Pharmacopoeial Standards and Analysis of Essential Oils with Respect to South African Essential Oils Prof

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CONFERENCE ON INDUSTRY MILESTONES, SHARING SUCCESSES AND DEMYSTIFYING MARKET REQUIREMENTS 25 - 26 July 2019 PRETORIA SA Pharmacopoeial Standards and Analysis of Essential oils with respect to South African Essential Oils Prof. Dr. K. Hüsnü Can Başer Near East University Faculty of Pharmacy Department of Pharmacognosy Lefkosa (Nicosia), N. Cyprus [email protected] www.khcbaser.com Quality control

• Due to volatile nature of their active ingredients, quality control of essential oils is extremely important. • In order to safeguard their quality, standards and specifications exist in national and international Standard monographs as well as pharmacopoeias and codices. European Pharmacopoeia Essential oils TESTS General Tests The essential oil complies with the prescribed limits for the following tests Relative density, refractive index, optical rotation, fatty oils and resinified essential oils, Supplementary Tests

If necessary, the essential oil complies with the prescribed limits for the following tests Freezing point, acid value, peroxide value, foreign esters, residue on evaporation, solubility in alcohol, water in essential oils. Water in Essential oils

• EP6-2.8.5 Mix 10 drops of the essential oil with 1 ml of carbon disulphide R. The solution remains clear on standing. Foreign Esters in Essential oils

• EP6-2.8.6 Heat 1 ml of the essential oil for 2 min on a waterbath with 3.0 ml of a freshly prepared 100 g/l solution of potassium hydroxide R in alcohol R. No crystals are formed within 30 min even after cooling. Fatty oils and resinified essential oils in Essential oils • EP6-2.8.7 Allow 1 drop of the essential oil to fall onto filter paper. The drop evaporates completely within 24 h without leaving any translucent or greasy spot. Odour and Taste of Essential oils

• EP6-2.8.8 Mix 3 drops of the essential oil with 5 ml of 90 per cent V/V alcohol R and stir in 10 g of powdered sucrose R. The odour and taste are similar to that of the plant or parts of the plant from which the essential oil has been obtained. Residue on evaporation of Essential oils • EP6-2.8.9 The residue on evaporation of an essential oil is the percentage by mass of the oil which remains after evaporation on a water bath under the conditions specified below. Apparatus: The apparatus consists of: - Waterbath with a cover having holes of 70 mm diameter, - Evaporating dish of heat-resistant glass which is inert to the contents, - Desiccator Residue on evaporation of Essential oils Residue on evaporation of Essential oils • Method: Weigh the evaporating dish after having heated it on the water-bath for 1 h and cooled it in the desiccator. Weigh into the evaporating dish 5.00 g of the essential oil, unless otherwise prescribed. Heat the oil on the vigourously boiling water-bath in a draught-free atmosphere for the prescribed time. Allow to cool in the desiccator and wait. During the test, the level of water in the bath is maintained about 50 mm beneath the level of the cover. Solubility in Alcohol of Essential Oils All essential oils are soluble in absolute alcohol and many are soluble in dilute alcohol. It is, therefore, possible to determine the number of volumes of dilute alcohol required for the complete solubility of one volume of oil. Sometimes, however, the solutions obtained are not clear, then, they are defined as “opalescent”. Solubility in Alcohol of Essential Oils EP6 - 2.8.10 • 1.0 ml of the essential oil in a 25 ml or 30 ml glass-stoppered cylinder. Place in a constant temperature device, maintained at a temperature of 20 ± 0.2 oC . Using a burette of at least 20 ml capacity, add the alcohol of the strength prescribed in the monograph by increments of 0.1 ml until solution is complete and then continue adding by increments of 0.5 ml to a total of 20 ml, shaking frequently and vigorously. Record the volume of alcohol added when a clear solution has been obtained and, if the solution becomes cloudy or opalescent before 20 ml of alcohol has been added, record the volume added when the cloudiness or opalescence appears and, where applicable, the volume added when the cloudiness and oplescence disappears. Solubility in Alcohol of Essential Oils • If a clear solution has not been obtained when 20 ml of alcohol of the prescribed strength has been added, repeat the test using the next highest concentration of alcohol. An essential oil is said to be “soluble in n volumes and more of alcohol of given strength l” when the clear solution in n volumes remains clear when compared with the undiluted oil after further addition of alcohol of the same strength up to a total of 20 volumes of alcohol. An essential oil is said to be “soluble in n volumes and more of alcohol of given strength l becoming cloudy when diluted”

when the clear solution in n volumes becomes cloudy in n1 volumes (n1 less than 20 ) and stays so after further gradual addition of alcohol of the same strength up to a total of 20 volumes of alcohol. Solubility in Alcohol of Essential Oils • An essential oil is said to be “soluble in n volumes and more of

alcohol of given strength l with cloudiness between n1 and n2 volumes” when the clear solution in n volumes becomes

cloudy in n1 volumes (n1 less than 20 ) and stays so after further gradual addition of alcohol of the same strength up to

a total of n2 volumes of alcohol and then becomes clear (n2 less than 20). • An essential oil is said to be “soluble with opalescence” when the alcoholic solution shows a bluish tinge similar to that of a standard of opalescence freshly prepared as follows: mix 0.5 ml of silver nitrate solution R2 and 0.05 ml of nitric acid R; add 50 ml of a 12 mg/l solution of sodium chloride R; mix and allow to stand protected from light for 5 min. Relative density

