Terpenoids

Dr. Amol Kharat Objectives

• To study the Terpenoids in the form of Meaning, Different types Properties, occurrences, uses Isolation Method General Biogenetic Pathway Pharmacognostic account of different drug con taining important constituents Terpenoids. INTRODUCTION

Terpenoids are the secondary metabolites synthesized by plants, marine organisms and fungi by head to tail joining of isoprene units . They are also found to occur in rocks, fossils and animal kingdom.

Isoprene The terpenoids , sometimes referred to as isoprenoids , are a large and diverse class of naturally-occurring organic chemicals similar to terpenes, derived from five-carbon isoprene units assembled and modified in thousands of ways. Most are multicyclic structures that differ from one another not only in functional groups but also in their basic carbon skeletons . These lipids can be found in all classes of living things, and are the largest group of natural products. CALASSFICATION

TYPE OF NUMBER OF ISOPRENE TERPENOIDS CARBON ATOMS UNITS hemiterpene C5 one monoterpenoid C10 two sesquiterpenoid C15 three diterpenoid C20 four sesterterpenoid C25 five triterpenoid C30 six tetraterpenoid C40 eight

NOTE hemi = half di = two : sesqui = one and a half tri = three tetra = four Mnonoterpenoids

Monoterpenes are a class of terpenes that consist of two isoprene units and have the molecular formula C 10 H16 . Monoterpenes may be linear (acyclic) or contain rings. Biochemical modifications such as oxidation or rearrange- ment produce the related monoterpenoids. Mnonoterpenoids

Acyclic monoterpenoid: Bicyclic monoterpenoid

CHO

CH 2 OH

n e ro l g era n ia l ¦Â-m y r c e n e ¦Á ---ppp i nene Monocyclic monoterpenodi

OH

O OH O d-borneol l- menthone cin eole Acyclic monoterpenoid Acyclic monoterpenoid

Biosynthetically, isopentenyl pyrophosphate( 异戊烯 焦磷酸) and dimethylallyl pyrophosphate ( 二甲基丙烯焦 磷酸酯) are combined to form geranyl pyrophosphate (牻牛儿醇焦磷酸酯).

Geranyl pyrophosphate Acyclic monoterpenoid

Elimination of the pyrophosphate group leads to the formation of acyclic monoterpenes such as ocimene and the myrcenes .

Myrcene ( 月桂烯) Acyclic monoterpenoid

Hydrolysis of the phosphate groups leads to the prototypical acyclic monoterpenoid geraniol.

geraniol （（香叶醇香叶醇） Acyclic monoterpenoid

Additional rearrangements and oxidations provide compounds such as citral ( 柠檬醛), citronellal( 香茅醛), citronellol( 香茅醇), and many others.

citral （柠檬醛） Acyclic monoterpenoid

Many monoterpenes found in marine organisms are halogenated( 卤代的), such as halomon .

Halomon is a polyhalogenated monoterpene first isolated from the marine red algae Portieria hornemannii . It has attracted research interest because of its promising profile of selective cytotoxicity ( 细胞毒) that Halomon suggests its potential use as an antitumor agent. Monocyclic monoterpenoid

In addition to linear attachments, the isoprene units can make connections to form rings. The most common ring size in monoterpenes is a six-membered ring. A classic example is the cyclization of geranyl pyrophosphate to form (柠檬烯）. Monocyclic monoterpenoid

The terpinenes, phellandrenes( 水芹烯), and terpinolene (异松油烯) are formed similarly. Hydroxylation of any of these compounds followed by dehydration can lead to the aromatic p-cymene( 百里香素). Important terpenoids derived from monocyclic terpenes are menthol ( 薄荷醇), ( 麝香草酚,百里酚), carvacrol( 香芹酚,甲基异 丙基苯酚) and many others.

β-phellandrene p-cymene menthol Menthol • waxy / crystalline substance • clear or white • solid at room temperature and melts slightly above.

