S. Maietti, M. Muzzoli and M. Scaglianti, Comparative evaluation of 11 essential oils of different origin as functional antioxidants, antiradicals and antimicrobials in foods. Food Chem., 2005; 91: 621-632. 13. S. Chanda and R. Dave, In vitro models for antioxidant activity evaluation and some medicinal plants possessing antioxidant properties. Afr. J. Microbiol. Res., 2009; 3: 981-996.

CHAPTER 4

OPTIMIZATION OF TOTAL ESSENTIAL OIL YIELD OF CINNAMOMUM ZEYLANICUM N. BY USING SUPERCRITICAL CARBON- DIOXIDE EXTRACTION

1. Introduction

The genus Cinnamomum includes around 250 species which are widely spread in China, India, Australia, Vietnam, Sri Lanka, Madagascar, Seychelles [1]. Cinnamon belongs to the Lauraceae family. Cinnamon has a long history of use as a significant traditional medicine. Cinnamon bark is used as a chief medicinal substance in China [2]. Cinnamomum cassia (Chinese Cinnamon) is also named Chinese Cinnamon, which has been found both wildly and cultivated in Southeast Asia since ancient age, then introduced into Indonesia, South America and Hawaii. Cinnamomum zeylanicum or true cinnamon is the inner bark of a small tropical evergreen tree native to Sri Lanka [3]. Cinnamomum tamala (Indian cassia) moderate sized evergreen tree which is native to India and have originated from the south slopes of Himalayas and it is spread up to an altitude of 900-2500 m in tropical and sub- tropical Himalayas [4]. Cinnamomum burmannii (Indonesian Cinnamon) is usually found in West Sumatra. Cinnamomum pauciflorum is cultivated in southwest China, north-eastern India, Assam, and Khassia hills. Among the major species of Cinnamomum, C. cassia, C. burmannii, C. zeylanicum, C. tamala and C. pauciflorum are of high commercial importance and have been used in the traditional medicines of India and China [5]. The most important use of cinnamon is as spice because of its distinct fragrant, sweet and warm odour [6]. Cinnamon has been used to cure blood circulation disturbance, dyspepsia, gastritis and inflammatory diseases in many countries since prehistoric period [7, 8].

Various studies have been done by researchers and have been found to possess significant as antipyretic, antiulcerogenic, anaesthetic, antiallergic, analgesic [9, 10], antioxidant [11], anticancer [12, 13] and antibacterial [14] activities. The in vitro studies on cinnamon extract have explored that it imitates the role of insulin, which enhances the action of insulin in inaccessible adipocytes and also improves the insulin receptor function [15, 16]. Essential oils are of great commercial value in case of aromatherapies which provides relief from anxiety, pain, irritability, tiredness etc. Essential oil obtained from the leaves of C. osmophloeum ct. contains about 90% linalool which has been found to be a commercial source for aromatherapies [17]. In this work, essential oil from dried bark of C. zeylanicum N. have been extracted by conventional methods (HD, SE, US) and by innovative technique (SC-CO2) in which

CO2 has been used as supercritical fluid as it is being non-toxic, inert and non-

ﬂammable, it is considered as a GRAS (Generally Recognized As Safe) solvent. Through extraction of essential oils by 4 methods the volatile compounds in the essential oils were identiﬁed by GC/MS, HPTLC and FTIR. Optimum conditions of pressure, temperature and flow rate were determined and their effects on yield as well.

2. Materials and methods

2.1. Plant Materials

The plant material (dried bark) was purchased from local market of Aligarh, India. The dried bark of C. zeylanicum was grounded in a mechanical grinder in order to get uniform particle size distribution. The grounded powder was sieved.

2.2. Solvents and reagents

All the ingredients taken were of pharmacopeial quality and quantity. Standards were obtained from Sami Labs Ltd., Bangalore, (India) as a gift samples and other chemicals and reagents used were of analytical grade (AR) and procured from Merck Ltd. India. Carbon-dioxide from Sigma gases, New Delhi, India.

2.3. Design of experiment

Full factorial design (FFD)

2.4. Hydro-distillation method

Hydro-distillation is most commonly used traditional methods for the extraction of volatile compounds from different matrix [18]. This extraction method have certain disadvantages as there is loss of volatile compounds, extraction efficiency is low, not suitable for thermolabile compounds [19]. The grounded powder (150 g) and 500 mL distilled water was mixed in 1L round bottomed flask and heated for 5.0 h to yield essential oil in a Clevenger-type apparatus, according to the method of Demirci [20]. The extract was centrifuged at 10,000 rpm for 10 min in

order to separate small water droplets present in the essential oil. The oil was kept at 4 °C until further use.

2.5. Solvent Extraction

The coarse powder (50 gm) was extracted with hexane (100 mL) was kept in conical flask in 1:2 w/v, ratio for 24 hrs. The extract was filtered and concentrated under reduced pressure in rotary vacuum evaporator at 40 °C. The essential oil obtained was weighed and kept at 4 °C until further analysis.

2.6. Ultrasonic assisted extraction

The coarse powder (50 gm) of plant material was taken in a 250 mL conical flask along with 100 mL hexane (1:2, w/v). The flask was covered and then placed in an ultrasound water bath apparatus for 30 min (frequency 33 kHz). The temperature of the water bath was held constant at 25 °C. The extract was filtered and concentrated under reduced pressure in rotary vacuum evaporator. The obtained oil was kept at 4 °C until further use.

2.7. Supercritical carbon-dioxide extraction

A SFE-1000M1- 2- C50 system (Pittsburgh, PA) was used for extractions. The extraction vessel was 200 mL stainless steel. Grounded plant material (50 g) was loaded in the extractor. The CO2 was allowed to pass through the matrix at desired pressure (100, 150 and 200 bar), temperature (40, 45 and 50 oC) and optimised flow rate (4, 8 and 10 g min-1). Total time of extraction was 30 min. After the completion of extraction, the extracted oil was collected from the collecting vessel [21].

2.8. Characterization of sample

Physicochemical characterization of the essential oil extracted from C. zeylanicum was carried out in order to determine its colour, odour, specific gravity, refractive index, optical rotation, solubility in alcohol, acid value, ester value, aldehyde content, phenolics and anthocyanin content. Procedures for all have already been given in chapter 3.

56 development was carried out in mobile phase composed of hexane: ethyl acetate: formic acid (7:2:1, v/v/v). The HPTLC plates were studied at 254 nm and 366 nm as well as in visible range (580 nm) after spraying with anisaldehyde sulphuric acid reagent.

Gas chromatography-mass spectrometry (GC–MS) was equipped with a DB-5 fused silica capillary tubes column (30 m × 0.25 mm × 0.25 μm). The injection volume was 1.0 μL using auto-sampler at a carrier gas (helium) flow of 2.0 μL min-1 with a split less mode. The initial oven temperature was 65 °C (3.0 min) then raised to 2.0 °C min-1 -114 °C, then to 4 °C min-1 - 160 °C, 6 °C min-1 - 302 °C, finally ramped to 310 °C at 15 °C min-1. Detector type was MS and its interface temperature was 250 °C. The essential oil obtained by different extraction techniques were diluted by adding 1998 µL of hexane to 2.0 µL oil (Hydro distilled oil) and 1990 µL hexane to 10 µL oil (other extracted oils).

Compounds were detected and identified by comparing with the National Institute of Standard and Technology (NIST) library [22].

Fourier transform infrared spectroscopy (FTIR) spectral analysis was done and FTIR spectrum was obtained using shimadzu BioRad FTIR (Kyoto, Japan). The samples (2µL) were dispersed and triturated with dry potassium bromide, grounded well in mortar and pestle and potassium bromide (KBr) disk were kept at a pressure of 1,000 psig. The disk was placed in the FTIR sample holder, where IR spectra in absorbance mode was obtained in the spectral region 4000 to 400 cm-1 using the resolution 4 cm-1.

2.9. Application

The antioxidant activity of essential oil sample extracted from bark of C. zeylanicum was determined by two methods; Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging [23] and Superoxide anionic scavenging [24]. Procedures for both the methods have already been given in Chapter 3.

2.10. Statistical analysis

3. Results and Discussion

3.1. Percentage yield (%v/w)# Specific yield of different extraction methods (% v/w) Different extraction techniques were carried out in order to obtain maximum yield of oil. The extract yields of essential oil of C. zeylanicum (dried bark) were

0.70%, 0.72%, 0.78 and 3.8%, v/w for HD, SE, US and SC-CO2, respectively.

3.2 Physiochemical Characterization of Essential Oil

Essential oil extracted from C. zeylanicum was in liquid form, yellowish red in color with characteristic odour having sweet taste. Other physicochemical characterizations are given in Table 1. Anthocyanin content was found to be maximum in the essential oil extracted by SC-CO2 extraction as it retains all the phytoconstituents with purity (Fig. 1). Percent of phenolic content was found to be maximum in SC-CO2 extracted essential oil as shown in Fig. 2, calibration curve is given in chapter 3.

Table 1. Physicochemical properties of C. zeylanicum essential oil Specific gravity 25°C C. zeylanicum (dried SC- Reported HD* SE* US* bark) oil sample CO2* value Specific gravity (25 °C) 0.987 0.967 0.963 0.999 1.000-1.034 Refractive index (25 °C) 1.562 1.568 1.568 1.571 1.573-1.600 Optical rotation (25 °C) 0.5° -1° -1.3° -4° 0 to -2° Clearly Clearly Clearly Clearly Soluble in 5 Solubility in alcohol (% soluble soluble soluble soluble ml of 70% v/v ethanol) in 7.0 in 6.0 in 6.0 in5.5 ethanol mL mL mL mL Acid value (Mean±SD*) 6.8±0.2 5.3±0.1 5.7±0.2 6.5±0.1 - Ester value (Mean±SD*) 9.8±0.1 10.4±0.1 10.2±0.2 9.2±0.08 - Aldehyde content (% 20.1 13.6 14.4 20.7 - w/v) Phenolics in %w/w 5.2±0.1 5.3±0.1 5.3±0.1 5.7±0.2 - (Mean ± SD*) Anthocyanin content 5.4±0.1 4.5±0.1 4.2±0.1 5.5±0.1 - %w/w (Mean ± SD*)

HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon- dioxide extraction, SD* Standard deviation

# Specific yield means it depends on polarity of solvent and neat SC-CO2 extracts mainly non-polar compounds.

58 6

5 4 3 2 C. zeylanicum 1 Anthocyanin % w/w Anthocyanin 0 HD SE US SC-CO2 Oil sample

Fig. 1. Comparative study of anthocyanin contents in essential oil of C. zeylanicum obtained by different extraction techniques

5.8 5.6 5.4 5.2 C. zeylanicum 5 4.8 Phenolic contentPhenolic % w/w HD SE US SC-CO2 Oil sample

Fig. 2. Comparative study of phenolic contents in essential oil of C. zeylanicum obtained by different extraction techniques

3.3. Development of solvent system for separation of oil sample by TLC

The development of solvent system was carried out by hit and trial method using different solvents considering the results of earlier reports. The solvent system composed of hexane: ethyl acetate: formic acid (7:2:1, v/v/v) with maximum separation of constituents and compactness of bands for all the oil samples.

3.4. Detection of spots of different samples

Spotted plates were observed before derivatization at UV 254 nm and 366 nm and after derivatization by spraying with anisaldehyde sulphuric acid reagent at 254 nm. The HPTLC plates were studied at 254 nm and 366 nm as well as in visible range (580 nm) after spraying with anisaldehyde sulphuric acid reagent. A good separation of constituents was observed at 254 nm and 580 nm. The comparative results of

different oils were showing a dominant constituent at Rf value 0.61 at 366 nm

whereas it was observed at 254 nm in only SC-CO2 extracted oil and not observed at 580 nm (sprayed). Track 1 and 2 of hydro distilled oil showing lesser no. of compounds with major part being intense blue spot. Solvent extracted and ultrasonic

assisted oils showed equal amount of separation. SC-CO2 extracted oil showing some new spots not present in other extracted oil as shown in Fig. 3.

Fig. 3. HPTLC fingerprint of different oils of C. zeylanicum extracted using different extraction techniques [track 1-2: Hydro-distilled oil, 3-4: Solvent extracted oil, 5-6: Ultra sonication oil, 7-8: SC-CO2 oil] visualized at A: 254 nm, B: 366 nm, C: Day light after derivatization using anisaldehyde sulphuric acid reagent.

The HPTLC fingerprinting of essential oil of C. zeylanicum extracted by different techniques showed A-O compounds with maximum no. of compounds seen at 366 nm (Figs. 4a, 5a, Table 2) and lesser at 254 nm (Figs. 4b, 5b Table 2).

Compound with Rf (0.61) had maximum area percentage at 366nm. Substance D, E, F were present in all extracted oils at 254 nm and substance F, I, J, K and N were present in all extracted oil samples at 366 nm. Substance A, B, C, G, J, K and L were

present in SC-CO2 extracted oil sample, absent in hydro-distilled oil at 254 nm.

Fig. 4a. 3D Chromatogram of 8 tracks of C. zeylanicum oil obtained by different extraction techniques at 366 nm

Fig. 5a. HPTLC fingerprint of different oils of C. zeylanicum extracted using different extraction techniques A: Hydrodistilled oil, B: Solvent extracted oil, C: Ultra sonication oil, D: SC-CO2 oil visualized at 366 nm with assigned substance

Fig. 4b. 3D Chromatogram of 8 tracks of C. zeylanicum oil obtained by different extraction techniques at 254 nm

Fig. 5b. HPTLC fingerprint of different oils of C. zeylanicum extracted using different extraction techniques A: Hydro-distilled oil, B: Solvent extracted oil, C: Ultra sonication oil, D: SC-CO2 oil visualized at 254 nm with assigned substance

Table 2. Substance A-O with their Rf and area percent of C. zeylanicum essential oil obtained by different extraction techniques

Area percentage(%) 254 nm Area percentage(%) 366 nm Substance (Rf) HD* SE* US* SC-CO2* HD* SE* US* SC-CO2* A(0.12) - - - 0.10 - - 0.18

B(0.14) - - - 0.19 - - 1.18

C(0.20) - 1.01 1.49 2.52 - 0.99 2.18 0.86 D(0.25) 0.23 2.24 2.58 2.41 - 0.82 1.43 - E(0.31) 2.56 3.87 5.20 6.72 - - - 4.09 F(0.36) 0.22 2.51 3.64 1.28 1.87 7.16 7.06 2.97 G(0.39) - 1.85 - 3.12 - - - 2.53 H(0.43) - - 1.11 - - 1.59 2.35 2.31 I(0.54) - 10.68 8.85 - 35.98 44.17 46.04 34.86 J(0.61) - - - 45.28 50.02 33.72 33.95 29.40 K(0.70) - - - 17.86 10.44 10.05 3.89 9.34 L(0.80) - - - 20.54 - - - 9.53 M(0.86) - - - - 0.61 0.33 - - N(0.93) - - - - 1.08 1.17 1.92 3.51 O(0.98) ------0.31

Rf* Retention factor, HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon-dioxide extraction

The HPTLC fingerprinting of essential oil of C. zeylanicum extracted by different extraction techniques showed A-R compounds at 580 nm (Figs. 4c, 5c).

Compounds A-R has maximum area percentage in SC-CO2 extracted oil. Details of the compounds have been given in table Table 3.