• The specific gravity of an oil is the weight of a given volume of the oil at a specific temperature compared with the weight of an equal volume of water at the same temperature, all weighings being taken in air. A pycnometer is used for this determination. Optical rotation

• The optical rotation ( ) of an oil is the angle through which the plane of polarization is rotated when polarized light passes through it. Results are expressed as dextrorotatory (+) or levorotatory (-) according to whether the plane of polarization is rotated chlockwise or anticlockwise, respectively, as determined by viewing towards the light source. Unless otherwise specified, the optical rotation is measured at a wavelength of the sodium D line (589.3 nm) at a temperature of 19.5 oC to 20.5 oC in a layer 1 dm thick. Polarimeters are used for the determination of optical activity. This technique provides useful data on the optical purity of the oil. Refractive index

• The refractive index ( ) of an oil with reference to air is the ratio of the sine of the angle of incidence to the sine of the angle of refraction of a beam of light passing from air into the oil. It varies with the wavelength of the light used in its measurement. Abbe type refractometers are widely used for the determination of the refractive index. Freezing point

• The freezing point is the highest temperature observed during the solidification of a supercooled liquid. Chemical tests Sensory evaluations • Sensory evaluations can be carried out only by expert noses. Such an ability can be gained after years-long tedious but systematic olfactive training. To an experienced nose, the evaporation pattern of an essential oil smeared on a smelling strip, over a period of time, gives information about its source, age, main components and even its authenticity. Smelling must be carried out at intervals immediately after dipping to 1,2 and 6 hours, and after standing overnight over a period not less than 18 hours. Comparison with an authentic sample of acceptable quality will help the assessor to make a correct judgement. As obvious, sensory evaluations are subjective and may vary from person to person. Therefore, such assesments are, in general, realized by a panel of experts and in all cases their assesments should be verified and documented by experimental proof. Quality control

• Gas chromatography (GC) is the most important technique for the separation of essential oil constituents and their quantitation. • Gas chromatography/mass spectrometry (GC/MS) may only be necessary to identify constituents which appear unexpectedly in a known essential oil of for the analysis of new essential oils.

KHC Baser, Analysis and quality assessment of essential oils. In: A Manual on the Essential Oil Industry, KT de Silva (Ed), UNIDO, Vienna (1995). Conformity to specifications • Essential oils are complex mixtures and their quality assessment requires multifarious analyses. The first and the simplest analysis is carried out by sensory organs, that is the smell, colour and viscosity means a lot to an experienced perfumer. Instrumental techniques developed in recent decades have revolutionized the advancements in the science and trade of essential oils for detecting adulteration, and for elucidating the structures of many hitherto unknown aroma chemicals occurring in essential oils. Such compounds have also been a model for new families of flavour and fragrance chemicals. It has also made quantification of even less than part per billion (ppb) amounts of essential oil constituents possible. With such a degree of sophistication, it is now a reality to assure the quality of an essential oil in almost absolute terms. Conformity to specifications • For known essential oils of commerce, specifications have been drawn and published by authoritative organizations, the most important for essential oils being TC54 Committee of the International Standards Organization (ISO). • Essential oil and aroma chemical monographs published by the Essential Oil Association of USA have now become obsolete but can still be safely used for referencing. • Nine volume set of Flavour and Fragrance Ingredient Data Sheets compiled by the Flavour and Extract Manufacturers’ Association (FEMA), the Research Institute for Fragrance Materials (RIFM) and the Fragrance Materials Association of the United States (FMA) contain safety information of over 1500 raw materials used in the fragrance and flavour industries. Conformity to specifications • International Fragrance Association (IFRA) and the International Organization of the Flavour Industry (IOFI) publish safety and use limits data on essential oils and aroma chemicals in their regularly updated “Code of Practice”. • FEMA produces FEMA GRAS list (GRAS stands for Generally Recognized As Safe). • AFNOR (Association Francaise de Normalisation) publishes standard monographs for essential oils. • Pharmacopoeias and Food Chemicals Codex provide monographs for essential oils used in food and pharmaceuticals. • International Federation of Essential Oils and Aroma Trades (IFEAT) has published a guideline for classification and labelling of essential oils for transport and handling. Flash points of essential oils are also indicated in the guideline. ESSENTIAL OIL MONOGRAPHS IN EUROPEAN PHARMACOPOEIA Aetheroleum

Matricariae aetheroleum Terebinthinae aetheroleum Foeniculi amari fructus aetheroleum Neroli aetheroleum Anisi aetheroleum