The main form of menthol occurring in nature is (-) - menthol, which is assigned the (1R, 2S, 5R) configuration.

(-) dextro- (+) laeotro- a colourless liquid at room temperatures Limonene with an extremely strong smell of oranges.

a chiral molecule (asymmetric carbon atoms) is common with such forms biological sources produce one enantiomer (对映体)

Limonene is common in cosmetic products. As the main odor constituent of citrus, D - limonene is used in food manufacturing and some medicines, e.g., bitter alkaloids, as a flavoring; it is also used as botanical insecticide Be increasingly used as a solvent for cleaning Limonene purposes, such as the removal of oil from machine parts, as it is produced from a renewable source (citrus oil, as a byproduct of orange juice manu- facture). Combustible, be considered as a biofuel

Limonene is common in cosmetic products. As the main odor constituent of citrus, D - limonene is used in food manufacturing and some medicines, e.g., bitter alkaloids, as a flavoring; it is also used a s botanical insecticide Limonene Chemistry relatively stable, can be distilled without decom- position oxidises easily in moist air to carveol and carvone Dehydration with sulfur gives to p-cymene, hydro gen sulfide, as well as some sulfides.

Carvone ( 香芹酮) Limonene Safety limonene and its oxidation products are skin and respiratory irritants. Although once thought to cause renal ( 肾脏的) cancer in rats, limonene now is considered by some researchers to be a significant chemopreventive agent with potential value as a dietary anticanc er tool in humans. Bicyclic monoterpenoid Bicyclic monoterpenoid

Geranyl pyrophosphate can also undergo two sequential cyclization reactions to form bicyclic monoterpenes, such as pinene( 蒎烯) which is the primary constituent of pine resin. Bicyclic monoterpenoid

Other bicyclic monoterpenes include carene ( 蒈烯) and camphene ( 樟脑萜). Camphor( 樟脑), borneol( 龙脑) and eucalyptol( 桉叶素) a re examples of bicyclic monoterpenoids containing ketone, alcohol, and ether functional groups, respectively.

carene ( 蒈烯) camphor( 樟脑) borneol( 龙脑) Bicyclic monoterpenoid

Role in Climate Monoterpenes are emitted by forests and form aerosols (气溶胶) that can serve as cloud condensation nuclei (CCN). Such aerosols can increase the brightness of clouds and cool the climate. Pinene There are two structural isomers found in nature: α-pinene and β-pinene. As the name suggests, both forms are important constituents of pine resin; they are also found in the resins of many other conifers, and more widely in other plants. Both are also used by many insects in their chemical communication system. Pinene Usage Selective oxidation of pinene with some catalysts in chemical industry give many components for pharmacy, artificial odorants and so on. The prima ry oxidation product is verbenone( 马鞭草烯酮) . It can form by simple air oxidation but a synthetic method employs lead tetraacetate . Eucalyptol a natural colorless liquid has a variety of synonyms the dominant portion of "Eucalyptus oil“ comprises up to 90 percent of Eucalyptus oil

Although it can be used internally as a flavoring and medi- cine ingredient at very low doses, typical of many essential oils (volatile oils), eucalyptol is toxic if ingested at high doses. Uses of eucalyptol flavorings, fragrances, and cosmetics. an ingredient of mouthwash and cough suppressant. Eucalyptol suppository( 栓栓栓剂), reducing inflammation and pain for the treatment of some respiratory ailments kill leukaemia cells in vitro

In a 1994 report released by five top cigarette companies, eucalyptol was listed as one of the 599 additives ( 添加剂) to c igarettes. It is added to improve the flavor. Sesquiterpenoids Sesquiterpenoids

Sesquiterpenes are a class of terpenes that consist of three isoprene units and have the

molecular formula C 15 H24 . Like monoterpenes, sesquiterpenes may be acyclic or contain rings, including many unique combinations. Biochemical modifications such as oxida- tion or rearrangement produce the related sesquiterpenoids. Sesquiterpenoids