Fig. 4c. 3D Chromatogram of 8 tracks of C. zeylanicum oil obtained by different extraction techniques at 580 nm

Fig. 5c. HPTLC fingerprint of different oils of C. zeylanicum extracted using different extraction techniques A: Hydrodistilled oil, B: Solvent extracted oil, C: Ultra sonication oil, D: SC-CO2 oil visualized at 580 nm with assigned substance

Table 3. Substance A-R with their Rf and area percent of C. zeylanicum essential oil obtained by different extraction techniques

Area percentage(%) 580 nm Substance (Rf) HD* SE* US* SC-CO2* A(0.06) - - 0.28 0.92 B(0.15) - 6.96 7.98 10.71 C(0.25) 0.62 9.89 8.67 10.47 D(0.32) 1.63 - 4.03 3.67 E(0.37) 0.70 5.48 5.66 2.17 F(0.43) 1.60 9.41 8.93 - G(0.46) - - - 9.53 H(0.51) 10.32 10.71 10.16 - I(0.55) - - - 11.91 J(0.58) 30.54 12.40 13.57 - K(0.61) - 16.33 13.33 13.60 L(0.64) 35.80 - - - M(0.68) - 10.79 12.83 23.36 N(0.77) 12.46 13.72 10.30 - O(0.8) - - - 4.39 P(0.85) 6.32 4.31 4.27 - Q(0.88) - - - - R(0.93) - - - 9.27 Rf* Retention factor, HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon-dioxide extraction

The HPTLC fingerprinting of essential oil of C. zeylanicum extracted by different technique showed A-R compounds. Substance C, E, F and H were present in all the four extracted oil samples. Substance G, M, O and R were present in oil

extracted by SC-CO2 extraction. Substance D was present in all the 3 extracted oil techniques except in solvent extracted oil. Substance L was present in only hydro distilled oil, absent in others. Substance A was present in oil extracted by ultrasonication and SC-CO2 extraction techniques.

3.5. Comparative GC-MS analysis of C. zeylanicum oil obtained by different extraction techniques

Comparative GC-MS chromatograms of essential oils of C. zeylanicum

extracted by traditional methods (HD, SE, US) and innovative SC-CO2 technique as shown in Fig. 6. A total of sixty compounds were identified with GC-MS analysis by using their retention time and mass from library (Nist and Wiley) (Table 4). The

majority of the identified oil compounds consisted of sesquiterpene hydrocarbon, fatty group and aromatic compounds. The most abundant component of the bark oils was trans-cinnamaldehyde (55.97%), α-humulene (8.21%), β-phellandrene (3.63%), eugenol (3.54%), caryophyllene (3.28%). Hexadecanoic acid (5.28%), 9, 12- Octadecadienoic acid (8.89%) were the fatty acid compounds present.

Fig. 6. Comparative GC-MS chromatograms of different oils of C. zeylanicum extracted using different extraction techniques A: Hydro-distilled oil, B: SC-CO2 oil, C: Solvent extracted oil, D: Ultra sonication oil.

Table 4. Results of GC-MS analysis of C. zeylanicum oil extracted by different techniques Area percent (%) SC- S. Component Name R * HD* SE* US* CO no. T 2 * Monoterpene hydrocarbon 1. α.-pinene, 5.43 0.48 - - - 2. l-Phellandrene 7.71 1.08 - 0.09 - 3. α-Terpinene 8.2 0.71 - - - 4. m-Cymene 8.53 0.71 - 0.4 - 5. β-Phellandrene 8.7 3.63 5.07 3.08 - 6. α-terpinolene 11.45 0.2 - - - Oxygenated monoterpene 7. Linalol 12.11 1.8 - 0.48 - 8. 4-Terpineol 16.15 0.37 - - - 9. trans-anethole 23.07 0.13 - - - 10. Carvacrol 24 0.03 - - - 11. Eugenol 27.12 3.54 1.6 1.04 0.3

12. Geranyl acetate 28.77 0.04 - - - 13. Methyleugenol 29.92 0.04 - - - 14. trans-Cinnamyl acetate 31.88 7.35 - 1.58 - 15. Cinnamyl alcohol, acetate 31.67 - 0.62 - - Sesquiterpene hydrocarbons 16. α-Copaene 27.85 0.44 0.61 0.45 0.32

17. Caryophylline-(I3) 29.56 0.17 - - - 18. Caryophyllene 30.18 3.28 6.16 2.73 3.34 19. α-Humulene 31.77 8.21 1.34 0.66 - 20. l-β-Bisabolene 34.24 0.06 - - - 21. β-Selinene 33.2 0.24 - - - 22. ValencenE 33.53 1.07 - - - 23. Longiborn-9-ene 34.38 0.26 - - - 24. (-)-.alpha.-Panasinsen 34.47 0.59 - - - 25. δ-Cadinene 34.77 0.1 - - - 26. Alloaromadendrene 38.71 0.11 - - - 27. γ-Gurjunene 39.38 0.16 - - - 28. β tumerone 39.66 0.08 - - - 29. Tumerone 39.77 0.24 - - - 30. Curloneα-tumerone 40.68 0.06 - - - Oxygenated sesquiterpenes derivatives 31. (-)-Caryophyllene oxide 36.86 0.15 - - - 32. Eudesm-4(14)-en-11-ol 39.12 0.04 - - - Aromatic compounds 33. Benzylacetaldehyde 15.44 0.51 - - - 34. Cinnamaldehyde, (E)- 20.94 0.18 55.9 52.6 22.4 35. Cinnamaldehyde 33.7 0.65 1.43 0.76 0.95 36. Myristyl aldehyde 37.96 0.11 - - - Non isoprenoid 37. Linalyl propionate 16.97 0.69 - - - 38. 3-Hexadecene, (Z)- 29.03 0.05 - - - 39. Octadecane 33.73 - 0.2 - - 40. Phenol, 2,4-bis(1,1-dimethylethyl) 34.62 0.19 - - - 41. 1-bromo-2-methyl-decane 36.95 0.15 - - - 42. Docosane 40.83 - 0.35 - - 43. Eicosane 41.31 - 0.28 2.48 0.29 44. Tricosane 41.94 - 0.17 - - 45. Benzoic acid, phenylmethyl ester 42.29 1.55 1.96 1.36 1.23 46. 1-Nonadecene 42.93 0.08 - 0.17 - 47. Pentacosane 45.26 - 0.3 0.18 0.23

48. Methyl palmitate 45.62 0.02 0.63 0.8 - 49. Pentadecanoic acid, 14-methyl-, methyl 45.61 - - - 0.99 ester 50. Hexadecanoic acid 46.43 - - - 5.28 51. Trifluoroacetoxy hexadecane 46.78 0.04 - - - 52. Nonacosane 46.91 - - 0.15 - 53. 9,12-Octadecadienoic acid (Z,Z)-, methyl 48.55 0.02 5.33 8.89 6.03 ester 54. 9-Octadecenoic acid (Z)-, methyl ester 48.64 - 2.13 3.19 2.5 55. Octadecanoic acid, methyl ester 49.06 - - 0.3 - 56. 1-Docosanol 50.03 0.02 - - - 57. Nonadecane 51.6 - - 0.2 - 58. n-Octadecane 52.99 - - 0.59 - 59. Butyl phthalate 55.05 0.02 2.61 3.33 1.76 60. Squalene 58.4 - - - 0.36 * RT Retention time, HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon-dioxide extraction

3.6. FTIR analysis

The interpretation of infrared spectra of C. zeylanicum essential oil was done by correlating of absorption bands in the spectrum with the known absorption frequency bands. Comparative spectra of essential oil extracted by different techniques are shown in Fig. 7. FTIR results are presented in Table 5.

The typical spectra of cinnamons were analyzed and several peaks were observed such as the peak at 1727 cm−1, corresponding to the aldehyde of a saturated fat and the peaks at 1678 cm−1 which correspond to the stretching vibration of an aldehyde carbonyl C-O. These main peaks correspond to high levels of cinnamaldehyde and aldehydes in the volatile oil of cinnamon bark.

Note on Table 4: Only non-polar compounds extracted as SC-CO2 is non polar

Fig. 7. Comparative spectra of different oils of Cinnamomum zeylanicum extracted using different extraction techniques A: Hydro-distilled oil, B: Ultra sonication oil, C: Solvent extracted oil, D: SC-CO2 oil.

Table 5. Results of FTIR analysis of C. zeylanicum oil obtained by different extraction techniques SC- S.no. Peak Functional group Intensity HD* SE* US* CO2* 1. 686 C-Cl(chloroalkane) Stretch,strong + - + + 2. 974 =C-H(alkene) Bending, strong + + + + 3. 1124 C-O(alcohol) Stretch, strong + + + + 4. 1246 C-O(acid) Stretch, strong + + + + 5. 1384 N-O(nitro) Stretch, strong + - + - 6. 1450 C=C(aromatic) Stetch,medium + - - + 7. 1678 C-O(aldehyde) Stretch, variable + + + + 8. 2268 -C=-C(alkyne) Stretch, variable - + - + 9. 3059 =C-C(alkene) Stretch, variable + + - - 10. 3408 O-H(alcohol,) Strong, broad - - + +

HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon- dioxide extraction

3.7. Antioxidant activity

The C. zeylanicum oil sample showed a significant concentration-dependent antioxidant activity by inhibiting DPPH free radical with an IC50 values of 70.55 μg -1 -1 mL , whereas, IC50 value of ascorbic acid was found to be 28.09 μg mL used as standard (Table 6, Fig. 8). It was found that the oil possesses hydrogen donating capabilities similar to ascorbic acid and acted as an antioxidant. The scavenging effect increased with increasing concentration of the extract and ascorbic acid (5.0-100 μg mL-1).

Table 6. Summary of % inhibition and IC50 values of C. zeylanicum oil sample by DPPH method (n=3)

Percentage inhibition % (Mean ± SD*) Conc. (µg mL-1) Ascorbic acid C. zeylanicum 100 92.5 ±1.1 55.6±0.8 50 64.3 ±0.9 43.6±0.4 25 45.7 ±1.0 32.8±0.8 12.5 29.5 ±0.54 29.4±0.8 6.25 20.6 ±0.8 20.5±1.2 3.125 12.1 ± 0.8 13.6±0.8 1.5625 8.45 ±1.0 4.3±1.2 0.781 5.85± 0.8 0.2±0.001

IC50 28.09 70.55 SD* Standard deviation

Fig. 8. Comparative dose response curve between percent inhibitions against log concentration by DPPH method

The superoxide scavenging activity of the drug was increased markedly with the increase in concentrations. A comparison of the antioxidant activity of the extract and ascorbic acid is shown in Table 7 and Fig. 9. The IC50 value of the essential oils -1 of C. zeylanicum was found to be 32.34 µg mL , whereas the IC50 value of ascorbic acid was 20.14 µg mL-1.

Table 7. Summary of % inhibition and IC50 values of C. zeylanicum oil sample by superoxide anion scavenging method (n=3) Percentage inhibition ± SD* Conc. (µg/ml-1) Ascorbic Acid C. zeylanicum 10 38.28±0.8 30.71±0.4 20 48.72±1.2 29.12±0.4 40 62.33±0.8 46.38±0.9 60 71.67±1.2 60.11±0.9 80 89.28±1.2 77.08±1.8 100 95.22±1.2 85.63±1.4

IC50 20. 14 32.34 SD* Standard deviation

Fig. 9. Comparative dose response curve between percent inhibitions against log concentration by superoxide scavenging method.

3.8. Factorial Design An experiment was run to study the effect of three factors (Pressure, temperature, flow rate) with three levels on response (Yield). The results were analyzed and are presented in Table 8. It can be seen that the pressure and flow rate have significant effect directly on the total yield of essential oil. The selected factors

explained 86.13 % fit model for the given data. The contour and surface plots have been shown in Figs. 10 & 11. Table 8. Estimated Effects and Coefficients for Yield Term Effect Coef SE Coef T P Constant 1.43791 0.07888 18.23 0.000 Pressure 2.10000 1.05000 0.08360 12.56 0.000 Temperature -0.07750 -0.03875 0.08360 -0.46 0.647 Flow rate 0.54777 0.27389 0.08309 3.30 0.003 Pressure*Temperature -0.17750 -0.08875 0.08360 -1.06 0.297 Pressure*Flow rate 0.28250 0.14125 0.08360 1.69 0.102 Temperature*Flow rate 0.16750 0.08375 0.08360 1.00 0.325 Pressure*Temperature*Flow 0.03750 0.01875 0.08360 0.22 0.824 rate

Contour Plots of Yield

Temperature*Pressure Flow rate*Pressure 50.0 10 Yield < 0.5 0.5 - 1.0 47.5 8 1.0 - 1.5 1.5 - 2.0 45.0 2.0 - 2.5 6 > 2.5 42.5 HoldHold Values Values Pressure 100 40.0 4 Temperature 40 100 125 150 175 200 100 125 150 175 200 Pressure 200 Flow rate 4 Flow rate*Temperature 10 Temperature 40 Flow rate 4 8

4 40.0 42.5 45.0 47.5 50.0

Fig. 10. Contour plots of yield vs pressure, temperature and flow rate

Surface Plots of Yield Hold Values Hold Values PressurePressure 200100 Temperature 40 TemperatureFlow rate 404 3 2.0 2 Yield 1.5 Yield Flow rate 4 1.0 1 0.5 200 0 200 500 150 10.00 150 45 Pressure 7.5 Pressure 440 10010 5.0 1001 Temmperaturet Floww rate

0.6 0.5 Yield 0.4 0.3 50 45 10.00 7.5 Tememperature 5.0 40 Floww rate

Fig. 11. Surface plots of yield vs pressure, temperature and flow rate

Pressure and flow rate have shown to have direct effect on yield as p<0.05, hence pressure and flow rate are significant factors for influencing yield. While temperature, combinations of pressure-flow rate, temperature-flow rate, pressure- temperature and pressure-temperature-flow rate have no significance on oil yield.

The optimum operating conditions for SC-CO2 extraction of essential oils from bark of C. zeylanicum, for high yield were pressure (200 bar), temperature (40 o C) and flow rate of CO2 (4 g/min). A confirmation experiment was run using the optimum conditions and the highest yield obtained was 3.8 %.

4. Conclusion In present work, essential oil was extracted via Hydro-distillation, Solvent

extraction, Ultrasonic assisted extraction and SC-CO2 extraction. Among all these

methods SC-CO2 gave the highest percentage yield (3.8 %) at optimum conditions of o pressure (200 bar), temperature (40 C) and flow rate of CO2 (4 g/min) by FFD. The analysis shows that pressure and flow rate have shown to have direct effect on yield as p<0.05, while temperature, combinations of pressure-flow rate, temperature-flow rate, pressure-temperature and pressure-temperature-flow rate have no significance on oil yield as p>0.05. Furthermore evidences from GC-MS as well as FTIR analysis

showed that SC-CO2 is best technique for extraction of essential oils. Essential oil obtained from bark of C. zeylanicum has shown to possess significant antioxidant activity which was investigated by DPPH and Superoxide anion scavenging methods and were found to be 70.55 and 32.34 µg mL-1, respectively. *Further it is best in terms of total yield as well as specific yield which accounts for yield of non-polar fraction. The antioxidant property of SC-CO2 extractant will naturally depend on non polar compounds present in it.

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CHAPTER 5

OPTIMIZATION OF SUPERCRITICAL CARBON - DIOXIDE EXTRACTION OF ESSENTIAL OIL FROM CINNAMOMUM TAMALA N. ALONG WITH ITS CHEMICAL COMPOSITION AND ANTIOXIDANT ACTIVITY

1. Introduction Supercritical fluid extraction (SFE) is a good technique for the production of flavours and fragrances from natural materials and can constitute a valid alternative to

conventional extraction techniques. In fact, compressed carbon dioxide, CO2, is able to solubilize hydrocarbons and oxygenated mono- and sesquiterpene [1], the main essential oil constituents. Cinnamon belongs to the Lauraceae family. The genus Cinnamomum includes around 250 species which are widely spread in China, India, Australia, Vietnam, Sri Lanka, Madagascar, Seychelles [2]. Cinnamon has a long history of use as a significant traditional medicine. Cinnamomum tamala (Indian cassia) moderate sized evergreen tree which is native to India and have originated from the south slopes of Himalayas and it is spread up to an altitude of 900-2500 m in tropical and sub-tropical Himalayas [3]. The most important use of cinnamon is as spice because of its distinct fragrant, sweet and warm odour [4]. Cinnamon has been used to cure blood circulation disturbance, dyspepsia, gastritis and inﬂammatory diseases in many countries since prehistoric period [5, 6]. Essential oils are of great commercial value in case of aromatherapies which provides relief from anxiety, pain, irritability, tiredness etc. Essential oil obtained from the leaves of C. osmophloeum ct. contains about 90% linalool which has been found to be a commercial source for aromatherapies [7]. In this work, essential oil from dried leaves of Cinnamomum tamala has been extracted by conventional methods (HD, SE, US) and by innovative technique SC-

CO2 extraction. The volatile compounds in the essential oils were identiﬁed by HPTLC, FTIR and GC/MS. 2. Materials and methods 2.1. Plant Materials The following plant material (dried leaves) was purchased from local market of Aligarh, India. As a ﬁrst step, dried cinnamon leaves were grounded in a mechanical grinder for a short but sufficient period of time (30 s) to get a uniform particle size distribution. The grounded powder was sieved. 2.2. Solvents and reagents All the ingredients taken were of pharmacopeial quality and quantity. Standards were obtained from Sami Labs Ltd., Bangalore, (India) as a gift samples. Other reagents and chemicals were purchased from sigma.