Foeniculi amari herbae aetheroleum Menthae piperitae aetheroleum

Foeniculi amari fructus aetheroleum Rosmarini aetheroleum Spicae aetheroleum Carvi aetheroleum Anisi stellati aetheroleum Cinnamomi cassiae aetheroleum Aurantii dulcis aetheroleum

Cinnamomi zeylanici corticis aetheroleum Pini pumilionis aetheroleum Melaleucae aetheroleum Cinnamomi zeylanici folium aetheroleum Eucalypti aetheroleum Thymi typo thymolo aetheroleum Citronellae aetheroleum Juniperi aetheroleum Limonis aetheroleum Salviae sclareae aetheroleum Lavandulae aetheroleum Salviae lavandulifoliae aetheroleum

Caryophylli aetheroleum Citri reticulatae aetheroleum Myristicae fragrantis aetheroleum Coriandri aetheroleum Menthae arvensis aetheroleum partim mentholi privum Pini silvestris aetheroleum

32 GLOBAL TRADE OF ESSENTIAL OILS

 Latest estimates suggest that the global production is more than 150.000 tonnes.  The global market of essential oils stands at $6.5 billion with a projected rise by 8.4 to 11.3% to $15.8 billion in 2024-2025.  Estimate for 2020 is 370.000 tonnes valued at over $10 billion.  Essential oil cultivation areas cover 600.000 hectares and 1 million farms are producers of essential oil bearing plants.

C. Barbieri, P. Borsotto, (2018) Essential oils: Market & legislation, Intech Open, Chapter 6. , 107-127. GLOBAL TRADE OF ESSENTIAL OILS

 Top four essential oils orange, eucalyptus, mint and lemon are produced in 100.000 tonnes which represent more than 2/3 of the total essential oil crop production.  European Union is the biggest importer of essential oils with France, Germany and UK being the major importing countries. GLOBAL TRADE OF ESSENTIAL OILS

In 2016, global exports of EOs amounted to $4.38 billion, and imports to $4.54 billion. Top exporters are USA, Germany, UK and France. Top importers are France, USA, Germany and Ireland. GLOBAL TRADE OF ESSENTIAL OILS

• Major producing countries of essential oils are China and India (Major), and Indonesia, Sri Lanka, Vietnam • In Africa, Morocco, Tunisia, Egypt, Algeria, the Ivory Coast, S. Africa, Ghana, Kenya, Tanzania, Uganda, Ethiopia • In Americas, USA, Canada and Mexico (Major); Argentina, Paraguay, Uruguay, Guatemala, Haiti • Others: France, Germany, Taiwan, Japan, Jamaica, Phillippines GLOBAL TRADE OF ESSENTIAL OILS The main markets are : • Food & drinks : 35% • Fragrance, cosmetics & aromatherapy : 29% • Household : 16% • Pharmaceuticals : 15% GLOBAL TRADE OF ESSENTIAL OILS

Oil Source Prodn. (Ton) Value $ (000) $/kg Producer country Orange Citrus sinensis 55.000 440.000 8 Brazil, USA, Israel, Argentina, Mexico, Spain, S. Africa Cornmint Mentha canadensis 40.000 720.000 18 India Eucalyptus oil – Eucalyptus globulus 11.000 330.000 30 China, India Cineole type Lemon Citrus limon 7.500 262.500 35 Italy, Spain, Argentina, Israel, USA Scotch spearmint Mentha x gracilis 3.650 - - USA Peppermint Mentha x piperita 3.300 132.000 40 USA, India Mustard (incl. cold- Brassica nigra, B. juncea 3.000 600.000 200 India pressed oil) Clove leaf Syzigium aromaticum 2.560 56.320 22 Indonesia, Madagascar, Zanzibar, Brazil, Sri Lanka

Lavender Lavandula officinalis 2.100 73.500 35 France, Bulgaria, Spain Lavandin grosso Lavandula x intermedia 2.100 73.510 35 France, Bulgaria, Spain Lime Citrus aurantiifolia 1.900 76.000 40 Mexico, Peru, Brazil Litsea cubeba Litsea cubeba 1.700 34.000 20 China Camphor Cinnamomum camphora 1.520 45.600 30 Taiwan Patchouli Pogostemon caplin 1.600 64.000 40 Indonesia, India Citronella Cymbopogon winterianus 1.100 33.000 30 China, Indonesia, India + C. nardus Cedarwood (Chinese) Chamaecyparis funebris 1.000 - - China GLOBAL TRADE OF ESSENTIAL OILS