• Acyclic sesquiterpenoids • Monocyclic sesquiterpenoids

O

姜 黄 酮 姜 稀 ¦Á-farnesene ¦Â-farnesene

• Bicyclic sesquiterpenoids

H

OH H

¦Á - eudesmol guaiazulene cadinene Acyclic Sesquiterpenoids

When geranyl pyrophosphate reacts with isopentenyl pyro- phosphate, the result is the 15-carbon farnesyl pyrophosphate (焦磷酸法尼酯) , which is an intermediate in the biosynthesis of sesquiterpenes such as farnesene( 金合欢烯) . Oxidation can then provide sesquiterpenoids such as farnesol( 金合欢醇) . Sesquiterpenes are found naturally in plants as defensive agents.

farnesene

farnesyl pyrophosphate farnesol Monocyclic Sesquiterpenoids

With the increased chain length and additional double bond, the number of possible ways that cyclization can occur is also in creased, and there exists a wide variety of cyclic sesquiterpenes. In addition to common six-membered ring systems such as is fo und in zingiberene( 姜烯), a consitituent of the oil from ginger, c yclization of one end of the chain to the other end can lead to m acrocyclic( 大环的)rings such as humulene( 葎草烯).

zingiberene姜 稀 Bicyclic Sesquiterpenoids

In addition to common six-membered rings such as in the cadinenes ( 杜松烯), one classic bicyclic sesquiterpene is caryophyllene( 丁香烯), from the oil of cloves( 丁香), which has a nine-membered ring and cyclobutane ( 环丁烷) ring. A dditional unsaturation provides aromatic bicyclic sesquiter - pe noids such as guaiazulene （（愈愈创木烷）.

H

H

cadinene caryophyllene guaiazulene Tricyclic Sesquiterpenoids

With the addition of a third ring, the possible structures become increasingly varied. Examples include longifolene (长松叶烯), copaene( 胡椒烯)and the alcohol patchoulol.

isomers of longifolene copaene patchoulol Example of Sesquiterpenoids

• Zingiberene

• is a monocyclic sesquitepenoid which is the predominant constituent of the oil of ginger (Zingiber officinale ), from which it gets its name .

• Zingiberene Chemical name 5-(1,5-di zingiberene姜 稀 m--ethyl-4-hexenyl)-2-methyl-1,3-cyclo

hexadieneChemical formulaC 15 H24 Example of Sesquiterpenoids

Artemisinin (Qing hao su) used to treat multi-drug resistant strains of falciparum malaria( 恶性 疟) . isolated from the plant Artemisia annua . Not all plants of this species con- tain artemisinin. can be synthesized from artemisi- nic acid Example of Sesquiterpenoids

History Artemisinin Artemisia has been used by Chinese herbalists for more than a thousand years in the treatment of many illnesses, such as skin diseases and malaria. The earliest record dates back to 200 BC , in the "Fifty two Prescriptions" unearthed from the Mawangdui Han Dynasty Tombs. Its anti- malarial application was first described in Zhouhou Beji Fang ("The Handbook of Prescriptions for Emergencies"), edited in 《肘后备急方》 the middle of fourth century by Ge Hong. Example of Sesquiterpenoids

Artemisinin In the 1960s a research program was set u p by the Chinese army to find an adequate treatment for malaria. In 1972, discovered artemisinin in the leav es of Artemisia annua It was one of 200 candidates tested for tre ating malaria and was the only one that w as effective Example of Sesquiterpenoids

Artemisinin

it was found that it cleared malaria parasites from their bodies faster than any other drug in history. Artemisia annua is a common herb and has been found in many parts of the world, including along the Potomac River, in Washington, D.C. Example of Sesquiterpenoids

Artemisinin Cancer treatment is under early research and testing peroxide lactone group

peroxide comes into contact with high iron concentrations (common in cancerous cells), the molecule becomes unstable and releases reactive oxygen species.It has been shown to reduce angio- genesis ( 血管生成的) and the expression of vascular endothelial growth factor( 血管内皮生长因 子)in some tissue cultures. Example of Sesquiterpenoids