2.3. Design of experiment

Full factorial design (FFD)

2.4. Hydro-distillation method

Hydro distillation is most commonly used traditional methods for the extraction of volatile compounds from different matrix [8]. This extraction method have certain disadvantages as there is loss of volatile compounds, extraction efficiency is low, not suitable for thermolabile compounds [9]. The grounded powder (150 gm) and 500 mL distilled water was mixed in 1 L round bottomed flask and heated for 5.0 h to yield essential oil in a Clevenger-type apparatus, according to the method of Demirci [10]. The extract was centrifuged at 10,000 rpm for 10 min in order to separate small water droplets present in the essential oil. The oil was kept at 4 °C until further use.

2.5. Solvent Extraction

2.6. Ultrasonic assisted extraction

2.7. Supercritical carbon-dioxide extraction

A SFE-1000M1- 2- C50 system (Pittsburgh, PA) was used for extractions. The extraction vessel was 200 mL stainless steel. Grounded plant material (50 gm) was loaded in the extractor vessel. The pressure in the extraction vessel was controlled by back pressure regulator. Heat exchangers were provided in system to maintain temperature in the extractor and separator vessel. Extraction pressure was varied from o 100-250 bar, temperature 60-80 C and CO2 flow rate was varied from 8 to 15 g/min. Total time of extraction was 45 min. After that oil was collected from the collecting vessel [11]. The extracted oil was collected in a glass vial and stored at 4 oC prior to analysis and its percentage yield was calculated.

2.8. Characterization of sample

In High performance thin layer chromatography (HPTLC) analysis, the samples were spotted in the form of band (3.0 mm) with a Camag microlitre syringe on TLC aluminium plate precoated with silica gel 60F-254 (20 x 10 cm with 0.2 mm thickness, E. Merck, Germany) using a Camag Linomat V sample applicator. The development was carried out in mobile phase composed of hexane: ethyl acetate: formic acid (7:2:1, v/v/v). The HPTLC plates were studied at 254 nm and 366 nm as well as in visible range (580 nm) after spraying with anisaldehyde sulphuric acid reagent.

Gas chromatography-mass spectrometry (GC-MS) was equipped with a DB-5 fused silica capillary tubes column (30 m × 0.25 mm × 0.25 μm). The injection volume was 1.0 μL using auto-sampler at a carrier gas (helium) flow of 2.0 μL min-1 with a split less mode. The initial oven temperature was 65 °C (3.0 min) then raised to 2.0 °C min-1 -114 °C, then to 4 °C min-1 - 160 °C, 6 °C min-1 - 302 °C, finally ramped to 310 °C at 15 °C min-1. Detector type was MS and its interface temperature was 250 °C. The essential oil obtained by different extraction techniques were diluted by adding 1998 µL of hexane to 2.0 µL oil (Hydro distilled oil) and 1990 µL hexane to 10 µL oil (other extracted oils).

Compounds were detected and identified by comparing with the National Institute of Standard and Technology (NIST) library [12].

Fourier transform infrared spectroscopy (FTIR) analysis was done and FTIR spectrum was obtained using shimadzu BioRad FTIR (Kyoto, Japan). The samples

(2µL) were dispersed and triturated with dry potassium bromide, grounded well in mortar and pestle and potassium bromide (KBr) disk were kept at a pressure of 1000 psig. The disk was placed in the FTIR sample holder, where IR spectra in absorbance mode was obtained in the spectral region 4000 to 400 cm-1 using the resolution 4 cm-1.

3. Antioxidant activity

The antioxidant activity of essential oil sample extracted from dried leaves of C. tamala was determined by two methods; Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging [13] and Superoxide anionic scavenging [14]. Procedures for both the methods have already been given in Chapter 3.

4. Statistical analysis

5. Results and Discussion

5.1. Percentage yield (% v/w)

Different extraction techniques were carried out in order to obtain maximum yield of oil. The extract yields of essential oil of C. tamala (dried leaves) were 1.80%,

2.5%, 2.9% and 3.64%, v/w for HD, SE, US and SC-CO2, respectively.

5.2. Physicochemical Characterization of Essential Oil

Essential oil extracted from C. tamala was in liquid form, colorless with characteristic odor and taste. Other physicochemical characterizations are given in the Table 1. Procedures to carry out them are given in chapter 3. Anthocyanin content was found to be maximum in the essential oil extracted by SC-CO2 extraction as shown in Fig. 1. Percent of phenolic content was found to be more in HD and SC-

CO2 extracted essential oils (Fig. 2), calibration curve is given in chapter 3.

Table 1. Physicochemical properties of C. tamala essential oil

Specific gravity 25°C C. tamala (dried leaves) SC- Reported HD* SE* US* oil sample CO2* value Specific gravity (25°C) 0.925 0.930 0.925 0.939 0.942-0.948 Refractive index (25°C) 1.520 1.519 1.525 1.531 1.529-1.535 Optical rotation (25°C) 1.9° 1.0° 1.0° 1.9° 1.6 to2.12 Soluble Soluble Soluble Soluble Soluble in 1.2 with with with with mL of 70% Solubility in alcohol (% slight slight slight slight ethanol v/v ethanol) haziness haziness haziness haziness in1.5 in1.5 in1.5 in 4.0 mL mL mL mL Acid value (Mean±SD*) 4.9±0.1 5.3±0.3 5.5±0.1 4.5±0.1 - Ester value (Mean±SD*) 6.6±0.1 7.0±0.2 7.5±0.2 6.4±0.2 - Aldehyde content (% 9.1 8.3 8.3 9.6 - w/v) Phenolics in %w/w 4.5±0.2 3.5±0.1 3.9±0.1 4.3±0.2 - (Mean ± SD*) Anthocyanin content 5.7±0.08 5.5±0.3 5.0±0.1 5.8±0.1 - %w/w (Mean ± SD*)

HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon- dioxide extraction, SD* Standard deviation

5.8 5.6 5.4

5.2 C. tamala 5 Anthocyanin % w/w Anthocyanin 4.8 4.6 HD SE US SC-CO2 Oil sample

Fig. 1. Comparative study of anthocyanin contents in essential oil of C. tamala extracted by different extraction techniques

4.5 4 3.5 3 2.5 2 C. tamala 1.5 1 Phenolic contentPhenolic % w/w 0.5 0 HD SE US SC-CO2 Oil sample

Fig. 2. Comparative study of phenolic contents in essential oil of C.tamala extracted by different extraction techniques

5.3. Development of solvent system for separation of oil sample by TLC

5.4. Detection of spots of different samples

The HPTLC plates were studied at 254 nm and 366 nm as well as in visible range (580 nm) after spraying with anisaldehyde sulphuric acid reagent. The

comparative results of different oils were showing a dominant constituent at Rf value

0.7 at 254 nm in hydro-distilled oil and at Rf 0.89 at 366 nm and not observed at 580 nm (sprayed). Track 1 and 2 of hydro-distilled oil showing lesser no. of compounds with major part being intense blue spot. Solvent extracted and ultrasonic assisted oil showed equal amount of separation. SC-CO2 extracted oil showing some new red

colour spots at Rf 0.89 not observed in other extracted oil as shown in Fig. 3.

Fig. 3. HPTLC fingerprint of different oils of C. tamala extracted using different extraction techniques [track 1-2: Hydrodistilled oil, 3-4: Solvent extracted oil, 5-6: Ultra sonication oil, 7-8: SC-CO2 oil] visualized at A: 254 nm, B: 366 nm, C: Day light after derivatization using anisaldehyde sulphuric acid reagent

The HPTLC fingerprinting of essential oil of C. tamala extracted by different

techniques showed A-T compounds. Substance M at Rf 0.61 was present in all the extracted oils with different area percent in the plate scanned at 254 nm (Figs. 4a, 5a

& Table 2) and 366 nm (Figs. 4b, 5b & Table 2). At Rf 0.83 substance R was present

in maximum amount in SC-CO2 extracted oil only scanned at 254 nm.

Fig. 4a. 3D Chromatogram of 8 tracks of C. tamala oil obtained by different extraction techniques at 254 nm

Fig. 5a. HPTLC fingerprint of different oils of C. tamala extracted using different extraction techniques A: Hydro-distilled oil, B: Solvent extracted oil, C: Ultra sonicated oil, D: SC-CO2 oil visualized at 254 nm with assigned substance

Fig. 4b. 3D Chromatogram of 8 tracks of C. tamala oil obtained by different extraction techniques at 366 nm

Fig. 5b. HPTLC fingerprint of different oils of C. tamala extracted using different extraction techniques A: Hydro-distilled oil, B: Solvent extracted oil, C: Ultra sonicated oil, D: SC-CO2 oil visualized at 366 nm with assigned substance

Table 2. Substance A-T with their Rf and area percent of C. tamala essential oil obtained by different extraction techniques Substance Area percentage (%) 254 nm Area percentage (%) 366 nm (Rf) HD* SE* US* SC-CO2* HD* SE* US* SC-CO2* A(0.01) 2.34 3.98 4.88 3.98

B (0.07) 1.99 0.43 4.80

C (0.1) 0.53 7.07

D (0.17) 5.86 6.63 5.86 5.34 17.57

E (0.2) 5.65 5.78 2.66 5.65 3.64

F (0.23) 4.56 4.45 2.11 4.56 3.26

G (0.28) 6.36 6.63 4.29 13.68 18.89 2.88

H (0.32) 9.25 9.80 1.70 9.25 26.21 7.54

I (0.37) 2.21 12.96

J (0.4) 15.05 13.95 2.10 15.05 6.11

K (0.46) 6.72 7.57 1.13 6.72

L (0.53) 13.68 11.75 13.28 2.19 17.78

M (0.61) 26.66 5.52 5.26 2.50 39.88 5.52 3.52 15.83 N (0.63) 15.80 3.09 3.69 6.85

O (0.66) 12.88 7.06 5.44 12.88 1.01 5.47

P (0.74) 30.72 8.90 12.31 9.16 8.90 0.54

Q (0.79) 0.99 1.01 0.99 3.48

R (0.83) 51.58 0.77 9.00

S (0.87) 0.71 13.62

T (0.89) 22.49 0.61 60.12

Rf* Retention factor, HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon-dioxide extraction

The HPTLC plate scanned at 580 nm (Figs. 4c, 5c & Table 3) showing maximum area percent at Rf (0.61) and Rf (0.55) in oil extracted by hydro distillation process. Substance N at Rf 0.61 was present in SC-CO2 extracted oil only. Substance

M at Rf 0.59 was showing maximum area percentage of all the extracted constituents signifying the major component eugenol. Substances K, M were present in only hydro-distilled and SC-CO2 extracted oil samples. Substance F, G, H, J and L were present in only solvent extracted and ultrasonicated oil samples. Substance P and Q were present in SC-CO2 extracted oil only.

Fig. 4c. 3D Chromatogram of 8 tracks of C. tamala oil obtained by different extraction techniques at 580 nm

Fig. 5c. HPTLC chromatograms of different oils of C. tamala extracted using different extraction techniques A: Hydro-distilled oil, B: Solvent extracted oil, C: Ultra sonication oil, D: SC-CO2 oil visualized at 580 nm with assigned substance

Table 3. Substance A-U with their Rf and area percent of C. tamala essential oil obtained by different extraction techniques Substance Area Percentage (%) 580 nm (Rf) HD* SE* US* SC-CO2* A (0.06) - - - - B (0.13) 3.21 - - 5.67 C (0.16) - - - 2.49 D (0.2) - 21.69 21.03 8.90 E (0.29) - - - 6.22 F (0.32) - 9.92 9.31 - G (0.4) - 14.06 16.06 - H (0.44) - 5.42 11.10 10.42 I (0.47) 21.81 5.79 - - J (0.52) - 6.75 8.34 - K (0.54) 9.80 - - 21.88 L (0.57) - 11.39 21.49 - M (0.59) 48.34 - - 17.53 N (0.61) - 18.56 - - O (0.64) - - 6.59 - P (0.68) - - - 3.75 Q (0.72) - - - 3.26 R (0.75) - 4.37 3.27 1.68 S (0.78) 14.03 - 2.80 - T (0.84) - 2.05 - 17.03 U (0.89) - 2.81 - - Rf* Retention factor, HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon-dioxide extraction

5.5. Comparative GC-MS analysis of C. tamala essential oil extracted by different extraction techniques

Comparative GC-MS chromatograms of essential oils of C. tamala extracted

by traditional methods (HD, SE, US) and innovative SC-CO2 technique as shown in Fig. 6.

A total of forty five compounds were identified with GC-MS analysis using their retention time and mass from library (Nist and Wiley) (Table 4). Among them 21 compounds were sesquiterpene hydrocarbons, non-isoprenoid compounds containing fatty acid. Trans-caryophyllene was present in maximum amount, of all the identified compounds in oil extracted by hydro-distillation. It was absent in oil extracted by solvent extraction and ultrasonication. Germacerene D was next highest compound

present in oil extracted by hydro-distillation. Present in lesser quantity in solvent

extracted and SC-CO2 extracted oil, absent in ultrasonicated oil.

Fig. 6. Comparative GC-MS chromatograms of different oils of C. tamala extracted using different extraction techniques A: SC-CO2 oil, B: Hydro-distilled oil, C: Solvent extracted oil, D: Ultrasonicated oil

Table 4. Results of GC-MS analysis of C. tamala oil extracted by different extraction techniques. Area percent (%)

S. SC- Component Name RT* HD* SE* US* no. CO2 * Monoterpene hydrocarbon 1. α-Ylangene 27.57 0.74 - -

Oxygenated monoterpene 2. Linalool 12.04 8.83 - - - 3. Apiol 38.45 - 0.77 0.63 - Sesquiterpene hydrocarbons 4. Bicycloelemene 25.59 2.1 4.29 - - 5. Copaene 27.84 0.82 1.77 - - 6. β- elemene 28.83 0.81 - - - 7. Caryophyllene 30.12 5.11 26.3 9.05 - 8. trans-Caryophyllene 30.13 26.2 - - 13.4 9. Germacrene B 30.94 0.74 - - - 10. Aromadendrene 31.4 1.12 2.09 1.3 - 11. α.-Humulene 31.74 1.54 3.44 1.4 -

12. α.-Caryophyllene 31.75 - 3.44 1.04 - 13. β- cubene 31.8 0.49 - 1.3 - 14. Bicyclogermacrene 33.64 6.17 20.9 5.48 - 15. α.-Amorphene 32.84 0.64 - - - 16. Germacrene D 32.98 13.5 8.32 - 1.61 17. γ-Cadinene 34.35 0.49 - - - 18. δ.-Cadinene 34.6 0.54 2.11 0.41 1.63 19. β.-Sesquiphellandrene 34.81 - - - 2.4 20. Germacrene B 35.91 0.37 - - - 21. (-)-Spathulenol 36.69 1.11 - - - Non isoprenoid 22. Isolongifolene 36.71 - 1.44 - - 23. 1-Dodecene 17.01 0.94 - - - 24. Cyclotetradecane 3-Hexadecene, (Z)- 28.98 1.72 - - - 25. Phenol,2,4-bis(1,1-dimethylethyl) 34.57 4.61 - - - 26. 1-Hexadecene 37.27 2.64 - - - Bicyclo[4.4.0]dec-1-ene,2-isopropyl-5- 27. 38.88 0.45 - - - methyl-9-methylene- 28. 1-Nonadecene 42.85 2.44 - - - 29. Docosane 44.69 - 1.96 0.36 1.59 30. Nonacosane 45.07 - 0.81 - - 31. Hexadecanoic acid, methyl ester 45.61 3.6 - - - 32. Methyl palmitate 45.62 - 1.72 3.97 - 33. 3-Eicosene, (E)- 46.79 1.34 - - - 9,12-Octadecadienoic acid (Z,Z)-, 34. 48.56 14.2 - 49.4 3.94 methyl ester 35. 9-Octadecenoic acid (Z)-, methyl ester 48.64 13.2 5.08 13 - 36. 10,13-Octadecadienoic acid 48.81 - 10.3 1.04 - 37. Octadecanoic acid, methyl ester 49.05 1.68 - 1.04 - 38. Diisooctyl maleate 49.47 0.28 - - - 39. 1-Docosene Behenic alcohol 50.04 0.57 - - - 40. Dotriacontyl pentafluoropropionate 52.91 0.31 - - - 41. Triacontane 52.98 0.34 - - - 42. Butyl pthalate 55.06 0.36 1.55 6.19 30.2 43. Eicosane 55.32 - 1.8 1.17 2.1 44. Hexatriacontane 55.59 - 0.76 - - 45. Heneicosane 59.13 - - - 2.12 RT* Retention time, HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon-dioxide extraction

5.6. FTIR analysis The interpretation of infrared spectra of C. tamala essential oil was done by correlating of absorption bands in the spectrum with the known absorption frequencies bonds. Comparative spectra of essential oil extracted by different techniques are presented in Fig. 7 and Table 5 shows the peak common to all the four extraction techniques.