Oil Source Prodn. (Ton) Value $ (000) $/kg Producer country Cornmint Mentha canadensis 40.000 720.000 18 India Sandalwood oil album Santalum album 300 690.000 2.300 India (ban but illegal cut), Australia, New Caledonia (lower quality) Mustard (incl. cold- Brassica nigra, B. juncea 3.000 600.000 200 India pressed oil) Orange Citrus sinensis 55.000 440.000 8 Brazil, USA, Israel, Argentina, Mexico, Spain, S. Africa Eucalyptus oil – Cineole Eucalyptus globulus 11.000 330.000 30 China, India type Lemon Citrus limon 7.500 262.500 35 Italy, Spain, Argentina, Israel, USA Vetiver Vetiveria zizanioides 500 200.000 400 Indonesia, Haiti, India, China Immortelle Helichrysum italicum 100 180.000 1.800 France, Italy, Croatia Peppermint Mentha x piperita 3.300 132.000 40 USA, India Lavender Lavandula angustifolia 750 127.500 170 Bulgaria, France, China, Russia Rose oil and Concrete Rosa damascena 14(C) 126.000 9000 (O) Bulgaria, Turkey 7(O) 63.000 Frankincense Boswellia serrata 400 120.000 300 India (Olibanum) Sandalwood oil Santalum lanceolatum, 100 120.000 1.200 New Caledonia, Australia, India spicatum S. spicatum Sandalwood oil Santalum spicatum 50 100.000 2.000 Australia Queensland Grapefruit Citrus paradisi 800 92.000 115 Mexico, Brazil, S. Africa Frankincense Boswellia sacra (B. 300 90.000 300 Somalia, Oman (Olibanum) carteri) EO production according to value (000 USD) 32 GLOBAL TRADE OF ESSENTIAL OILS

Oil Source Prodn. (Ton) Value $ (000) $/kg Producer country

Agarwood Aquilaria spp. - - 42.000 China, India, Malaysia, (e.g., A. Bangladesh malaccensis) Rose oil and Rosa damascena 14(C) 126.000 9000 (O) Bulgaria, Turkey Concrete 7(O) 63.000 Neroli Citrus aurantium 10 45.000 4.500 Egypt, Morocco

Immortelle Helichrysum 100 180.000 1.800 France, Italy, Croatia italicum Sandalwood oil Santalum album 300 690.000 2.300 India (ban but illegal album cut), Australia, New Caledonia (lower quality) Sandalwood oil Santalum 50 100.000 2.000 Australia Queensland spicatum

Sandalwood oil Santalum 100 120.000 1.200 New Caledonia, spicatum lanceolatum, Australia, India S. spicatum Most valued essential oils (000 USD) Future Prospects

• Low Price Oils (<20 US$) IN • Heavy, Animalic, Musky Notes OUT • China & India – Prospective consumer Societies • Shortages in suppy of Essential oils • New prospective production areas in subsaharan Africa, Australia, South America • Essential Oil Producing Developing Countries will turn to isolation of Aroma-chemicals • Major Flavour & Fragrance houses are shifting to Asia and South America Criteria for finding New Sources of Natural Isolates

• Very strong and characteristic aroma with high added value • High Enantiomeric Purity • Not-so-complex oil composition • No components causing off-odour/off-flavour or toxicity Essential Oil exports/imports of Turkey (2018) • Exports Oregano 65.9 tons USD 5.255.103 Rose+conc. +Stearopten 22.9 tons USD 16.627.214 Other EOs 102 tons USD 15.126.530 TOTAL USD 47.480.760 • Imports TOTAL USD 33.545.630

I shall give two examples of important commercial essential oils from

Turkey 36 ROSE (Rosa damascena)

 Rose oil is produced in Turkey and Bulgaria by water distillation of the fresh flowers of Rosa damascena Miller.  This species is a natural hybrid of Rosa gallica L. and Rosa phoenicia Boiss. both of which are found in Turkey.  The variety cultivated is “trigintipetala” (30 petalled).

Rosa gallica Rosa phoenicia

Rosa damascena ROSE OIL (Gül yağı)

Turkey is one of the two major producers of rose oil together with Bulgaria. Rose oil by water distillation and rose concrete by solvent extraction are produced from fresh flowers of Rosa damascena (Rosaceae). ROSE OIL (Gül yağı)

 Annually, 7000 tons of roses are worked up to produce 1600 kg of rose oil and 2400 kg of rose concrete*. Rose absolute** is also produced by some companies in limited amounts.

* Dried n-hexane extract of fresh roses ** Ethanolic extract of rose concrete – a dark coloured oil ROSE OIL (Gül yağı)

 The price of Turkish rose oil varies in the range of € 10.000 per kg. Rose concrete usually sells for € 1000 per kg. Annually, Turkey exports around $ 10 million worth of rose oil. ROSE OIL (Gül yağı)

 Rose is an entirely cultivated crop and 3500 - 4000 kg of fresh roses are required to produce 1 kg of rose oil, while 400 kg of fresh roses are sufficient to produce 1 kg of rose concrete.