Bisabolol (没药醇) • a natural monocyclic sesquiterpene alcohol • a colorless viscous oil • almost insoluble in water • well soluble in ethanol

α-(-)-bisabolol Example of Sesquiterpenoids

Bisabolol has a weak sweet floral aroma and (没药醇) is used in various fragrances. Be used for hundreds of years in cosmetics because of its perceived skin healing properties. anti-irritant anti-inflammatory α-(-)-bisabolol anti-microbial Diterpen oids Diterpenoids

Diterpenes are composed for four isoprene units and have the molecular 焦磷酸牻 formula C 20 H32 . They derive from geranylgeranyl pyrophosphate ( 牛儿基牻牛儿酯). Examples of diterpenes are cafestol (咖啡醇[抗炎药]), kahweol (咖啡白脂), cembrene (西柏烯)and taxadiene (precursor of taxol).

geranylgeranyl pyrophosphate

cafestol cembrene Diterpenoids

Diterpenes also form the basis for biologically important compounds such as retinol (维生素A), retinal (维生素A醛), and phytol (叶绿醇). They are kn own to be antimicrobial and antiinflammatory. The herb Sideritis( 唇形科毒 马草属) contains diterpenes.

retinol retinal Structure ----Diterpenoids

Acyclic diterpenoids Monocyclic diterpenoids

CH OH CH 2OH 2

phyt ol vi t amin A

Bicyclic diterpenoids Tricyclic diterpenoids Tetracyclic diterpenoids

O H HO O

H COOH HO H CH2OH pi mar i c aci d

andrographolide kaurene Example of Diterpenoids

Phytol • is a a key acyclic diterpene alcohol that is a

precursor for vitamins E and K 1. • is an extremely common terpenoid, found in all plants esterified to Chlorophyll ( 叶绿素) to confer lipid solubility. • is found in petroleum sediments. Example of Diterpenoids

Cembrene

• a natural monocyclic diterpenoid • a colorless oil with a faint wax-like odor.

• Cembrene itself has little importance as chemical entity, being a trail pheromone for termites (白蚁);

• the chemical structure of cembrene is central to a very wide variety of other natural products found both in plants and in animals. Example of Diterpenoids

Taxol (Paclitaxel)

Overview • a mitotic inhibitor (有丝分裂抑制剂) used in cancer chemotherapy • 1967, Wall and Wani • Pacific yew tree, Taxus brevifolia • Bristol-Myers Squibb (BMS) • taxol ---- paclitaxel ---- trademark 'Taxol‘ Example of Diterpenoids

Taxol (Paclitaxel) Overview

Paclitaxel is now used to treat patients with • Lung cancer • Ovarian ( 卵巢的) cancer • breast cancer • head and neck cancer, Example of Diterpenoids

Overview

Taxol (Paclitaxel) Paclitaxel works by interfering with normal microtubule ( 微管) breakdown during cell division. Together with docetaxel ( 多西紫杉醇), it forms the drug category of the taxanes ( 紫杉 烷类 ). Example of Diterpenoids

Overview As well as offering substantial improvement Taxol (Paclitaxel) in patient care, paclitaxel has been a relatively controversial drug. There was originally concern because of the environmental impact of its original sourcing, no longer used, from the Pacific yew. In addition, the assignment of rights, and even the name itself, to Bristol-Myers Squibb were the subject of public debate and Congressional hearings. Example of Diterpenoids

Taxol (Paclitaxel) Undisturbed Pacific Yew bark contains paclitaxel and related chemicals

The bark is peeled and processed to provide paclitaxel Example of Diterpenoids

Taxol (Paclitaxel)

From 1967 to 1993, almost all paclitaxel produced was derived from bark from the Pacific yew, the harvesting of which kills the tree in the process. While there was considerable uncertainty about how large the wild population of Taxus brevifola was and what the eventual demand for taxol would be, it had been clear for many years that an alternative, sustainable source of supply would be needed. Example of Diterpenoids