IR spectra of C. tamala oil is showing peak at 2858 cm-1 of strong stretching vibration of alkane common to all four extraction process. Strong stretching vibration of nitro group at wave number 1375 cm-1 is not present in hydro-distilled oil. Aromatic peak of strong to medium intensity and strong stretching vibration of ester

was present in solvent and SC-CO2 extracted oils only.

Fig. 7. Comparative spectra of different oils of C. tamala extracted using different extraction techniques A: Hydro-distilled oil, B: Ultra sonication oil, C: Solvent extracted oil, D: SC-CO2 oil Table 5. Results of FTIR analysis of C. tamala oil extracted by different techniques S.no Peak Functional group Intensity HD* SE* US* SFE* 1. 839.03 =C-H(alkene) Sretch, strong - + + - 2. 983.7 =C-H(alkene) Sretch, strong - + + - 3. 1101.35 C-O(alcohol) Sretch, strong + + + - 4. 1163.08 N-H(amine) Stretch, medium - + + - 5. 1375.25 N-O(nitro) Sretch, strong - + + + 6. 1452.4 C=C(aromatic) Strong, medium - + - + 7. 1735 C=O(ester) Stretch, strong - + - + 8. 2231.64 C-N(nitrile) Stretch , medium - + + - 9. 2858.51 C-H(alkane) Stretch, strong + + + + 10. 3446.79 O-H(alcohol) Stretch, strong + - - - 11. 3398.64 O-H(alcohol) Stretch, strong - + + -

HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon- dioxide extraction

5.7. Antioxidant activity

The C. tamala oil sample showed a significant concentration-dependent antioxidant activity by inhibiting DPPH free radical with an IC50 values of 89.4μg -1 -1 mL , whereas, IC50 value of ascorbic acid was found to be 28.09 μg mL used as standard (Table 6 and Fig. 8). It was found that the oil possesses hydrogen donating capabilities similar to ascorbic acid and acted as an antioxidant. The scavenging effect increased with increasing concentration of the extract and ascorbic acid (5.0-100 μg mL-1).

Table 6. Summary of % inhibition and IC50 values of C. tamala oil sample by DPPH method (n=3)

Percentage inhibition ± SD* Conc. (µg/mL-1) Ascorbic Acid C. tamala 100 92.5 ±1.1 51.9±0.8 50 64.3 ±0.9 41.9±0.4 25 45.7 ±1.0 14.6±1.2 12.5 29.5 ±0.54 11.9±0.8 6.25 20.6 ±0.8 11.0±0.8 3.125 12.1 ± 0.8 7. 3±1.2 1.5625 8.45 ±1.0 6.2±0.8 0.781 5.85± 0.8 1.7±0.8

IC50 28.09 89.4 SD* Standard deviation

Fig. 8. Comparative dose response curve between percent inhibitions against log concentration by DPPH method

The superoxide scavenging activity of the drug was also determined. The IC50 value of the essential oil of C. tamala was found to be 32.24 µg mL-1, whereas the -1 IC50 value of ascorbic acid was 20.14 µg mL as shown in Table 7 and Fig. 9.

Table 7. Summary of % inhibition and IC50 values of C. tamala oil sample by superoxide anion scavenging method (n=3) Percentage inhibition ± SD* Conc. (µg/mL-1) Ascorbic Acid C. tamala 10 38.28±0.8 32.38±2.0 20 48.72±1.2 35.30±2.02 40 62.33±0.8 43.03±1.6 60 71.67±1.2 68.66±2.9 80 89.28±1.2 75.93±2.4 100 95.22±1.2 88.66±3.2

IC50 20.14 32.24 SD* Standard deviation

log-dose vs response 100

40 std Ascorbic acid C. tamala 20 Percentage Inhibition Percentage 0 0 0.5 1 1.5 2 2.5 log concentration

Fig. 9. Comparative dose response curve between percent inhibitions against log concentration by superoxide scavenging method

An experiment was run to study the effect of with three factors (Pressure, temperature, flow rate) with three levels on response (Yield). The results were analyzed and are presented in Table 8.

Table 8. Estimated Effects and Coefficients for Yield Term Effect Coef SE Coef T P Constant 1.22614 0.07399 16.57 0.000 Pressure 2.17500 1.08750 0.07845 13.86 0.000 Temperature 0.07817 0.03909 0.07728 0.51 0.617 Flow rate 0.73625 0.36812 0.07845 4.69 0.000 Pressure*Temperature 0.03712 0.01856 0.07845 0.24 0.815 Pressure*Flow rate 0.72000 0.36000 0.07845 4.59 0.000 Temperature*Flow rate 0.31938 0.15969 0.07845 2.04 0.051 Pressure*Temperature*Flow 0.31188 0.15594 0.07845 1.99 0.057 rate

It can be seen that the selected factors showed significant effect either directly or through the interaction with other factors. The selected factors explained 89.69% fit model for the given data. The contour and surface plots have been shown in Fig. 10 & 11.

Contour Plots of Yield

Temperature*Pressure Flow rate*Pressure 55.0 15.0 Yield < 0.5 0.5 - 1.0 52.5 12.5 1.0 - 1.5 1.5 - 2.0 50.0 10.0 2.0 - 2.5 2.5 - 3.0 47.5 7.5 > 3.0

Hold Values 45.0 5.0 Pressure 200 100 125 150 175 200 100 125 150 175 200 Temperature 55 Flow rate*Temperature 15.0 Flow rate 15

12.5

10.0

7.5

5.0 45.0 47.5 50.0 52.5 55.0

Fig. 10. Contour plots of yield vs pressure, temperature and flow rate

Surface Plots of Yield

Hold Values Pressure 200 Temperature 55 Flow rate 15 3 3 Yield 2 Yield 2

1 1

0 45 0 5 200 50 200 10 150 Temeemperature 1500 FFlow rate Pressure 100 55 Pressssure 100 15

3.0 Yield 2.4 1.8

1.2 5 55 50 10 Temperature 45 15 FFlow rate

Fig. 11. Surface plots of yield vs pressure, temperature and flow rate

Pressure, flow rate and combination of pressure-flow rate have shown to have direct effect on yield as p<0.05, hence pressure, flow rate and combination of pressure-flow rate are significant factors for influencing yield. While temperature, combinations of pressure-temperature, temperature-flow rate and pressure- temperatute-flow rate have no significant effect on yield as p>0.05.

The optimum operating conditions for SC-CO2 extraction of essential oils from leaves of C. tamala for high yield were pressure (200 bar), temperature (55 oC)

and flow rate of CO2 (15 g/min). A confirmation experiment was run using the optimum conditions and the highest yield obtained was 3.64 %. 6. Conclusions In present work, essential oil from leaves of C. tamala was extracted via HD,

SE, US and SC-CO2, out of all these methods SC-CO2 gave the highest percentage yield (3.64%) using FFD with optimum conditions of pressure (200 bar), temperature o (55 C) and flow rate of CO2 (15 g/min).The analysis shows that pressure, flow rate and interaction of pressure-flow rate have significant effect on oil yield while temperature, combinations of pressure-temperature, temperature-flow rate and

pressure-temperatute-flow rate have no significant effect on yield as p>0.05. SC-CO2

is a green, environmentally friendly and sustainable extraction technique. Forty five compounds were identified by GC/MS. Essential oil obtained from leaves of C. tamala has shown to possess significant antioxidant activity which was investigated by DPPH and Superoxide anion scavenging methods and was found to be 89.4 and 32.24 µg mL-1, respectively.

References:

1. E. Sthal and D. Gerard, Solubility behaviour and fractionation of essential oils in dense carbon dioxide. Perfum. Flavor., 1985; 10: 29-37.

2. G.K. Jayaprakasha, L.J.M. Rao and K.K. Sakariah, Volatile constituents from Cinnamomum zeylanicum fruit stalks and their antioxidant activities. J. Agric. Food Chem., 2003; 51: 4344-4348.

3. A. Ahmed, M.I. Choudhary, A. Farooq, B. Demirci, F. Demirci and K.H.C. Baser, Essential oil constituents of the spice Cinnamomum tamala (Ham.) Nees & Eberm. Flavour Frag J., 2000; 15: 388-390.

4. R. Lee and M.J. Balick, Sweet wood-cinnamon and its importance as a spice and medicine. Explore-NY., 2005; 1: 61-64.

5. H.S. Yu, S.Y. Lee and C.G. Jang, Involvement of 5-HT1A and GABAA receptors in the anxiolytic-like effects of Cinnamomum cassia in mice. Pharmacol Biochem Behav., 2007; 87: 164-170.

6. L.K. Chao, K.F. Hua, H.Y. Hsu, S.S. Cheng, J.Y. Liu and S.T. Chang, Study on the antiinﬂammatory activity of essential oil from leaves of Cinnamomum osmophloeum. J. Agric. Food Chem., 2005; 53: 7274-7278.

7. B.H. Cheng, C.Y. Lin, T.F. Yeh, S.S. Cheng and S.T. Chang, Potential source of S-(þ)-linalool from Cinnamomum osmophloeum ct. linalool leaf: essential oil proﬁle and the enantiomeric purity. J Agr Food Chem., 2012; 60: 7623- 7628.

8. M.T. Golmakani and K. Rezaei, Comparison of microwave-assisted hydrodistillation with the traditional hydrodistillation method in the extraction of essential oils from Thymus vulgaris L. Food Chem., 2008; 109: 925-930.

9. B. Bayramoglu, S. Sahin and G. Sumnu, Solvent-free microwave extraction of essential oil from oregano. J. Food Eng., 2008; 88: 535-540.

10. F. Demirci, K. Guven, B. Demirci, M.Y. Dadandi and K.H.C. Baser, Antibacterial activity of two Phlomis essential oils against food pathogens. Food Control., 2008; 19: 1159-1164.

11. M. Khajeh, Y. Yamini, N. Bahramifar, F. Sefidkon and M.R. Pirmoradei, Comparison of essential oils compositions of Ferula assa-foetida obtained by supercritical carbon dioxide extraction and hydrodistillation methods. Food Chem., 2005; 91: 639-644.

12. E. Derwich, Z. Benziane, R. Taouil, O. Senhaji and M. Touzani, Aromatic Plants of Morocco: GC/MS Analysis of the Essential Oils of Leaves of Mentha piperita. Adv Environ Biol., 2010; 4: 80-85.

13. G. Sacchetti, S. Maietti, M. Muzzoli and M. Scaglianti, Comparative evaluation of 11 essential oils of different origin as functional antioxidants, antiradicals and antimicrobials in food. Food Chem., 2005; 91: 621-632.

14. S. Chanda and R. Dave, In vitro models for antioxidant activity evaluation and some medicinal plants possessing antioxidant properties. Afr. J. Microbiol. Res., 2009; 3: 981-996.

CHAPTER 6

SUPERCRITICAL CARBON-DIOXIDE EXTRACTION OF ESSENTIAL OIL FROM LEAVES OF EUCALYPTUS GLOBULUS L., ANALYSIS AND THEIR APPLICATION

1. Introduction

The plants contain a variety of compounds which are of great medicinal value for mankind. In order to extract these compounds in pure form, researchers are working over it extensively. Various conventional techniques (hydro-distillation, solvent extraction, soxhlet extraction) have been used for the extraction of natural compounds [1]. Hydro distillation is a simple and regarded as low cost extraction process, but its disadvantage is that some of the highly volatile and hydro soluble phytoconstituents will be lost permanently [2-5]. Solvent extraction is a simple process without any complex equipment having large selectivity and flexibility with controlled recovery. There are certain drawbacks of this solvent extraction which includes formation of emulsion, ineffective, loss of compounds, laborious, lengthy, pre-concentration step is required, it leaves traces of toxic solvents in the solute. This is a concern for food and medicinal extracts [6, 7]. UAE can be carried out at lower temperatures, avoiding thermal damage and the loss of volatile compounds in boiling. It is an economical, effortless and effective alternative to traditional techniques. It gives better extraction yield. Adverse effects of this process are: the ultrasound wave attenuation occurs due to the presence of dispersed phase and the dynamic part of ultrasound inside the extractor is limited to a zone situated in the vicinity of the ultrasonic emitter. Thus, these two factors must be considered in the design of ultrasound-assisted extractors [8, 9].

The concentrations of the essential oils and bioactive compounds in plants are usually very low, so it is necessary to apply a technique which is more effective, selective and environmentally friendly. SFE is an advance technique which offers numerous potential advantages over conventional extraction processes, including: reduced extraction time, solvent free, provides high solubility, improved mass transfer rates, better selectivity by changing temperature and pressure of the process and gives pure compound in less time [10-12]. Carbon dioxide which is non-toxic, low cost, easily available in high purity, non-corrosive, inert, non-ﬂammable and non- explosive, is most widely used supercritical fluid and considered as a GRAS (Generally Recognized As Safe) solvent [13-16]. Thermolabile compounds can also o be extracted by using CO2 due to its low critical temperature (31.1 C). Carbon dioxide is recycled in industries and hence provides clean and compact operations [10, 15, 17, 18].

The Eucalyptus is an evergreen tall tree which is a single group of plants belonging to Myrtaceae family includes around 900 species and subspecies [19, 20]. Over 300 species of this genus are known to contain volatile oils in their leaves, though fewer than 20 of these have ever been exploited commercially for the production of essential oils rich in 1,8-cineole (more than 70%) which are used in the pharmaceutical and cosmetic industries [21]. Essential oil extracted from various varieties of eucalyptus is beneficial in treatment of several infections and also as natural food preservatives [22, 23]. Apart from its extensive use in pulp industry, Oleum Eucalypti (eucalyptus oil) which is extracted on commercial scale in many countries like China, India, South Africa, Portugal, Brazil and Tasmania is used as raw material in perfumery, cosmetics, food beverage, aromatherapy and phytotherapy. This oil plays a vital role in several biological functions (antibacterial, antifungal, antihyperglycaemic and antioxidant activities) [24]. Eucalyptus oil is now considered under the GRAS category by the U.S. Food and Drug Administration and classiﬁed as nontoxic [25, 26].