 Rose distillation season is between mid-May and mid-June and 100 tons of rose water is also produced annually. 30 YEARS OF GÜLBİRLİK ROSE OIL (1986-2016)

Main components (%) Min. Max. Citronellol 30.9 43.9 Geraniol 9.3 20.8 Nonadecane 8.2 14.7

Citronellol Nerol 5.2 10.7 1- Nonadecene 2.0 4.9 Methyl eugenol 2.0 4.0 Heneicosane 2.5 4.2 Geranyl acetate 1.0 2.3 Linalool 0.3 2.1 Phenylethyl alcohol 1.2 2.0

Geraniol b-Caryophyllene 0.4 1.6 Citronellyl acetate 0.5 1.4 Germacrene D 0.7 1.4 (2E,6E)-Farnesol 0.6 1.6

1-Phenylethyl alcohol ROSE OIL (2017)

Production Kg Price ($/kg)

Rose oil 1.350 10.000- 11.000 Rose concrete 10.000 1.100-1.150

Rose absolute 2.500 2.200-2.600

Rose water 300.000 2-3 ROSE PRODUCTS

• Ancient texts mention that rose is good for disorders of the brain and the heart1. • Several recent studies have provided scientific evidence for this information. The hydroalcoholic extract of R. damascena flowers has been shown to potentially increase heart rate and contractility in isolated guinea pig heart, possibly via a stimulatory effect on b- adrenergic receptors2. • Rosa damascena has beneficial effects on brain function and has potential applications for the treatment of dementia. • A recent study suggested that rose oil and a main component of the rose fragrance, phenylethyl alcohol, significantly inhibits acetylcholine esterase (AChE) and butyrylcholine esterase (BChE)3.

1KHC Başer, A. Altıntaş, M. Kürkçüoğlu, TURKISH ROSE: A Review of the History, Ethnobotany, and Modern Uses of Rose Petals, Rose Oil, Rose Water, and Other Rose Products, HerbalGram (in press). 2MH Boskabady, A. Vatanprast , H. Parsee , M. Ghasemzadeh .Effect of aqueous-ethanolic extract from Rosa damascena on guinea pig isolated heart. Iranian J Basic Med Sci. 2011;14:116- 121. 3FS Senol, I Orhan, M Kurkcuoglu, MH Khan, A. Altintas, B. Sener, KHC Baser, An in vitro approach to neuroprotective activity of Rosa damascena Mill, a medieval age traditional medicine used for memory enhancement. Planta Med. 2011;77(12):1440-1440. ROSE PRODUCTS

 Rose products found in the market are  Rose oil  Rose water  Rose concrete  Rose absolute  Rose jam  Rose lokum  Rose soap  Rose syrup  Dried rose flowers Kekik (Oregano): A panacea OREGANO (Kekik)

Coridothymus capitatus Origanum husnucan-baseri  Oregano is a collective term referring to members of several genera common feature being that they all contain carvacrol as main constituent in their essential oils.  Such genera include Origanum, Thymbra, Satureja, Thymus and Coridothymus of the family Lamiaceae (or Labiatae) and Lippia graveolens among others.  Plants smelling like Oregano or Thyme are called as “kekik” in Turkish. Origanum species constitute the largest portion of Oregano traded around the world.

Thymus kotschyanus Thymbra spicata

Satureja cuneifolia Origanum vulgare ssp. hirtum KEKİK (= Oregano )

 Kekik is the most important wild crop of Turkey.  Turkey is the world’s biggest supplier of oregano.  In 2018, dried oregano exports reached $ 57 million for 17.666 tons with a unit value of $3.2 per kg. In 2005, it was $17.9 million for 10.400 tons, with a unit value of $1.7 per kg.  Oregano is cultivated in 104.863 da to yield 13.000 tons  ca. 1.000 tons consumed domestically.  The rest is either exported or used in essential oil production.  In 2018, Turkey exported 66 tons of oregano oil for a return of $5.5 M ($1.5M-2011). K.H.C.Baser, The Turkish Origanum Species, In: Oregano, The Genera Origanum and Lippia, Ed.: S.E.Kintzios, Taylor and Francis, UK (2002). KEKİK (Oregano )

Majority of kekik exports from Turkey comprises the following five Origanum sp. (in order of significance ): O. onites (Turkish oregano, Bilyalı kekik), O.vulgare subsp. hirtum (= O.heracleoticum ) (Greek oregano, İstanbul kekiği), O.minutiflorum (Yayla kekiği, Sütçüler kekiği)[endemic], O. majorana (= O dubium ) (Beyaz kekik, white oregano), O.syriacum var. bevanii (Tarsus kekiği, Israeli oregano) Oregano – PhEur 2005 OREGANO WATER (Kekik Suyu )

 A herbal drink favoured in Western and Southern Turkey.  The distillate of oregano plants left out of the removal of essential oil. It is a hydrosol or aromatic water.  It contains 0.12% oil with carvacrol (ca.70%) and rare p-menthen-diols (ca.10%) as main OH OH components. OH OH OH

carvacrol cis-p-menth-4-en-1,2-diol cis-p-menth-3-en-1,2-diol OREGANO WATER (Kekik Suyu )

 Oregano water is produced using very simple make- shift distillation stills of ingenious design in house kitchens.  The oil which floats on top is scooped with a spoon and kept in a separate bottle for rubbing on skin as a remedy for rheumatism.  Some distillation companies market oregano water in 1 L or 5 L plastic bottles diluting it 1:1 with water in urban centres in Turkey. OREGANO (KEKİK) WATER

Folk medicine: Antiulcer, digestive, antidiabetic, GIS regulation, general prophylaxy.