Taxol (Paclitaxel)

Initial attempts used needles from the tree, or material from other related Taxus species, including cultivated ones. But these attempts were bedevilled by the relatively low and often highly variable yields obtained. It was not until the early 1990s, at a time of increased sensitivity to the ecology of the forests of the Pacific North West, that it was successfully extracted on a clinically useful scale from these sources. Example of Diterpenoids

Taxol (Paclitaxel)

From the late 1970s, chemists in the US and France had been interested in taxol. A number of US groups attempted a total synthesis of the molecule, starting from petrochemical -derived starting materials. This work was primarily motivated as a way of generating chemical knowledge, rather than with any expectation of developing a practical production technique. By contrast a French group of Pierre Potier quickly recognized the problem of yield. They populated by the related yew Taxus baccata , so that needles were available locally in large quantity. Example of Diterpenoids

Taxol (Paclitaxel) By 1981 they had shown that it was feasible to isolate relatively large quantities of the compound 10-deacetylbaccatin, a plausible first step for a semi-synthetic production route to taxol. By 1988 they co-published such a semi-synthe- tic route from needles of Taxus baccata . The view of the NCI, however, was that even this route was not practical.

10-deacetylbaccatin Example of Diterpenoids

Taxol (Paclitaxel)

By 1988, and particularly with Potier's publication, it was clear to Holton as well that a practical semi- synthetic production route would be important. By late 1989, Holton's group had developed a semi- synthetic route to paclitaxel with twice the yield of the Potier process. Florida State University, where Holton worked, signed a deal with Bristol-Myers Squibb to license this and future patents. Example of Diterpenoids

Taxol (Paclitaxel) In 1992, Holton patented an improved process with an 80% yield. BMS took the process in-house and started to manufacture paclitaxel in Ireland from 10- deacetylbaccatin isolated from the needles of the Euro pean yew. In early 1993, BMS were able to announce that they would cease reliance on Pacific yew bark by the end of 1995, effectively terminating the ecological contro- versy over its use. This announcement also made good their commitment to develop an alternative supply route, made to the NCI in their CRADA application of 1989. Example of Diterpenoids

Taxol (Paclitaxel) Currently, all paclitaxel production for BMS uses plant cell fermentation (PCF) technology developed by the biotechnology company Phyton Biotech, Inc and carried out at their plant in Germany. This starts from a specific taxus cell line propagated in aqueous medium in large fermentation tanks. Pacli- taxel is then extracted directly, purified by chromato- graphy and isolated by crystallization. Compared to the semi-synthesis, PCF eliminates the need for many hazardous chemicals and saves a considerable amount of energy. Example of Diterpenoids

Taxol (Paclitaxel) In 1993 it was discovered that taxol was coinciden tally produced in a newly described fungus living in the yew tree. It has since been found in a number of other endophytic fungi, opening the possibility of taxol production by culturing one of these fungal species. The initial motivation for synthetic approaches to paclitaxel included the opportunity to create closely related compounds. Indeed this approach led to the development of docetaxel. Example of Diterpenoids

Ginkgolides

Ginkgo is a genus of highly unusual non-flowering plants with one extant species, G. biloba, which is regarded as a living fossil.

Ginkgolides are biologically active terpenic lactones present in Ginkgo biloba . They are bicyclic diterpenoids, and there are six lactone-rings in their skeletons. Example of Diterpenoids

Ginkgolide B is an extract from the cultivated leaves of the Ginkgo Tree. Clinical studies have shown ginkgo to benefit short term memory, cardiovas- cular health, respiratory health and gastrointestinal health.

Ginkgolide B is an active platelet aggregating factor antagonist( 对抗物).