The aim of this study was to extract essential oils from the dried leaves of

Eucalyptus globulus L. via four extraction methods (HD, SE, UAE and SC-CO2) in order to compare yield and to determine the composition of essential oils via HPTLC, GC-MS and FTIR along with its antioxidant activity.

2. Materials and methods

2.1. Plant Material

The plant material (dried leaves of Eucalyptus) was collected from Aligarh Muslim University (campus), Aligarh. As a ﬁrst step, dried eucalyptus leaves were grounded in a mechanical grinder for a short but sufficient period of time (30 s) to get a uniform particle size distribution. Grounded powder was then sieved (50 mesh).

2.2. Solvents and reagents

All the ingredients taken were of pharmacopeial quality and quantity. Standards were obtained from Sami Labs Ltd., Bangalore, (India) as a gift samples. Potassium hydroxide (KOH), Ethanol, ether, Hydrochloric acid (HCl), Ethanolic hydroxylamine hydrochloride solution (90%), Ethanolic Potassium hydroxide solution

(60%), 10 % Folin-Ciocalteu, Na2CO3 (Sodium carbonate), Standard (Gallic acid), methanol, DPPH (Diphenyl-1-picrylhydrazyl), Standard (Ascorbic acid), Phenazine

methosulfate (PMS), Nitroblue tetrazolium (NBT) and Nicotinamide adenine dinucleotide (NADH) used were of analytical grade (AR) and were procured from Merck Ltd. India. Carbon-dioxide was obtained from Sigma gases, New Delhi, India.

2.3. Design of experiment

Full factorial design (FFD)

A factorial design is type of designed experiment that lets us study the effects that several factors can have on a response. When conducting an experiment, varying the levels of all factors at the same time instead of one at a time lets us study the interactions between the factors. The most fundamental structures experimental design is FFD. In FFD, responses are measured at all combinations of experimental factor levels [27]. Each experimental condition is called a “run” and each run represents variation of one variable. Each response will be measured for an observation. The entire set of runs is the “design”. Since, we consider three parameters with three levels, the total run with full factorial design is 33 (27) number of trials. The variation levels for parameters were pressure (100, 200 and 350 bar), temperature (40, 60 and 80 oC) and flow rate (8, 10 and 12 g/min).

2.4. Hydro-distillation Method

The grounded powder (150 g) and distilled water (500 mL) were mixed in 1L round bottomed flask and heated for 5.0 h to yield essential oil [28]. The extract was centrifuged at 10,000 rpm for 10 min in order to separate small water droplets present in the essential oil. The oil was kept at 4 °C until further quantification and percent yield was calculated.

2.5. Solvent Extraction

The coarse powder (50 g) was extracted with hexane (100 mL) in 1:2 w/v, ratio and was kept in conical flask for 24 h [6, 7]. After that extract was filtered and concentrated under reduced pressure in rotary vacuum evaporator at 40 °C. The oil so obtained was stored at 4 °C prior to analysis.

2.6. Ultrasonic Assisted Extraction

The coarse powder (50 g) of plant material was taken in a 250 mL conical flask along with 100 mL hexane (1:2, w/v). The flask was covered and then placed in an ultrasound water bath apparatus for 30 min (frequency 33 kHz). The temperature

100

of the water bath was held constant at 25 °C. The extract was filtered and concentrated under reduced pressure in rotary vacuum evaporator [8, 9]. The obtained oil was stored at 4 °C prior to analysis and its percentage yield was calculated.

2.7. Supercritical CO2 Extraction

A SFE-1000M1- 2- C50 system (Pittsburgh, PA) was used for extractions. The extraction vessel was 200 mL stainless steel. Grounded plant material (50 g) was loaded in the extractor and the CO2 was allowed to pass through the seed matrix at desired pressure (100, 200 and 350 bar), temperature (40, 60 and 80 oC), flow rate (8, 10 and 12 g/min) for 45 mins. After that oil was collected from the collecting vessel [29]. The extracted oil was stored at 4 oC prior to analysis and its percentage yield was calculated.

2.8. Characterization

Physicochemical characterization of the essential oil extracted from E. globulus was carried out in order to determine its colour, odour, specific gravity, refractive index, optical rotation, solubility in alcohol, acid value, ester value, aldehyde content, phenolics and anthocyanin content. Procedures for all have already been given in chapter 3.

The surface morphology of oily and non-oily biomass of eucalyptus leaves before and after SC-CO2 extraction was examined by scanning electron microscopy (SEM) using Model JSM-6510LV, JEOL, (Japan). The SEM was operated at 10 kV, working distance of 11 and 12 mm, at magnification of 1000x and 2000x.

In High performance thin layer chromatography (HPTLC) analysis, the samples were spotted in the form of band (3.0 mm) with a Camag microlitre syringe on TLC aluminium plate precoated with silica gel 60F-254 (20 × 10 cm with 0.2 mm thickness, E. Merck, Germany) using a Camag Linomat V sample applicator. The development was carried out in mobile phase composed of hexane: ethyl acetate: formic acid (7:2:1, v/v/v). The HPTLC plates were studied at 254 nm and 366 nm as well as in visible range (580 nm) after spraying with anisaldehyde sulfuric acid reagent. A good separation of constituents was observed at 254 nm and 580 nm.

Gas chromatography-mass spectrometry (GC–MS) was equipped with a DB-5 fused silica capillary tubes column (30 m × 0.25 mm × 0.25 μm). The injection

101

volume was 1.0 μL using auto-sampler at a carrier gas (helium) flow of 2.0 μL min-1 with a split less mode. The initial oven temperature was 65 °C (3.0 min) then raised by 2.0 °C min-1 to 114 °C, then by 4 °C min-1 to 160 °C and 6 °C min-1 to 302 °C and then finally ramped to 310 °C at 15 °C min-1. Detector type was MS and its interface temperature was 250 °C. The essential oil obtained by different extraction techniques were diluted by adding 1998 µL of hexane to 2.0 µL oil (Hydro distilled oil) and 1990 µL hexane to 10 µL oil (extracted by other techniques). Compounds were detected and identified by comparing with the National Institute of Standard and Technology (NIST) library [30]. Fourier transform infrared (FTIR) spectral analysis was performed by using shimadzu BioRad FTIR (Kyoto, Japan). The samples (2 µL) were dispersed and triturated with dry KBr and grounded well in mortar and pestle to make (KBr) disk at a pressure of 1,000 psig. Then disk was placed in the FTIR sample holder, where spectra in absorbance mode was obtained in the spectral region 4000 to 400 cm-1 using the resolution 4 cm-1. 3. Application

Antioxidant activity The antioxidant activity of essential oil sample extracted from leaves of E. globulus was determined by two methods; Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging [31] and Superoxide anionic scavenging [32]. Procedures for both the methods have already been given in Chapter 3. 4. Statistical analysis

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observed. The characteristic odour was determined by sniffing and to determine its characteristic feel to touch, it was rubbed between fingers. It was found to be pale yellow liquid having aromatic and camphoraceous odour with pungent taste.

Other physicochemical characterizations of essential oil extracted from E. globulus were determined and have been mentioned in the Table 1. Anthocyanin

content was found to be maximum in the essential oil extracted by SC-CO2 extraction

as shown in Fig. 1. Percent of phenolic content was found to be maximum in SC-CO2 extracted essential oils (Fig. 2) as it retains all the phytoconstituents with purity, calibration curve is given in chapter 3.

Table 1. Physicochemical characterization of essential oil

E. globulus HD* SE* US* SC-CO2* Reported (dried value leaves) oil specific gravity 0.899 0.880 0.878 0.899 0.906-0.925 (25°C) refractive 1.44 1.460 1.459 1.65 1.458-1.470 index (25°C) Optical rotation -3.9° -4° -7.1° -3.9° -2° to +5° (25°C) Solubility in Clearly Clearly Clearly Clearly Soluble in 4 alcohol (% soluble in soluble in soluble in soluble in mL of 70% v/v ethanol) 5.0 mL 5.0 mL 4.5 mL 4.4 mL ethanol

Acid value (Mean±SD*) 5.8±0.2 6.0±0.5 5.9±0.2 5.4±0.1 -

Ester value 9.1±0.1 9.9±0.2 9.6±0.1 8.5±0.1 - (Mean±SD*) Aldehyde content 2.5 3 3.1 3.4 - (% w/v) Phenolics in %w/w 5.5±0.2 5.8±0.1 6.0±0.08 6.3±0.1 - (Mean ± SD*) Anthocyanin content - %w/w 5.4±0.1 5.7±0.1 6.0±0.1 6.7±0.08 (Mean ± SD*) HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon- dioxide extraction

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8 7 6 5 4 3 E. globulus 2

Anthocyanin % w/w Anthocyanin 1 0 HD SE US SC-CO2 Oil sample

Fig. 1. Comparative study of anthocyanin contents in essential oil of E. globulus extracted by different extraction techniques

6 5 4 3 2 E. globulus 1 Phenolic contentPhenolic % w/w 0 HD SE US SC-CO2 Oil sample

Fig. 2. Comparative study of phenolic contents in essential oil of E. globulus extracted by different extraction techniques

5.2. Percentage yield (% v/w)

By using extraction techniques, HD, SE, UAE and SC-CO2 percent yield of essential oil was found to be 2.0%, 2.2%, 2.6% and 3.6%, v/w, respectively. 36 experiments were run at pressure (100, 200 and 350 bar), temperature (40, 60 and 80 oC), flow rate (8, 10 and 12 g min-1) at various combinations in order to study direct effect of each parameter and the effect of interactions between parameters

on the yield of the essential oil extracted by SC-CO2 extraction technique. The yield of the essential oil extracted, increases with increasing pressure as the density of SC-

CO2 increases at higher pressure and hence the solvent power to dissolve the oil inside the matrix of the powdered plant material increases. Increasing temperature increases the solubility of the essential oil, which results in higher yields. Instead of that the increasing temperature contributed to

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damage the particle cell walls and as the result availability of essential oil for extraction was increased.

With the increase in the CO2 flow rate, the intermolecular interaction between

CO2 and the essential oil increases, hence increasing the solute dissolution. Amount and time have no significant effect on oil yield.

Experimental result shows that the increase in CO2 flow rate has enhanced the higher extraction efficiency. Extraction of essential oil can thus be conducted within short duration of time with higher extraction yield. Highest yield of essential oil was obtained at pressure (350 bar), temperature (80 oC) and flow rate (12 g/min). 5.3. SEM

SEM images of oily and non-oily biomass before and after SC-CO2 extraction were observed at 1000 and 2000 magnification. Fig. 3 shows that oily biomass has smoother surfaces of oil glands filled with oil before SC-CO2 extraction while non-

oily biomass appears smashed with rugged features and rough edges after SC-CO2 extraction. *More explanation is given on page 131.

Fig. 3. SEM images of Eucalyptus leaves biomass: (A) before SC-CO2, (B) after SC- CO2 at 1000x magnification and (C) before SC-CO2, (D) after SC-CO2 at 2000x magnification

5.4. Development of solvent system for separation of oil sample by TLC

TLC was done to separate the constituents of essential oil obtained by various extraction techniques. Number of solvent systems in different proportions were tried for the separation of phytoconstituents present in the essential oils. The solvent

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system composed of hexane: ethyl acetate: formic acid (7:2:1, v/v/v) resulted in maximum separation of constituents and compactness of bands for all the oil samples.

5.5. HPTLC The spotted HPTLC plates were studied at 254 nm, 366 nm and in visible range (580 nm) after spraying with anisaldehyde sulphuric acid reagent Fig. 4.

Fig. 4. HPTLC fingerprint of different oils of E. globulus extracted using different extraction techniques [track 1-2: Hydro-distilled oil, 3-4: Solvent extracted oil, 5-6: Ultra sonicated oil, 7-8: SC-CO2 oil] visualized at A: 254 nm, B: 366 nm, C: Day light after derivatization using anisaldehyde sulphuric acid reagent

A good separation of constituents was observed at 254 nm. The comparative

results of different oils showing a dominant constituent at Rf value 0.5 at 254 nm and 580 nm (sprayed) whereas it was not observed at 366 nm. Track 1 and 2 of hydro distilled oil showing lesser no. of compounds with major part being intense blue spot. Solvent extracted and ultrasonic assisted oil showed equal amount of separation. SC-

CO2 extracted oil showing some new spots not present in other extracted oils. 3D Chromatogram and HPTLC fingerprinting of the oils extracted from different methods scanned at 254 and 366 nm are shown in Fig. 5a, 5b and Fig 6a, 6b, respectively. The comparative results of different oils are given in Table 2.

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Fig. 5a: 3D Chromatogram of 8 tracks of E. globulus oil obtained by different extraction techniques at 254 nm

Fig. 5b: 3D Chromatogram of 8 tracks of E. globulus oil obtained by different extraction techniques at 366 nm

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Fig. 6a: HPTLC fingerprint of different oils of E. globulus extracted using different extraction techniques A: Hydro-distilled oil, B: Solvent extracted oil, C: Ultra sonicated oil, D: SC-CO2 oil visualized at 254 nm with assigned substance

Fig. 6b: HPTLC fingerprint of different oils of E. globulus extracted using different extraction techniques A: Hydro-distilled oil, B: Solvent extracted oil, C: Ultra sonicated oil, D: SC-CO2 oil visualized at 366 nm with assigned substance

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Table 2. Substance A-T with their Rf and area percent of E. globulus essential oil obtained by different extraction techniques Area percentage (%) 254 nm Area percentage (%) 366 nm Substance (Rf) HD* SE* US* SFE* HD* SE* US* SC-CO2* A(0.03) 1.37 6.10 4.11 0.90 3.00 2.31 1.75 1.14 B(0.05) - - - 0.70 - 2.41 1.75 - C(0.07) - 2.04 2.44 - - 2.65 3.05 0.76 D(0.08) - 2.53 2.88 1.31 - 3.59 7.91 2.68 E(0.11) - - 2.23 2.04 - - - - F(0.13) - 3.68 - 1.58 - 3.92 - - G(0.16) - 14.63 15.26 - - 8.46 2.89 - H(0.2) - - - 14.42 - - 5.85 5.50 I(0.27) 1.17 3.54 16.44 - - - - 8.80 J(0.3) - 13.52 - 6.19 - 5.10 5.30 - K(0.34) - - - 4.05 - 4.15 - 7.25 L(0.37) 12.09 - 5.85 - - 16.86 20.44 8.98 M(0.41) 7.43 5.12 3.11 4.67 - - - - N(0.46) - - - 5.48 37.27 5.83 5.78 3.97 O(0.5) 72.61 - 11.22 - 31.61 - - - P(0.55) - 9.79 - - - 7.15 8.47 4.98 Q(0.59) - 5.31 - 18.40 14.48 - - - R(0.68) - 28.95 25.02 - 13.64 30.92 28.99 55.32 S(0.73) - - 6.14 35.81 - 6.66 7.84 - T(0.78) 5.32 4.79 5.32 4.45 - - - 0.61 Rf* Retention factor HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon-dioxide extraction

The HPTLC plate scanned at 580 nm (Fig. 7a and 7b) showing maximum

area percent at Rf (0.41) in SC-CO2 extracted oil, with lesser area percent at 254 nm, not visible at 366 nm. Substance with maximum area percent in plate scanned at 254

nm at Rf (0.5) was showing lower concentration at 580 nm with its presence in solvent extracted oil also. Substance B, C, D, F and G were present in all extracted oil samples except hydro distilled oil. Substance O and I were present in all the extracted oil samples with different area percentage. Substance L was present in all except SC-

CO2 extracted oil. The comparative results of different oils are presented in Table 3.