Pharmacological studies: Bileflow, barbiturate sleeping time, isolated ileum and aorta experiments, inhibition of gastrointestinal contractions, antihypertensive activity… My joint studies on South African essential oils with SA scientists (Profs. Viljoen, Van Wyk, Van Vuuren, Kamatou et al. ) 27 Papers published on the essential oils of 82 species belonging to 23 genera in 11 families: Myrothamnus flabellifolius (Myrothamnaceae), Siphonochilus aethiopicus (Zingiberaceae), Osmitopsis asteriscoides (Asteraceae), Artemisia afra (Asteraceae), Pteronia (adenocarpa, camphorata, elongata, empetrifolia, fasciculata, flexicaulis, glauca, glomerata, pallens, paniculata, viscosa) (Asteraceae), Helichrysum (cymosum, dasyanthum, felinum, excisum, petiolare) (Asteraceae), Pegolettia (baccharidifolia, retrofracta) (Asteraceae), Pentzia (incana, punctata) (Asteraceae), Lippia javanica (Verbenaceae), Vitex (poorara, rehmannii, obovata ssp. obovata, obovata ssp. wilmsii, zeyheri) (Lamiaceae), Salvia (repens, runcinata, stenophylla) (Lamiaceae), Plectranthus (ciliatus, zuluensis, hybrids) (Lamiaceae), Agathosma (arida, bathii, betulina, capensis, collina, crenulata, hirsuta, lanata, namaquensis, ovalifolia, ovata, parva, pubigera, pungens, roodebergensis, stipitata, zwartbergense) (Rutaceae), Diosma prama (Rutaceae), Coleonema (album, aspalathoides, calycinum, pulchellum, virgatum) (Rutaceae), Adenandra (obtusata, villosa) (Rutaceae), Euchaetis albertiniana (Rutaceae),Acmadenia (alternifolia, obtusata, sheilae) (Rutaceae), Pelargonium (betulinum, capitatum, citronellum, glutinosum, graveolens, hispidum, panduriforme, papilionaceum, quercifolium, radens, scabrum, tomentosum, vitifolium) (Geraniaceae), Heteropyxis natalensis (Heteropyxidaceae), Sclerocarya birrea (Anacardiacaea), Gethylis (afra, ciliaris) (Amaryllidaceae), Widdringtonia cedarbergensis (Cupressaceae) The top 10 South African essential oils identified by UNIDO*: • Pelargonium var rose (Rose geranium) • Eriocephalus spp - (Cape chamomile) • Helichrysum spp - (Everlastings) • Lippia spp - (Lemonbush) • Agathosma spp (Buchu) • Sclerocarya birrea (Marula) ------• Adansonia digitata (Baobab) (fixed oil) • Ximenia species (Sour plum) (fixed oil) • Schinziophyton rautanenii ( Manketti/ Mongongo) (fixed oil) • Citrullus lanatus (Kalahari melon) (fixed oil)

*Kindly supplied by Ms. Karen Swanepoel Genera in red have been studied by our group. Helichrysum Helichrysum dasyanthum, Helichrysum felinum, Helichrysum excisum and Helichrysum Petiolare

The acetone and methanol extracts as well as the essential oils exhibited activity against Gram-positive bacteria, while both the methanol and acetone extracts of all four species were active in the anti-oxidant assay. The essential oils, on the other hand, displayed activity in the 5-lipoxygenase assay, which was used as an indication of anti-inflammatory activity. Two extracts exhibited promising activity in the antimicrobial assay, the acetone extract of Helichrysum dasyanthum with a MIC value of 15.63 mg/ml and the methanol extract of Helichrysum excisum with a MIC value of 62.5 mg/ml. The acetone extract of

Helichrysum dasyanthum was the most active free radical scavenger in the DPPH assay (IC50 of 9.53 mg/ml) while values for the anti-inflammatory activity of the essential oils ranged between 25 and 32 mg/ml. Hd Hf He Hp

a-pinene 16.6 2.8 - 6.8

b-pinene 6.2 1.1 - 0.4

limonene 6.1 2.2 - 3.1

1,8-cineole 20.6 34.0 - 22.4

p-cymene 5.5 6.5 - 9.8

a-copaene 0.1 2.5 4.0 1.3

b-caryophyllene 13.3 5.7 27.6 14.0

a-dihydroagarofuran 3.4 - - -

10-hydroxy-b-caryophyllene 5.2 - - -

caryophyllene oxide 1.3 2.1 6.9 2.5

a-humulene 0.6 0.2 9.4 2.0

alloaromadendrene - 0.7 7.3 -

In vitro biological activity and essential oil composition of four indigenous South African Helichrysum species, A.C.U. Lourens, D.Reddy, K.H.C. Baser, A.M. Viljoen, S.F. Van Vuuren, J. Ethnopharmacol., 95, 253-258 (2004). Helichrysum

Helichrysum cymosum Asteraceae.