All ginkgo formulas must have ade- quate levels of ginkgolide B in order to successfully increase c erebral blood flow. Example of Diterpenoids

Tanshinones

Tanshinones are diterpene quinones isolated from the roots of Radix Salvia miltiorrhiza (Danshen), a traditional Chin ese medicine used in the treatment of car- diovascular diseases and recently reported to exhibit anti-tumour effects. The anti-tumour potential of Danshen was investi-gated by studying the cytoto- xicity of the major diterpenes, including tanshinone IIA, cryptotanshinone( 隐丹参 Tanshinone II A 酮), tanshinone I and dihydrotanshinone on a human HepG2 cell line in this study. Triterpenoids Triterpenoids

Triterpenes consist of six isoprene units and have the molecular formula 角 C30 H48 . The linear triterpene ( 鲨烯), the major constituent of s hark liver oil, is derived from t he reductive coupling of two molecules of fa rnesyl pyrophosphate ( 焦磷酸法尼酯). Squalene is then processed biosynthet ically to generate either lanosterol ( 羊毛甾醇)or cycloartenol ( 环阿乔醇), the structural precursors to all the steroids.

squalene

farnesyl pyrophosphate

lanosterol cycloartenol Structure ----Triterpenids

• Acyclic triterpenids squalene

• Bicyclic triterpenids

α-carotene

β-carotene Structure ----Triterpenids

• Tetracyclic triterpenids Dammarane( 达玛烷) Lanostane( 羊毛甾烷) Tirucallane( 甘遂醇)

H H H

H H

H H dammarane H lanostane tirucallane H H

Cycloartane( 环木菠萝烷) Cucurbitane( 葫芦烷)

H

H H H

cycloartane cucurbitane H Structure ----Triterpenids

• Pentacyclic triterpenoids

Oleanane (齐墩果烷) Ursane(乌苏烷) Lupane (羽扇豆烷)

H

H H

H H

H H H ursane lupane oleanane H H H Example of Triterpenoids

Squalene

Squalene is a natural organic compound originally obtained for commercial purposes primarily from shark liver oil, though there are botanic sources as well, including amaranth ( 苋菜) seed, rice bran ( 米 糠), wheat germ ( 小麦胚芽), and olives ( 橄榄). All higher organisms p roduce squalene, including humans. It is a hydrocarbon and a tri-te rpene. Squalane ( 角鲨烷,异三十烷) is a saturated form of squalene in w hich the double bonds have been eliminated by hydrogenation. Be cause it is less susceptible to oxidation, it is more commonly used in pe rsonal care products than squalene. Example of Triterpenoids

Squalene Squalene is a low density compound often stored in the bodies of cartilaginous fishes ( 软骨鱼, their skeletons made of c artilage ( 软骨)rather than bone) such as s harks, which lack a swim bladder 鳔(动) a nd must therefore reduce their body d ensity with fats and oils. Squalene, w hich is stored mainly in the shark's liver, i s lighter than water with a specific g ravity of 0.855. Environmental and other concerns over shark hunting have mo- tivated its extraction from vegetable sources instead. Example of Triterpenoids

Squalene

Recently it has become a trend for sharks to be hunted to process their livers for the purpose of making squalene health capsules. However, there is little clinical evidence to prove that, taken internally, squalene does anything to increase an individual's quality of life. Example of Triterpenoids

Squalene

Use as a moisturizer Squalene is used in cosmetics as a natural moisturizer. It p enetrates the skin quickly, does not leave a greasy feeling on t he skin and blends well with other oils and vitamins. Example of Triterpenoids

Squalene

Role in steroid synthesis Squalene is the biochemical precursor to the whole family of steroids. Oxidation (via squalene monooxygenase 角鲨烯单加氧 酶) of one of the terminal double bonds of squalene yields 2, 3- squalene oxide, which undergoes enzyme-catalyzed cyclization to afford lanosterol, which is then elaborated into cholesterol ( 胆 甾醇) and other steroids. Example of Triterpenoids