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Fig. 7a. 3D Chromatogram of 8 tracks of E. globulus oil obtained by different extraction techniques at 580 nm

Fig. 7b. HPTLC fingerprint of different oils of E. globulus extracted using different extraction techniques A: Hydro-distilled oil, B: Solvent extracted oil, C: Ultra sonicated oil, D: SC-CO2 oil visualized at 580 nm with assigned substance

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Table 3. Substance A-O with their Rf and area percent of E. globulus essential oil obtained by different extraction techniques Area percentage(%) 580 nm Substance (Rf) HD* SE* US* SC-CO2* A(0.01) 0.09 0.74 - - B(0.04) - 2.81 1.77 5.52 C(0.11) - 1.98 1.78 2.37 D(0.11) - 4.04 1.82 3.49 E(0.15) - - 0.57 0.98 F(0.2) - 2.07 0.81 2.05 G(0.27) - 8.06 6.17 6.17 H(0.34) 16.13 - - - I(0.41) 35.05 24.70 26.69 40.33 J(0.5) - 6.28 5.81 8.33 K(0.55) - 4.02 4.36 - L(0.6) 12.08 6.22 3.60 - M(0.67) - 13.93 19.87 18.14 N(0.71) - 5.36 5.29 - O(0.78) 14.59 19.79 21.47 12.62 Rf* Retention factor HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon-dioxide extraction

5.6. GC-MS Comparative GC-MS chromatograms of essential oils of E. globulus extracted

by traditional methods (HD, SE and UAE) and innovative SC-CO2 technique are shown in Fig. 8. Chromatographic analysis (GC/MS) of the essential oil allowed the separation and identification of 53 components by using their retention time and mass from library (Nist and Wiley) (Table 4). The major terpenoidal compounds in oil extracted by HD method, include cymene (26.39%), β-pinene (15.21%) and eudesmol

(12.64%). The oil extracted by SC-CO2 contained eudesmol (12.64%) and 1, 2 benzenedicarboxylic acid (19.28%). The fatty acids were also present in significant amount as 9, 12, 15-octadecatrienoic acid and methyl ester (56.70%) in ultrasonicated oil and 9, 12-octadecadienoic acid (34.43%) in solvent extracted oil. In addition to these α- selinene (9.83%), β- selinene (2.16%), 4- terpineol (1.95%), borneol (1.33%), thymol (0.45%) and carvacol (0.64%) were also present.

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Fig. 8. Comparative GC-MS chromatograms of different oils of E. globulus extracted using different extraction techniques A: Hydro-distilled oil, B: SC-CO2 oil, C: Solvent extracted oil, D: Ultra sonicated oil

Table 4. Results of GC-MS analysis of E. globulus oil extracted by different techniques Area percent (%) S. SC- Component Name RT* HD* SE* US* no. CO2* Monoterpene hydrocarbon 1. α.-pinene 5.45 10.6 0.21 0.65 - 2. (-)-β-Pinene 6.77 15.21 3.29 2.82 1.86 3. l-Phellandrene 7.76 10.29 4.01 4.16 11.2 4. Cymene 8.59 26.39 2.35 2.04 0.82 5. γ.Terpinene 10.03 1.33 1.34 - - 6. α.-terpinolene 11.45 0.5 - - - Oxygenated monoterpene 7. D-Fenchyl alcohol 12.68 0.79 - - - 8. Linalool 14.48 0.21 - - - 9 Borneol 15.5 1.32 - - - 10. 4-Terpineol 16.19 1.95 - 0.35 - 11. β-fenchyl alcohol 16.97 - - - 3.14 12. Myrtenol 17.33 0.65 - - -

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13. α-phellandrene epoxide 19.71 0.47 - - - 14. Thymol 23.34 0.45 - - - 15. Carvacrol 23.86 0.64 - - - Sesquiterpene hydrocarbons 16. Copaene 27.83 0.15 - - - 17. Caryophyllene 30.11 0.14 - -

18. α.-Humulene 31.73 0.07 - - - 19. α-amorphene 34.36 0.03 - - - 20. Alloaromadendrene 34.92 0.04 - 0.14 - 21. β-Selinene 36.19 0.26 2.16 - - 22. Valencene 38.47 - 0.32 0.14 1.44 23. (+)-α.-Selinene 39.22 - 2.36 - 9.83 24. β-Maaliene 40.96 - 0.61 0.8 2.95 Oxygenated sesquiterpenes derivatives 25. 3-Cyclohexene-1-methanol 17 4.03 - 0.35 - 26. 2-Cyclohexen-1-one 20.24 0.4 - - - 27. Phellandral 21.82 0.05 - - - 28. Elemol 35.75 0.06 - - - 29. Champacol 37.46 0.05 - - - 30. Eudesm-4(14)-en-11-ol 40.23 11.41 2.73 0.44 12.64 Non isoprenoid 31. 1-Tetradecene 28.98 0.08 - - - Phenol, 2,4-bis(1,1- 32. 34.6 0.16 - - - dimethylethyl 33. Dodecanoic acid, methyl ester 34.95 0.11 - 0.3 - 1,6,10-Dodecatrien-3-ol, 3,7,11- 34. 36.35 0.85 - - - tr imethyl 35. 2-Tetradecene, (E)- 37.28 0.07 - - - 36. Methyl tetradecanoate 41.29 0.04 - 0.11 - 37. Methyl palmitate 45.61 0.42 4.57 - - 38. Hexadecanoic acid, methyl ester 45.62 - - - 1.71 39. 5-Eicosene, (E)- 46.78 0.06 - - - 40. 1-Nonadecene 48.47 0.04 0.13 - - 41. 9,12-Octadecadienoic acid (Z,Z)- 48.58 - 34.43 8.71 6.37 9-Octadecenoic acid (Z)-, methyl 42. 48.69 1.71 0.55 - 5.44 ester 9,12,15-Octadecatrienoic acid, 43. 48.71 - - 56.7 - methyl ester 44. Octadecanoic acid, methyl ester 49.05 0.15 1.61 3.95 - 45. Arachidic acid methyl ester 50.04 0.05 0.32 - - 46. cis-11-Eicosenoic acid, methyl 51.67 0.02 - - -

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ester 47. Docosanoic acid 54.74 0.08 0.25 - - 48. 1,2-benzenedicarboxylic acid 55.06 - 27.91 0.3 19.28 49. Hexatriacontane 55.47 - 0.15 0.18 0.79 50. Tetracosanoic acid, 57.22 0.02 - - - 51. Squalene 58.41 - - 0.69 - 52. Eicosane 59.13 - - 0.3 2.5 53. Vitamin E 61.83 - - 0.16 -

RT* Retention time, HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon-dioxide extraction

5.7. FTIR

The interpretation of infrared spectra of E. globulus L. essential oil was done by correlating of absorption bands in the spectrum with the known absorption frequency bands. Comparative spectra of essential oil extracted by different techniques are shown in Fig. 9 and FTIR results are presented in Table 5.

Spectra showed the presence of sharp peak of aromatic para-disubstituted benzene ring in solvent extracted and ultrasonicated oil samples. A prominent peak of ether was present in all extracted oil samples except in hydro-distilled oil. Peaks at 858.32 cm-1 (Aromatic p-disubstituted benzene), 1629.85 cm-1 (conjugated diene), 2229.43 cm-1 (alkyne), 2926 cm-1(methylene) and 3342.64 cm-1 (O-H (alcohol)) are -1 absent in hydro distilled and SC-CO2 extracted oil samples. Peak at 1452.4 cm (C=C (aromatic)) was absent in solvent and ultra sonicated extracted oil. Peak at 1375.95 cm-1 was absent in ultra sonicated extracted oil which lacks presence of nitro compounds.

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Fig. 9. Comparative spectra of different oils of E. globulus extracted using different extraction techniques A: Solvent extracted oil, B: Ultra sonicated oil, C: Hydro- distilled oil, D: SC-CO2 oil

Table 5. Results of FTIR analysis of E. globulus oil extracted by different techniques SC- S.no Peak Functional group Intensity HD* SE* US* CO2* Aromatic p- Strong 1. 858.32 - + + - disubstituted benzene 2. 1174.65 C-F(fluoroalkane) Strong, broad - + + + 3. 1301.95 C-O( ether) Strong,stretch - + + + 4. 1375.95 N-O(nitro,aliphatic) weaker + + - + C=C(aromatic) Stretch,medium 5. 1452.4 + - - + - strong C-C(conjugated Strong 6. 1629.85 - + + - diene) 7. 2229.71 -C=-C(alkyne) Stretch,strong - + + - methylene Medium - 8. 2926.43 - + + - strong 9. 3342.64 O-H(alcohol) Strong, broad - + + -

HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon- dioxide extraction

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5.8. Antioxidant activity

The E. globulus oil sample showed a significant concentration-dependent antioxidant activity by inhibiting DPPH free radical with an IC50 values of 47.61 μg -1 -1 mL , whereas, IC50 value of ascorbic acid was found to be 28.09 μg mL used as standard (Table 6 and Fig. 10). It was found that the oil possesses hydrogen donating capabilities similar to ascorbic acid and acted as an antioxidant. The scavenging effect increased with increasing concentration of the extract and ascorbic acid (5.0-100 μg mL-1).

Table 6. Summary of % inhibition and IC50 values of E. globulus oil sample by DPPH method (n=3) Percentage inhibition % (Mean ± SD*) Conc. (µg mL-1) Ascorbic acid E. globulus 100 92.5 ±1.1 55.5±0.8 50 64.3 ±0.9 43.5±0.8 25 45.7 ±1.0 30.7±0.8 12.5 29.5 ±0.54 25.8±1.2 6.25 20.6 ±0.8 21.1±0.8 3.125 12.1 ± 0.8 15.8±0.9 1.5625 8.45 ±1.0 5.8±0.8 0.781 5.85± 0.8 0.5±0.8

IC50 28.09 47.61 SD* Standard deviation

log-dose vs response

100 80 60 40 std Ascorbic acid 20 E. globulus 0 Percentage Inhibition Percentage -1 0 1 2 3 Log concentration

Fig. 10. Comparative dose response curve between percent inhibitions against log concentration by DPPH method

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The superoxide scavenging activity of the oil sample was increased markedly with the increase in concentrations. A comparison of the antioxidant activity of the

extract and ascorbic acid is shown in Table 7 and Fig. 11. The IC50 value of the -1 essential oils of E. globulus was found to be 26.05 µg mL , whereas the IC50 value of ascorbic acid was 20.14 µg mL-1.

Table 7. Summary of % inhibition and IC50 values of E. globulus oil sample by superoxide anion scavenging method (n=3) Percentage inhibition ± SD* Conc. (µg/mL-1) Ascorbic Acid E. globulus 10 38.28±0.8 35.76±2.5 20 48.72±1.2 40.35±1.5 40 62.33±0.8 54.06±0.2 60 71.67±1.2 72.88±0.9 80 89.28±1.2 80.07±2.5 100 95.22±1.2 87.26±1.5

IC50 20.14 26.05 SD* Standard deviation

log-dose vs response 100

40 std Ascorbic acid E. globulus 20 Percentage inhibition Percentage 0 0 1 2 3 log concentration

Fig. 11. Comparative dose response curve between percent inhibitions against log concentration by superoxide scavenging method

5.9. Full factorial design (FFD) An experiment was run to study the effect of three factors (Pressure, temperature, flow rate) with three levels on response (Yield). The results were analyzed and are presented in Table 8. It can be seen that the selected factors showed significant effect either directly or through the interaction with other factors. The

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selected factors explained 86.69% fit model for the given data. The contour and surface plots have been shown in Figs. 12 & 13.

Table 8. Estimated Effects and Coefficients for Yield Term Effect Coef SE Coef T P Constant 1.25434 0.08354 15.01 0.000 Pressure 2.10096 1.05048 0.08835 11.89 0.000 Temperature 0.08048 0.04024 0.08724 0.46 0.648 Flow rate 0.74569 0.37284 0.08855 4.21 0.000 Pressure*Temperature 0.05230 0.02615 0.08850 0.30 0.770 Pressure*Flow rate 0.74556 0.37278 0.08855 4.21 0.000 Temperature*Flow rate 0.30381 0.15191 0.08855 1.72 0.097 Pressure*Temperature*Flow 0.27244 0.13622 0.08855 1.54 0.135 rate

Contour Plots of Yield

Temperature*Pressure Flow rate*Pressure 80 12 Yield < 0.5 0.5 - 1.0 70 11 1.0 - 1.5 1.5 - 2.0 60 10 2.0 - 2.5 2.5 - 3.0 50 9 > 3.0

Hold Values 40 8 Pressure 350 100 200 300 100 200 300 Temperature 80 Flow rate*Temperature 12 Flow rate 12

8 40 50 60 70 80

Fig. 12: Contour plots of yield vs pressure, temperature and flow rate

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Surface Plots of Yield

Hold Values Pressure 350 Temperature 80 Flow rate 12

3 3 Yield 2 Yield 2 1 1 0 40 0 8 60 10 300 TeTemperature 300 Flow rate 200 808 20000 121 Pressure 1000 Prressure 1000

3.0 Yield 2.424 1.8 1.2 8 80 10 60 112 Flow rate Temperature 440

Fig. 13. Surface plots of yield vs pressure, temperature and flow rate

The optimum operating conditions for SC-CO2 extraction of essential oils from leaves of E. globulus for high yield were pressure (350 bar), temperature (80 oC) and flow rate of CO2 (12 g/min). A confirmation experiment was run using the optimum conditions and the highest yield obtained was 3.6 %.

6. Conclusion

In present work, essential oil was extracted via HD, SE, UAE and SC-CO2

extraction. Among all these methods SC-CO2 gave the highest percentage yield (3.6%) at optimum conditions of pressure (350 bar), temperature (80 oC) and flow rate

of CO2 (12 g/min) as determined by FFD. Pressure, flow rate and combination of pressure-flow rate have shown to have direct effect on yield as p<0.05, hence pressure, flow rate and combination of pressure-flow rate are significant factors for influencing yield. While temperature, combinations of pressure-temperature, temperature- flow rate and pressure-temperatute-flow rate have no significant effect on yield as p>0.05.

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Increasing the CO2 flow rate results in better oil yield due to enhanced interaction

between CO2 and essential oil. Physicochemical characterization of essential oil

extracted by HD, SE, UAE and SC-CO2 shows that the aldehyde, phenolic and

anthocyanin content are highest in SC-CO2 extracted oil as all the phytoconstituents are retained in the extracted oil without any loss or addition of any adulterant and also

free from any toxic solvents as no organic solvents are used in the SC-CO2 extraction

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22. B. Tepe, D. Daferera, M. Sökmen, M. Polissiou and A. Sökmen, In vitro antimicrobial and antioxidant activities of the essential oils and various extracts of Thymus eigii M Zohary et PH Davis. J. Agric. Food Chem., 2004; 52: 1132-1137.

23. K.M. Shuenzel and M.A. Harisson, Microbial antagonists of food borne pathogen on fresh minimally processed vegetables. J. Food Protect., 2002; 65: 1909-1915.

24. T. Takahashi, R. Kokubo and M. Sakaino, Antimicrobial activities of Eucalyptus leaf extracts and ﬂavonoids from Eucalyptus maculate. Lett Appl Microbiol., 2004; 39: 60-64.

25. USEPA (United States Environment Protection Agency), R.E.D. FACTS, Flower and Vegetables Oil. U.S. EPA, Ofﬁce of Pesticide Programs, Special Review and Reregistration Division, Washington D.C. 1993.

26. M. Mahboubi, N. Kazempour and M. Valian, Antimicrobial activity of natural respitol-b and its main components against poultry microorganisms. Pak. J. Biol. Sci., 2013; 16: 1065-1068.

27. Z. Solati, B.S. Baharin and H. Bagheri, Supercritical carbon dioxide (SC-CO2) extraction of Nigella sativa L. oil using full factorial design. Ind. Crop. Prod., 2012; 36: 519-523.

28. A. Elaissi, K.H. Salah, S. Mabrouk, K.M. Larbi, R. Chemli and F. Harzallah- Skhiri, Antibacterial activity and chemical composition of 20 Eucalyptus species’ essential oils. Food Chem., 2011; 129: 1427-1434.

29. M. Khajeh, Y. Yamini, N. Bahramifar, F. Sefidkon and M.R. Pirmoradei, Comparison of essential oils compositions of Ferula assa-foetida obtained by supercritical carbon dioxide extraction and hydrodistillation methods. Food Chem., 2005; 91: 639-644.