1,8-cineole (20.4%), a-pinene (12.4%), b-caryophyllene (10.8%), (Z)-b-ocimene (9.5%), limonene (7.2%). The essential oil displayed the highest efficacy against the malaria parasite, which was comparable to the classical antimalarial chloroquine and quinine. Helihumulone a non-volatile phloroglucinol derivative of the acetone extract showed the highest antimicrobial activity.

The antimicrobial, antimalarial and toxicity profiles of helihumulone, leaf essential oil and extracts of Helichrysum cymosum (L.) D. Don subsp. cymosum, S.F. van Vuuren, A.M. Viljoen, F.R. van Zyl, F.R. van Heerden, K.H.C. Baser, South African Journal of Botany, 72, 287-290 (2006) Agathosma (Buchu) A. arida, A. bathii, Agathosma betulina, A. capensis, A..collina, Agathosma crenulata, A. hirsuta, A. lanata, A. namaquensis, A. ovalifolia, A. ovata, A. parva, A. pubigera, A. pungens, A. roodebergensis, A. stipitata, A. zwartbergense Rutaceae.

Constituents over 10% are indicated in the following list: a-pinene (par:10.4), b-pinene (ari:11.4; lan:16.9; par:14.3), sabinene (ova:32.2; pub:22.9), myrcene (cap[B]:26.5; cap[G]: 19.3; col:14.8; ova:13.9; pub:18.1), limonene (bat:25.6; bet:23.7; cap[B]:12.3; col:30.9; cre:13.4; pub:12.1; roo:11.6), 1,8-cineole (nam:22.1), b-phellandrene (nam:10), geijerene (roo:27.9), menthone (bet:29.2; cre:16.6), citronellal (hir:72.5; zwa:64.7), isomenthone (bet:14.2), linalool (ari:10.1; cap[B]:17.1; cap[G]:33.3; par:28.9; pun:15.4), methyl citronellate (nam:10.0), pulegone (bat:28.7; cre:34.9), neral (sti:34.8), geranial (sti:16.1), methyl eugenol (oval:23.0), dictamnol (roo:14.2) trans-8-mercapto-p-menthan-3-one (bet:0.1; cre:0.1) cis-8-mercapto-p-menthan-3-one (Bet:0.1; cre:t) trans-8-methylthiomenth-3-one (bet:t; cre:0.5) cis-8-methylthiomenth-3-one (bet:t; cre:t) trans-p-mentha-8-methylthiomenth-3-one (bat:2.1) cis-p-mentha-8-methylthiomenth-3-one (bat:0.5) 1) The Biological Activity and Essential Oil Composition of 17 Agathosma (Rutaceae) Species, A. M. Viljoen, A. Moolla, S.F. van Vuuren, R. L. van Zyl, K.H.C. Baser, B. Demirci, T. Ozek, T. H. Trinder-Smith, J. Essent. Oil Res. , 18 , 2-16 (Special Edition 2006); 2) A Seasonal Variation Study of the Chemical Composition and Antimicrobial Activity of the Essential Oil of Agathosma ovata (Thunb.) Pillans (Rutaceae), A. M. Viljoen, A. Molla, S. F. van Vuuren, K.H.C. Baser, B. Demirci, T. Ozek, J. Essent. Oil Res., 18 , 30-36 (Special Edition 2006) Sclerocarya birrea (Marula)

Anacardiaceae

Head-space volatiles of the fruit pulp and the whole fruits (skin volatiles) were investigated using solid phase micro-extraction (SPME) and GC–MS. The two major compounds in the fruit pulp were β-caryophyllene (91.3%) and a-humulene (8.3%). Thirty volatiles representing 88.7% of the total composition were identified in the head-space of the whole fruits. Heptadecene (16.1%); benzyl 4-methylpentanoate (8.8%), benzyl butyrate (6.7%), (Z)-13-octadecenal (6.2%) and cyclo- pentadecane (5.7%) were present in levels higher than 5%. The major alcohol detected in the head-space of the whole intact fruits was (Z)-3-decen-1-ol (8.4%).

Head-space volatiles of marula (Sclerocarya birrea subsp. caffra), A.M. Viljoen, G.P.P. Kamatou, K.H.C. Baser, South African J. Bot., 74, 325-326 (2008) Lippia javanica

Verbenaceae

From 16 samples (representing five natural populations), 5 chemotypes were identified; a myrcenone rich-type (36–62%), a carvone rich-type (61–73%), a piperitenone rich-type (32–48%), an ipsenone rich-type (42–61%) and a linalool rich-type (>65%). The myrcenone and linalool chemotypes have been mentioned in the literature but the carvone, ipsenone and piperitenone chemotypes have not previously been reported for Lippia javanica. Time kill studies were performed on three microbial respiratory isolates to document the scientific rationale of using Lippia to treat respiratory complaints in traditional herbal medicine. Klebsiella pneumoniae, Cryptococcus neoformans and Bacillus cereus showed reduction in microbial populations with the strongest bacteriostatic effect observed for Klebsiella pneumoniae.