Ginsenosides

Ginsenosides are a class of triterpene saponins, found exclusively in the plant genus Panax (ginseng). Ginsenosides have been the target of research, as they are viewed as the active compounds behind the claims of ginseng's efficacy. Because ginsenosides appear to affect multiple pathways, their effects are complex and difficult to isolate. Ginsenosides are separated by column chromatography. Ginsenoside content can vary widely depending on species, location of growth, and growing time before harvest. Example of Triterpenoids

Ginsenoside Rb 1 • most abundant in Panax quinquefolius (American Ginseng). • affect the reproductive system in animal testicles. • recent research ---- affects rat embryo( 胚胎) development. • may increase testosterone ( 睾酮)production in male rats indirectly through the stimulation of the luteinizing hormone ( 促黄体生成激素). • inhibits chemoinvasion and angiogenesis. Example of Triterpenoids

Ginsenoside Rc

• is recognized for producing more sedative (镇静的) related results than other ginsenosides • In one particular study on breast cancer and different ginsenosides, it was found that only ginsenoside-Rc was capable of inhibiting the growth of these cancer cells. This suggests that there is a possibility that ginsenoside-Rc may have effects that prevent or limit the develop- ment of breast cancer. Example of Triterpenoids

Ginsenoside Rc

• A further study was also able to demonstrate a possible effect of ginsenoside -Rc on the motility of sperm ( 精子活动能力). Data from this experimentation showed a significant increase in motility when the sperm was in a ginsenoside-Rc solution. Example of Triterpenoids

Ginsenoside Rg1

• most abundant in Panax ginseng (Chinese/ Korean Ginseng). • Improves the capability of spatial learning for mice, plus estrogen-like (雌激素) activity (which could account for the boosting of "yin" theory) Essential Oils Essential Oils

• Terpenes and terpenoids are the primary constituents of the essential oils of many types of plants and flowers.

• Essential oils are used widely as natural flavor additives for food, as fragrances in perfumery, in aroma therapy, and in traditional and alternative medicines. Synthetic variations and derivatives of natural terpenes and ter- penoids also greatly expand the variety of aromas used in perfumery and flavors used in food additives. ISOLATION & SEPARATION TECHNIQUES

Essential oils containing mono- and sesquiterpenoids are obtaind by water and or steam distillation of the part such as flowers, lea- -ves of stems, where the essential oils occur in more concentrated form. Due to the heat lability of certain constituents of essential oils different distillation methods have to be used for different raw m- -erials which are briefly described below:

Distillation

Expression

Solvent extraction Distillation

Today, most common essential oils, such as lavender 熏衣草, p eppermint 薄荷油, and eucalyptus, are distilled. Raw plant material, c onsisting of the flowers, leaves, wood, bark, roots, seeds, or peel, is p ut into an alembic 蒸馏器(distillation apparatus) over water. As the wa ter is heated the steam passes through the plant material, vaporizing th e volatile compounds. The vapors flow through a coil where they co ndense back to liquid, which is then collected in the receiving vessel. Distillation

Most oils are distilled in a single process. One exception is Ylang- ylang (Cananga odorata 依兰香水树), which takes 22 hours to complet e through a fractional distillation. Distillation

The recondensed water is referred to as a hydrosol 水溶胶, herbal d istillate 馏出物 or plant water essence, which may be sold as another fr agrant product. Popular hydrosols are rose water, lavender water , lem on balm 香脂 , and orange blossom water . The use of herbal distillates i n cosmetics is increasing. Some plant hydrosols have unpleasant smells and are therefore not sold. Expression

Most citrus peel oils are expressed mechanically, or cold-pressed. Due to the large quantities of oil in citrus peel and the relatively low cost to grow and harvest the raw materials, citrus-fruit oils are cheaper than most other essential oils. Lemon or sweet orange oils that are obtained as by-products of the citrus industry are even cheaper. Prior to the discovery of distillation, all essential oils were extracted by pressing. Solvent extraction