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31. G. Sacchetti, S. Maietti, M. Muzzoli and M. Scaglianti, Comparative evaluation of 11 essential oils of different origin as functional antioxidants, antiradicals and antimicrobials in foods. Food Chem., 2005; 91: 621-632.

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CHAPTER 7

OPTIMIZATION OF SUPERCRITICAL CO2 EXTRACTION, CHEMICAL COMPOSITION AND

ANTIOXIDANT ACTIVITY OF ESSENTIAL OIL FROM

TRACHYSPERMUM AMMI L.

1. Introduction Trachyspermum ammi L. is an annual herb belonging to family Apiaceae. It is also known by other common names as, Sanskrit: Yamini, Yaminiki, Yaviniki, Assamese: Jain, Bengali: Yamani, Yauvan, Yavan, Javan, Yavani, Yoyana, English: Bishop's weed, Gujrati: Ajma, Ajmo, Yavan, Javain, Hindi: Ajwain, Jevain, Kannada: Oma, Yom, Omu, Malayalam: Oman, Ayanodakan, Marathi: Onva, Oriya: Juani, ajwan [1]. It is known to be native of اﺟﻮاﺋﻦ :Tamil: Omam, Telugu: Vamu, Urdu Egypt and is also cultivated in Iraq, Iran, Afghanistan, Pakistan, and India (Madhya

pradesh, Uttar pradesh, Gujarat, Rajasthan, Maharashtra, Bihar and West Bengal) [115T ]15T . Gujarat and Rajasthan are its major cultivators in India. Thymol (2-isopropyl-5-

methylphenol) is a natural monoterpene phenol derivative of cymene, CR10RHR14RO, isomeric with carvacrol. It has been reported in many plants which contains thymol as major component likes Trachyspermum ammi (Ajwain), Monarda didyma, Monarda fistulosa, Origanum dictamnus, Origanum compactum, Origanum dictamnus, Origanum onites, Origanum vulgare, Thymus glandulosus, Thymus hyemalis, Thymus vulgaris, Thymus zygis, Satureja hortensis etc [2-5]. Trachyspermum ammi (L) Sprague is a Greek word Trachy means rough and spermum means seeded, whereas ammi is name of plant in Latin Syn. Carum copticum, commonly known as Joan belonging to family Apiaceae or Umbelliferae [6]. It has been found to be medicinally valued seed and have shown various pharmacological activities like antioxidant, antinociceptive, cytotoxic, antiviral [7], anti-inflammatory [8], antifungal [9-13], molluscicidal [14-16], antihelminthic (in sheep) [17, 18], plant nematicidal [17, 19], antipyretic [20], antiaggregatory [21] and antimicrobial activity [22-24], hypolipidemic [25], antihypertensive, antispasmodic, broncho-dilating actions, antilithiasis, diuretic, abortifacient, antitussive [26] and antifilarial [27]. The objective of present study was to extract essential oil from the seeds of T.

ammi via four extraction methods (HD, SE, US and SC-CO2) in order to compare

yield, optimize SC-CO2 extraction process via FFD and to determine the composition of essential oils via HPTLC, GC-MS and FTIR along with its antioxidant activity. 2. Materials and methods 2.1. Plant Materials The plant material (seeds of T. ammi) was collected from local market of Aligarh. Dried T. ammi seeds were grounded in a mechanical grinder for a short but

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sufficient period of time (30 s) to get a uniform particle size distribution. The grounded powder was sieved.

2.2. Solvents and reagents

All the ingredients taken were of Pharmacopeial quality and quantity. Standards were obtained from Sami Labs Ltd., Bangalore, (India) as a gift samples. Potassium hydroxide (KOH), Ethanol, ether, Hydrochloric acid (HCL), Ethanolic hydroxylamine hydrochloride solution (90%), Ethanolic Potassium hydroxide solution

(60%), 10% Folin-Ciocalteu, Na2CO3 (Sodium carbonate), Standard (Gallic acid), methanol, DPPH (Diphenyl-1-picrylhydrazyl), Standard (Ascorbic acid), Phenazine methosulfate (PMS), Nitroblue tetrazolium (NBT) and Nicotinamide adenine dinucleotide (NADH) used were of analytical grade (AR) and were procured from Merck Ltd. India. Carbon dioxide was obtained from Sigma gases, New Delhi, India.

2.3. Design of experiment

Full factorial design (FFD)

A factorial design is type of designed experiment that lets us study the effects that several factors can have on a response. When conducting an experiment, varying the levels of all factors at the same time instead of one at a time lets us study the interactions between the factors. The most fundamental structures experimental design is FFD. In FFD, responses are measured at all combinations of experimental factor levels [28]. Each experimental condition is called a “run” and each run represents variation of one variable. Each response will be measured for an observation. The entire set of runs is the “design”. Since, we consider three parameters with three levels, the total run with full factorial design is 33 (27) number of trials. The variation levels for parameters are shown in Table 1.

Table 1. Parameters and levels used in experimental design

FACTORS Pressure (bar) Temperature CO2 Flow rate LEVEL (oC) (g/min) A B C 1. 150 25 5 2. 175 30 10 3. 300 40 15

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2.4. Extraction methods 2.4.1. Hydro-distillation Method The grounded powder (150 g) and distilled water (500 mL) was mixed in 1L round bottomed flask and heated for 5.0 h to yield essential oil [29]. The extract was centrifuged at 10,000 rpm for 10 min in order to separate small water droplets present in the essential oil. The oil was kept at 4 °C until further quantification and percent yield was calculated. 2.4.2. Solvent Extraction The coarse powder (50 g) was extracted with hexane (100 mL) in 1:2 w/v, ratio and was kept in conical flask for 24 h. After that extract was filtered and concentrated under reduced pressure in rotary vacuum evaporator at 40 °C. The oil so obtained was stored at 4 °C prior to analysis. 2.4.3. Ultrasonic Assisted Extraction The coarse powder (50 g) of plant material was taken in a 250 mL conical flask along with 100 mL hexane (1:2, w/v). The flask was covered and then placed in an ultrasound water bath apparatus for 30 min (frequency 33 kHz). The temperature of the water bath was held constant at 25 °C. The extract was filtered and concentrated under reduced pressure in rotary vacuum evaporator. The obtained oil was stored at 4 °C prior to analysis and its percentage yield was calculated.

2.4.4. Supercritical CO2 Extraction A SFE-1000M1- 2- C50 system (Pittsburgh, PA) was used for extractions. The extraction vessel was of 200 mL stainless steel. Grounded plant material (50 g) was

loaded in the extractor and the CO2 was allowed to pass through the seed matrix at desired pressure (150-300 bar), temperature (25-40 °C) and optimised flow rate (5-15 g min-1) [30]. Total time of extraction was 30 min. After that oil was collected from the collecting vessel [31, 32]. The extracted oil was stored at 4 oC prior to analysis and its percentage yield was calculated. 2.5. Analytical methods 2.5.1. Scanning Electron Microscopy (SEM) Analysis The surface morphology of oily and non-oily biomass of T. ammi seeds before

and after SC-CO2 extraction was examined by SEM using Model JSM-6510LV,

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JEOL, (Japan). The SEM was operated at 10 kV, working distance of 11 and 12 mm, at magnification of 1000x and 2000x. 2.5.2. High Performance Thin Layer Chromatography (HPTLC) The samples were spotted in the form of band (3.0 mm) with a Camag microlitre syringe on TLC aluminium plate precoated with silica gel 60F-254 (20×10 cm with 0.2 mm thickness, E. Merck, Germany) using a Camag Linomat V sample applicator. A constant application rate of 120 nL sec-1 was employed and space between two bands was 8.0 mm. The slit dimension was kept 3.0×0.20 mm and scanning speed of 20 mm sec-1 was employed. The development was carried out in linear ascending manner in twin trough glass chamber (20 × 10 cm) saturated with mobile phase composed of hexane: ethyl acetate: formic acid in ratio (7:2:1, v/v/v). The optimized chamber saturation time for mobile phase was 20 min at room temperature and the chromatogram was developed up to the length of 85 mm. Subsequent to the development TLC plate was air dried. The HPTLC plates were studied at 254 nm and 366 nm as well as in visible range (580 nm) after spraying with anisaldehyde sulfuric acid reagent. A good separation of constituents was observed at 580 nm. 2.5.3. Gas chromatography-mass spectrometry (GC-MS) The essential oil obtained by different extraction techniques were analyzed using GC (Agilent Technologies, USA) interfaced with a MS ion-trap detector. The separation was obtained using 5% phenyl polymethyl siloxane capillary column with (30 m × 0.25 mm i.d., thickness 0.25 µm). Helium was used as the carrier gas (1 ml/min). Carrier gas temperature was kept 65 oC. Injector was maintained at 65 oC. The column temperature was raised at a rate of 10 oC/min from 160 oC to 302 oC. Splitless sample injection volume 2 µl. The ionization energy for the mass spectrometer was 70 eV. The essential oil obtained by different extraction techniques were diluted by adding 1998 µl of hexane to 2 µL oil (Hydro-distilled oil) and 1990µL hexane to 10 µL oil (extracted by other techniques) under optimized operating condition. The compounds were detected and identified by comparing with the standards available in National Institute of Standard and Technology (NIST) library [33].

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2.5.4. Fourier transform infrared spectroscopy (FTIR) analysis Fourier transform infrared (FTIR) spectral analysis was performed using Shimadzu BioRad FTIR (Kyoto, Japan). The samples (2 µL) were dispersed and triturated with dry KBr and grounded well in mortar and pestle to make (KBr) disk at a pressure of 1,000 psig. Then disk was placed in the FTIR sample holder, where spectra in absorbance mode was obtained in the spectral region 4000 to 400 cm-1 using the resolution 4 cm-1. 3. Antioxidant activity The antioxidant activity of essential oil sample extracted from dried seeds of T. ammi was determined by two methods; Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging [34] and Superoxide anionic scavenging [35]. Procedures for both the methods have already been given in Chapter 3.

4. Statistical analysis

The data obtained from SC-CO2 extraction were subjected to analysis of variance (ANOVA) to determine significant difference among all extract yields. P- value less than 0.05 were considered significant. All statistical analysis were performed using MINITAB 14 statistical software package. 5. Results and Discussion 5.1. Percentage yield (% v/w) Different extraction techniques were carried out in order to obtain maximum yield. Oil obtained by SC-CO2 (2.64%) gave maximum yield, whereas 1.20%, 1.82% and 2.30% were obtained from HD, SE and US methods, respectively. 5.2. Physicochemical Characterization of Essential Oil Essential oil extracted from T. ammi was in liquid form, yellowish-brown in color with characteristic odor and taste of thymol. Other physicochemical characterizations are given in the Table 2. Procedures to carry out them are given in chapter 3. Anthocyanin content was found to be maximum in the essential oil extracted by SC-CO2 extraction as it retains all the phytoconstituents with purity (Fig. 1). Percent of phenolic content was found to be more in HD and SC-

CO2 extracted essential oils as shown in Fig. 2, calibration curve is given in chapter 3.

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Table 2. Physicochemical characterization of essential oil of T. ammi

T. ammi HD* SE* US* SC-CO2* Reported (seeds) oil value specific gravity 0.850 0.854 0.848 0.849 0.910-0.930 (25°C) refractive index (25°C) 1.489 1.499 1.494 1.481 1.498-1.505 Optical rotation 7.7° 8.0° 8.0° 7.5° Up to 5° (25°C) Solubility in Clearly Clearly Clearly Clearly Soluble in 4 alcohol (% soluble in soluble in soluble in soluble in mL of 70% v/v ethanol) 5.0 mL 5.2 mL 5.2 mL 4.2 mL ethanol

Acid value (Mean±SD*) 3.5±0.1 3.7±0.3 3.9±0.1 3.1±0.1 -

Ester value (Mean±SD*) 4.7±0.2 5.0±0.1 4.8±0.2 4.3±0.2 - Aldehyde content 2.2 1.8 1.8 2.3 - (% w/v) Phenolics in %w/w - (Mean ± 7.7±0.1 6.5±0.2 6.4±0.08 7.4±0.1 SD*) Anthocyanin content %w/w 8.59±0.09 8.0±0.4 7.9±0.1 8.9±0.2 - (Mean ± SD*)

HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon- dioxide extraction, SD* Standard deviation

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9 8.8 8.6 8.4 8.2 8 T. ammi 7.8

Anthocyanin % w/w Anthocyanin 7.6 7.4 HD SE US SC-CO2 Oil sample

Fig. 1. Comparative study of anthocyanin contents in essential oil of T. ammi extracted by different extraction techniques

8 7 6 5 4 3 T. ammi 2

Phenolic contentPhenolic % w/w 1 0 HD SE US SC-CO2 Oil sample

Fig. 2. Comparative study of phenolic contents in essential oil of T. ammi extracted by different extraction techniques

5.3. SEM Analysis

oily biomass appears smashed with rugged features and rough edges after SC-CO2 extraction. *Thus images shows that most of the oily fraction has been extracted

which are in conformity with high yield with SC-CO2. CO2 being a gas was not retained by the matrix and thus SEM offers more depth profile than conventional light microscope to understand sample morphology.

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Fig. 3. SEM images of T. ammi seeds biomass: (A) before SC-CO2, (B) after SC-CO2 at 1000x magnification and (C) before SC-CO2, (D) after SC-CO2 at 2000x magnification

5.4. Development of solvent system for separation of oil sample by TLC TLC was done to separate the constituents of essential oil obtained by various techniques. Number of solvent systems in different proportions was tried for the separation of phytoconstituents present in the essential oils. The solvent system composed of hexane: ethyl acetate: formic acid (7 : 2 : 1, v/v/v) resulted in maximum separation of constituents and compactness of bands for all the oil samples. 5.5. Detection of spots of different samples The HPTLC plates were studied at 254 nm and 366 nm as well as in visible range (580 nm) after spraying with anisaldehyde sulphuric acid reagent which has been shown in Fig. 4. A good separation of constituents was observed at 580 nm.

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Fig. 4. HPTLC fingerprint of different oils of T. ammi extracted using different extraction techniques [track 1-2: Hydro-distilled oil, 3-4: Solvent extracted oil, 5-6:

Ultra sonication oil, 7-8: SC-CO2 oil] visualized at A: 254 nm, B: 366 nm, C: Day light after derivatization using anisaldehyde sulphuric acid reagent

The comparative results of different oils were showing a dominant constituent at Rf value 0.58 at 254 nm in oil extracted by different extraction techniques at different area percentage. Substance N at Rf 0.66 was showing a higher area percentage in the oil extracted by SC-CO2 extraction technique at 366 nm as compared to other extraction techniques. Substance M was present in maximum

amount in all the oil samples at 254 nm and in only solvent extracted and SC-CO2

extracted oil at 366 nm. Substance A, E, G, I were present in SC-CO2 extracted oil only at 254 nm. 3D Chromatogram and HPTLC fingerprinting of the oils extracted from different methods scanned at 254 and 366 nm are shown in Figs. 5a, 5b and Figs. 6a, 6b, respectively. The comparative results of different oils are given in Table 3.