The composition, geographical variation and antimicrobial activity of Lippia javanica (Verbenaceae) leaf essential oils, A.M. Viljoen, S. Subramoney, S.F. Van Vuuren, K.H.C. Baser, B Demirci, J. Ethnopharmacol., 96, 271–277 (2005) Pelargonium Geraniaceae Pelargonium betulinum, P. capitatum, P.citronellum, P. glutinosum, Pelargonium graveolens, P. hispidum, P. panduriforme, P. papilionaceum, P. quercifolium, P. radens, P. scabrum, P. tomentosum, P. vitifolium Over 10% constituents only

yield% p- menthone isomenthone viridiflorol T- citronellic decanoic cymene cadinol acid acid Pbe 0.04 0.2 - - 0.3 18.9 - - Pca 0.02 0.0-0.1 0.0-0.9 0.0-1.9 0.0-24.0 0.0-0.5 t 0.1-0.3 Pci*,**** 0.36 0.1 - 0.1 t - 0.1 - Pglu 0.02 0.5 0.7 1.4 8.9 - - - Pgra 0.02- 0.9 1.0-5.1 65.8-83.3 - t t-0.1 0.4-12.9 0.22 Phis*** 0.02 0.1 0.1 0.3 - t 0.8 47.0 Ppan 0.35- 37.6- - 0.0-0.3 7.9 - - - 0.48 45.4 Ppap 0.17 t - t - - 96.2 - Pquer 0.07 6.2- - - 13.0- 0.0-0.9 - - 54.9 36.6 Pra 0.69 1.1 1.9 84.5 - - - 0.1 Psca** 0.12 1.0 - t - 6.1 t 6.4 Pto 0.21- 0.8-1.1 0.0-41.1 49.3-56.6 t - t - 0.27 Pvi 0.05 0.2 36.1 - - 0.9 74.7 - *geranic acid (36%), **14-hydroxy-b-caryophyllene (27.9%), ***2-decenoic acid (31.3%), ****geranial (27.2%)

The Essential Oil Composition and Chemotaxonomical Appraisal of South African Pelargoniums (Geraniaceae), J.Y.Y. Lalli, A.M. Viljoen, K.H.C. Baser, B. Demirci, T.Ozek, J. Essent. Oil Res., 18 , 89-105 (Special Edition 2006) Main component (%) Chinese Egyptian Bourbon

Citronellol 38-40 31-33 19-21

Geraniol 7-10 13-15 16-19

Linalool 2-3 5-6 7-10

Linalool + Geraniol /Citronellol ratio 0.3 0.6 1.4 Distinguishing chemicals Guaia-6,9-diene 1-7 - 1-7

10-epi-gamma-eudesmol - 3-7 -

Refs. K. F. El-Fadly, An overview of best practices for production of Egyptian Geranium oil, EMAP (2012); K.V. Peter, Handbook of Herbs and Spices 2, CRC Press (2004) p. 164. South African Oils have development potential

• South Africa with the richness and diversity of its flora offers opportunities to the World in terms of novel flavours and fragrances. • Supplying genuine, standardized, unadulterated essential oils is a must in the highly conservative F&F trade. • Cultivation of the aromatic plants in high demand is recommended in order to conserve diversity and to get steady and quality supplies. • For standardization, pharmacopoeial or standard monographs must be prepared for the selected essential oils and also for others with development potential. • European Pharmacopoeia, ISO, AFNOR, etc. can be taken as examples for developing monographs.

ABC James A. Duke Excellence in Botanical Literature Award for 2016 HANDBOOK OF ESSENTIAL OILS: Science, Technology and Applications Second Edition

Edited by: K. Hüsnü Can Başer Gerhard Buchbauer

CRC Press Taylor & Francis

ca. 1200 pages 1st Edn. : January 2010 2nd Edn. : January 2016 3rd Edn. : Expected 2020 A Quarterly Open Access Scientific Journal

Editor in Chief K. Hüsnü Can Başer

Associate Editor Fatih Demirci

Editorial Secretary Gökalp İşcan

Editorial Board Yoshinori Asakawa (Japan) Gerhard Buchbauer (Austria) Jan Demyttenaere (Belgium) Ana Cristina Figueiredo (Portugal) Chlodwig Franz (Austria) Jan Karlsen (Norway) Karl-Heinz Kubeczka (Germany) Stanislaw Lochynski (Poland) Agnieszka Ludwiczuk (Poland) Patrizia Rubiolo (Italy) Luigi Mondello (Italy) Johannes Novak (Austria) Alvaro Viljoen (South Africa) Éva Németh-Zámboriné (Hungary) Nurhayat Tabanca (USA) Sandy van Vuuren (South Africa) Salvador Canigueral (Spain) Temel Özek (Turkey) Massimo Maffei (İtaly) www.nveo.org Thanks for your kind attention