Most flowers contain too little volatile oil to undergo expression and their chemical components are too delicate and easily denatured 变性的 by the high heat used in steam distillation. Instead, a solvent such as hexane or supercritical 超临界的carbon dioxide is used to extract the oils. Extracts from hexane and other hydrophobic solvent are called concretes 凝结物 , which is a mixture of essential oil, waxes, resins, a nd other lipophilic (oil soluble) plant material. Solvent extraction

Although highly fragrant, concretes contain large quantities of non-fragrant waxes and resins. As such another solvent, often ethyl alcohol, which only dissolves the fragrant low-molecular weight compounds, is used to extract the fragrant oil from the concrete. T he alcohol is removed by a second distillation, leaving behind the a bsolute. ISOLATION & SEPARATION TECHNIQUES

• Terpenoids • Following methods are employed for the extraction of mono-, sesqui-, di-, tri-, and tetraterpenoids.

• Air dried powdered part of the plant is extracted by percolation or soxhlet extraction successively with organic solvents with increasing polarity such as petroleum ether, benzene, diethyl ether, chloroform, ethyl acetate, acetone, ethanol, methanol and water. The extraction efficiency can be increased with the decrease in the time of the process by stirring the pulverized plant material using mechanical stirrer with the chosen solvent and filtering it to obtain the extract. STRUCTURE ELUCIDATION

• Physical Mehtods 1. Molecular formula 2. Specific rotation 3. Refractive index • Spectral Methods for Structure Determination 1. UV 2. IR 3. MS 4. NMR Physical Mehtods

1. Molecular formula Determination of the molecular formula of an isolated pure terpenoid is done by finding out the empirical formula and molecular weight. Empirical formula can be found out by elemental analsis .While molecular weight can be determined by vapour density, elevation of boiling point and depression of freezing point.

2. Specific rotation Specific rotation of a compound is measured to ascertain the optical activity exhibited by it. It helps to distinguish between optical isomers.

3. Refractive index It is measured to calculate the value of molecular refraction, which is useful to find out the nature of the carbon skeleton especially in the case of sesquiterpenoids . Spectral Methods

1. UV Functional groups, present in terpenoids , which absorb in the UV range between 200-350nm are termed as chromophores. However UV data becomes valuable only when the terpenoid molecule contains conjugated double bonds and/or α,β-unsaturated carbonyl group.

2. IR This method is routinely used for the identification as well as the structure elucidation of new terpenoids.

3. MS FAB-MS affords the e xact molecular ion peak along with diagnostic fragmentation patterns of the terpenoid molecule. It is an important tool for the structure determination . Spectral Methods

4. NMR • NMR spectroscopy comprising of both PMR and CMR is in fact one of the Most important tools furnishing a good teal of information required for the structure elucidation.

The combination of 1D selective and 2D NMR techniques such as COSY, TOCSY, ROESY,2D IN-ADEQUATE, HMQC, HMBC COLOC, HOHAHA , HETCOR and selective INEPT are of great value for the structure elucidation of various terpenoids including the saponins and glyosides of a number of sugar moieties. EXAMPLE

• C10 H16 O • b.p.77 ℃℃℃ • UV :236nm • IR :1665 ，1625 ，1603 ，1398 ，1190 ， 1117cm -1. • MS :m/z 69(100),41,84,94,109,67,83,81 CHO • 1H-NMR :1.65(6H ，d， C-7 methyls) ， 2.15(3H ，s，C-3 Me) ，5.0(1H ，t， H-6) ， 5.8(1H ，d，H-2) ， 9.84(1H ， d，H-1). • 13 C-NMR :190(C-1) ，127.5(C-2) ，16 2.1(C-3) ，40.5(C-4) ，26.5(C-5) ，123. 5(C-6) ，132.3(C-7) ，25.3(C-8) ，17.4 geranial (C-9) ，17.0(C-10). Types of Terpenoids Diterpenoids

Outcome

• Student understood the terpenoids in the form of Meaning, Different types Properties, occurrences, uses Isolation Method General Biogenetic Pathway Pharmacognostic account of different drug con taining important constituents Terpenoids.