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Fig. 5a. 3D Chromatogram of 8 tracks of T. ammi oil obtained by different extraction techniques at 254 nm

Fig. 5b. 3D Chromatogram of 8 tracks of T. ammi oil obtained by different extraction techniques at 366 nm

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Fig. 6a. HPTLC fingerprint of different oils of T. ammi extracted using different extraction techniques A: Hydro-distilled oil, B: Solvent extracted oil, C: Ultra sonication oil, D: SC-CO2 oil visualized at 254 nm with assigned substance

Fig. 6b. HPTLC fingerprint of different oils of T. ammi extracted using different extraction techniques A: Hydro-distilled oil, B: Solvent extracted oil, C: Ultra sonication oil, D: SC-CO2 oil visualized at 366 nm with assigned substance

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Table 3. Substance A-Q with their Rf and area percent of T. ammi essential oil obtained by different extraction techniques Area percentage (%) 254nm Area percentage (%) 366nm Substance (Rf) HD* SE* US* SC-CO2* HD* SE* US* SC-CO2* A (0.01) - 6.57 3.55 0.44 - - - - B (0.01) - - - - - 14.93 11.36 - C (0.09) 1.17 - - 0.66 - - - 0.78 D (0.15) ------3.01

E (0.2) - - - 4.52 - - - - F (0.24) - 1.74 - - - 20.90 18.16 - G (0.3) - - - 4.86 - - - 3.93 H (0.35) - 1.88 2.00 - - 18.96 21.78 4.45 I (0.37) - - - 1.30 - 8.11 - - J (0.41) - - 1.35 - - 3.25 -

K (0.44) - 0.98 - 3.20 28.37 19.60 30.43 10.82 L (0.46) 3.50 - 5.09 - - 11.14 15.02 - M (0.58) 79.34 73.80 73.62 53.11 71.63 - - 8.03 N (0.66) 8.49 5.19 - - - 6.36 - 68.99 O (0.71) - 3.74 - 29.99 - - - - P (0.76) 5.31 6.10 14.39 - - - - - Q (0.83) 2.20 - - 1.91 - - -

Rf* Retention factor, HD* Hydro-distillation, SE*Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon-dioxide extraction

The HPTLC plate scanned at 580 nm (Figs. 5c, 6c) showing maximum area

percent at Rf (0.59) in oil extracted by hydro distillation process. Substance K and J were present with maximum area percent in all the extracted techniques, as given in Table 4.

Fig. 5c. 3D Chromatogram of 8 tracks of T. ammi oil obtained by different extraction techniques at 580 nm

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Fig. 6c. HPTLC fingerprint of different oils of T. ammi extracted using different extraction techniques A: Hydro-distilled oil, B: Solvent extracted oil, C: Ultra sonication oil, D: SC-CO2 oil visualized at 580 nm with assigned substance

Table 4. Substance A-P with their Rf and area percent of T. ammi essential oil obtained by different extraction techniques

Area Percentage (%) 580 nm Substance (Rf) HD* SE* US* SC-CO2* A(0.02) - 0.59 2.21 - B (0.06) 1.20 0.28 - 4.12 C (0.11) - 0.41 - 1.66 D (0.16) 0.52 0.66 0.92 3.33 E (0.18) 0.82 - - - F (0.25) - 1.43 1.64 3.99 G (0.31) - 9.29 - 7.98 H (0.33) - - 8.03 - I (0.39) 18.80 6.42 4.88 - J (0.44) 9.25 12.36 12.59 21.41 K (0.59) 61.94 27.17 30.34 28.94 L (0.72) - 34.46 - 8.68 M (0.76) - - 30.23 12.75 N (0.78) 3.76 6.92 - - O (0.82) 4.52 - 9.14 7.02 P (0.92) - - - 0.13

Rf* Retention factor, HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon-dioxide extraction

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5.6. GC-MS analysis Comparative GC-MS chromatograms of essential oils of T. ammi extracted by traditional methods (HD, SE, US) and innovative SC-CO2 technique are shown Fig. 7. Chromatographic analysis (GC/MS) of the essential oil allowed the separation and identification of 49 compounds using their retention time and mass from library (Nist and Wiley) (Table 5).

Fig. 7. Comparative GC-MS chromatograms of different oils of T. ammi extracted using different extraction techniques A: Hydro-distilled oil, B: SC-CO2 oil, C: Solvent extracted oil, D: Ultra sonication oil

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Table 5. Results of GC-MS analysis of T. ammi oil extracted by different techniques Area percent (%) 580 nm S. SC- Component name RT* HD* SE* US* no CO2* Monoterpene hydrocarbon 1. 3-Thujene 5.42 1.20 0.51 - - 2. (-)-.β-Pinene 6.76 - - 2.31 0.62 3. l-Phellandrene 7.77 - - - 0.41 4. α-Terpinene 8.28 0.39 0.16 - - 5. p-Cymene 8.54 - 21.62 4.83 11.06 6. γ-Terpinene 10.07 23.62 20.90 5.07 11.56 7. α.-terpinolene 11.46 0.15 - - - 8. Cis-sabinene hydrate 11.95 - - - 0.21 Oxygenated monoterpene 9. Cymol 8.67 32.78 10. 4-Thujanol 11.95 - - 0.15 0.21 11. Terpineol 16.984 - 11.01 - 12. Thymol 23.58 35.63 49.33 69.94 66.25 13. Carvacrol 23.98 0.29 - - 0.48 Sesquiterpene hydrocarbons 14. Trans-Caryophyllene 30.12 - - - 0.09 15. β-Selinene 33.17 0.02 0.07 - 0.11 16. α.-selinene 33.55 0.01 - - 0.10 17. Eudesma-3,11-Diene 33.54 - 0.06 - - 18. Tumerone 39.76 - - 2.25 - 19. Curlone.α. 40.68 - - 1.18 - Oxygenated sesquiterpenes derivatives 20. γ.-Eudesmol 38.54 - - - 0.29 21. Eudesm-4(14)-en-11-ol 39.11 - - - 1.28 22. Dill-Apiol 38.44 0.02 - 0.32 - Aromatic compound Bicyclo[3.1.1]heptane, 6,6-dimethy 23. 6.78 3.54 - - - l-2-methylene-, (1s)- Mentha-1,4,8-triene 1,5,8-p- 24. 14.12 0.04 - - - menthatriene 3-Cyclohexen-1-ol, 4-methyl-1-(1- 25. 16.17 0.50 - 0.15 0.25 m ethylethyl) Non isoprenoid 26. Linalyl propionate 16.99 0.26 - - 0.26 27. Heneicosane 33.72 0.01 - - - 28. Pentadecane 33.90 - - 0.13 0.06 29. Phenol, 2,4-Bis(1,1-Dimethylethyl 34.58 0.01 - - - 30. Dodecanoic acid, methyl ester 34.95 - - - 0.09 31. Dodecane 35.45 - - - 0.05 1,6,10-Dodecatrien-3-ol, 3,7,11-tr 32. 36.33 - - - 0.07 imethyl-, [S-(Z)]- 33. Hexadecane 37.52 0.01 0.03 0.12 0.09 34. n-Heptadecane 40.57 - - 0.08 0.15

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35. 3 n -butyl Phthalide 41.56 - - - 0.16 36. Octadecane 41.92 0.01 0.03 0.27 - Pentadecanoic acid, 14-methyl-, 37. 45.61 - - 0.85 - methyl ester 38. Heneicosane 46.91 - - 0.42 0.10 39. Docosane 47.59 - 0.11 - 0.07 9,12-Octadecadienoic acid (Z,Z)-, 40. 48.55 - 0.39 4.27 0.53 methyl ester 41. Octadecanoic acid, methyl ester 49.05 - - 0.64 - 42. 9-Octadecenoic acid 49.304 - 0.83 - - 43. Nonadecane 50.14 0.03 0.05 0.06 0.17 44. Hexatriacontane 50.12 - - 0.78 0.14 45. Eicosane 50.97 - 0.08 0.44 0.22 46. Pentacosane 51.597 - 0.09 - 0.06 47. Tricosane 52.00 - - 0.07 0.28 48. Butyl pthalate 55.05 0.18 - 0.91 0.37 49. Squalene 58.41 - 0.08 0.17 - RT* Retention time, HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon-dioxide extraction

Thymol (69.25%) and γ terpinene (23.62%) were main constituents of all the samples. Other components present were eudesmol (1.28%), 3-Thujene (1.20%). Fatty acids were also present in significant quantity.

5.7. FTIR analysis

The interpretation of infrared spectra of T. ammi essential oil was done by correlating of absorption bands in the spectrum with the known absorption frequencies bands. Comparative spectra of essential oil extracted by different techniques are shown in Fig. 8. FTIR results are presented in Table 6.

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Fig. 8. Comparative spectra of different oils of T. ammi extracted using different extraction techniques A: Solvent extracted oil, B: Ultra sonication oil, C: Hydro- distilled oil, D: SC-CO2 oil

Table 6. Results of FTIR analysis of T. ammi oil extracted by different techniques SC- S.no Peak Functional group Intensity HD* SE* US* CO2* 1. 810.1 =C-H(alkene) Sretch, strong + - - + 2. 945.12 =C-H(alkene) Sretch, strong + - - + 3. 1155.36 C-O(alcohol) Sretch, strong + + - + 4. 1224.8 C-O( aldehyde) Sretch, medium + + - + 5. 1284.59 C-O( aldehyde) Sretch, medium - + - + 6. 1458.18 C=C(aromatic) Sretch, medium + + - + 7. 1514.12 N-O(nitro) Sretch, strong + - - - 8. 1616.35 N-O(nitro) Strtch, strong + - - + 9. 2729.27 =C-H(aldehyde) Sretch, medium + + - + 10. 2926.01 methylene Medium - strong - + - + 11. 3007.02 C-H(aromatic) Sretch, medium - - - + 12. 3429.43 N-H(amine) Sretch, medium + - - +

HD* Hydro-distillation, SE* Solvent extraction, US* Ultrasonication, SC-CO2* Supercritical carbon- dioxide extraction

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IR spectrum for T. ammi oil exhibits a medium intensity band at 3429 cm-1 assigned to the stretching vibration of amine group. Peak at 2962-2729 cm-1 was due -1 to stretching of CH3 group, 1458 cm presents C=C aromatic ring stretching. Peak at 810 cm-1 was due to out-of-plane alkene C-H stretching. C-H stretching medium intensity peak of aldehydic group present in HD, SE and SC-CO2 oil samples. Band appearing at 1155.36 cm-1 representing strong stretching vibration of alcoholic group present in all except ultrasonicated oil sample.

5.8. Antioxidant activity

The T. ammi oil sample showed a significant concentration-dependent antioxidant activity by inhibiting DPPH free radical with an IC50 values of 36.41 μg -1 -1 mL , whereas, IC50 value of ascorbic acid was found to be 28.09 μg mL used as standard (Table 7 and Fig. 9). It was found that the oil possesses hydrogen donating capabilities similar to ascorbic acid and acted as an antioxidant. The scavenging effect increased with increasing concentration of the extract and ascorbic acid (5.0-100 μg mL-1).

Table 7. Summary of % inhibition and IC50 values of T. ammi oil sample by DPPH method (n=3)

Percentage inhibition ± SD* T. ammi Conc. (µg mL-1) Ascorbic Acid 100 92.5 ±1.1 64.1±0.8 50 64.3 ±0.9 56.7±0.8 25 45.7 ±1.0 41.5±0.8 12.5 29.5 ±0.54 34.1±0.8 6.25 20.6 ±0.8 28.9±0.8 3.125 12.1 ± 0.8 20.9±4.7 1.5625 8.45 ±1.0 12.9±0.8 0.781 5.85± 0.8 1.5±0.8

IC50 28.09 36.41 SD* Standard deviation

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log-dose vs response

100 80 60 40 std Ascorbic acid 20 T. ammi L.

Percentage inhibition Percentage 0 -1 0 1 2 3 log concentration

Fig. 9. Comparative dose response curve between percent inhibitions against log concentration by DPPH method

The superoxide scavenging activity of the drug was increased markedly with the increase in concentrations. Comparison of the antioxidant activity of the extracts and ascorbic acid is shown in Table 8 and Fig. 10. The IC50 values of the essential -1 oils of T. ammi were found to be 20.55 µg mL , whereas the IC50 value of ascorbic acid was 20.14µg mL-1.

Table 8. Summary of % inhibition and IC50 values of T. ammi oil sample by superoxide anion scavenging method (n=3)

Percentage inhibition ± SD* Conc. (µg/mL-1) Ascorbic Acid T. ammi 10 38.28±0.8 39.18±0.8 20 48.72±1.2 43.88±0.8 40 62.33±0.8 57.69±0.8 60 71.67±1.2 77.03±0.8 80 89.28±1.2 85.76±0.8 100 95.22±1.2 93.00±0.8

IC50 20.14 20.55 SD* Standard deviation

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log-dose vs response

100 80 60 40 std Ascorbic acid 20 T. ammi L. Percentage Inhibition Percentage 0 0 1 2 3 log concentration

Fig. 10. Comparative dose response curve between percent inhibitions against log concentration by superoxide scavenging method

5.9. Full factorial design (FFD)

An experiment was run to study the effect of three factors (Pressure, temperature and flow rate) with three levels on response (Yield). The results were analyzed and presented in Table 9. It can be seen that the selected factors showed significant effect either directly or through the interaction with other factors. The selected factors explained 85.42% fit model for the given data. The contour and surface plots have been shown in Figs. 11 & 12.

Table 9. Estimated Effects and Coefficients for Yield

Term Effect Coef SE Coef T P Constant 1.09433 0.05154 21.23 0.000 Pressure 1.07339 0.53670 0.05318 10.09 0.000 Temperature 0.42795 0.21397 0.05414 3.95 0.000 Flow rate 0.67875 0.33937 0.05446 6.23 0.000 Pressure*Temperature 0.15887 0.07943 0.05431 1.46 0.155 Pressure*Flow rate 0.04000 0.02000 0.05446 0.37 0.716 Temperature*Flow rate 0.21875 0.10938 0.05446 2.01 0.054 Pressure*Temperature*Flow -0.00250 -0.00125 0.05446 -0.02 0.982 rate

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Contour Plots of Yield

Temperature*Pressure Flow rate*Pressure 40 15.0 Yield < 0.3 0.3 - 0.6 12.5 35 0.6 - 0.9 0.9 - 1.2 10.0 1.2 - 1.5 30 1.5 - 1.8 7.5 > 1.8

Hold Values 25 5.0 Pressure 225 150 200 250 300 150 200 250 300 Temperature 32.5 Flow rate*Temperature 15.0 Flow rate 10

12.5

10.0

7.5

5.0 25 30 35 40

Fig. 11. Contour plots of yield vs pressure, temperature and flow rate

Surface Plots of Yield

Hold Values Pressure 225 Temperature 32.5 Flow rate 10 2.0 2.0

1.5 1.5 Yield Yield 1.0 101.0 0.5 0.5 320 320 42 36 240 15 240 30 1660 Pressure 100 1660 Pressure Tempeeraturet Flow ratt e 5

1.5 Yielded 1.0

0.5 42 15 36 100 30 Temmperature Flow rat e 5

Fig. 12. Surface plots of yield vs pressure, temperature and flow rate

All the three factors (Pressure, temperature and flow rate) have shown to have direct effect on yield as p<0.05. Hence pressure, temperature and flow rate are significant factors for influencing yield. While combination of temperature- flow rate effects yield to a certain extent as p<0.05, on the other hand pressure-flow rate, pressure-temperature and pressure-temperatute-flow rate combinations have no significance on oil yield as p>0.05.

The optimum operating conditions for SC-CO2 extraction of essential oil from seeds of T. ammi for high yield were pressure (225 bar), temperature (32.5 oC) and

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flow rate of CO2 (10 g/min). A confirmation experiment was run using the optimum conditions and the highest yield obtained was 2.64 %.

6. Conclusion

In present work, essential oil from seeds of T. ammi was extracted via HD, SE,

UAE and SC-CO2 extraction. Out of all these methods SC-CO2 gave the highest percentage yield (2.64%) as compared to other extraction techniques. FFD was used to determine the optimum conditions for SC-CO2 extraction of essential oil were o pressure (225 bar), temperature (32.5 C) and flow rate of CO2 (10 g/min). The analysis shows that pressure, temperature and flow rate effect yield of oil and combination of temperature-flow rate also shows significant influence on yield while pressure-flow rate, pressure-temperature and pressure-temperature and flow rate combinations have no effect on oil yield. Furthermore evidences from HPTLC, GC-

MS as well as FTIR analysis showed that SC-CO2 technique is best technique for

extraction of essential oils. SC-CO2 is a green, environmentally friendly and

sustainable extraction technique. Essential oil obtained from seeds of T. ammi has shown to possess significant antioxidant activity which was investigated by DPPH and Superoxide anion scavenging methods and was found to be 36.41 and 20.55 µg mL-1, respectively.

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