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Vanilla is a vine-like tropical orchid, from the Orchidaceous family. Among the orchids, apart from the ornamental orchids; grown for their flowers, vanilla is the only genus of economic importance. Vanilla is chiefly cultivated for its sweet aroma and delicate flavour, much prized for various dishes all over the world. It is known to be the second most expensive flavouring spice after saffron in the world.
Vanilla originates from the tropically-humid regions of Mexico and Central America, as well as the forests of South America..
There are about 110 species of vanilla which have been reported but among these, only three species are cultivated for commercial use. These are Vanilla planifolia (Vanilla fragrans), Vanilla pompana and Vanilla tahitensis.
Cultivation of Vanilla pompana and Vanilla tahitensis are only done occasionally as they yield inferior quality products as compared to Vanilla planifolia.
1.1 - Structure of Vanilla Plants
The vanilla plants mostly grow on the edges of the tropical forests. Vanilla plants consist of long vines of about 35 metres, and they have alternate leaves spread along its length. The leaves are short, oblong and dark green in colour. Vanilla leaves are also thick, leathery and fleshy.
The flowers of the plant are green, white, cream or greenish-yellow. These flowers are hermaphrodites; meaning that they possess both male and female organs. Thus, pollination of the flower merely requires the transfer of pollen from the anther to the stigma. However, the particular tubular shape of the flower and the presence of rostellum which is a membrane separating the stamen and the stigma in the flower, prevent the flower from being self-pollinated. Only one specific pollinator is known to exist for Vanilla planifolia and they are bees of the genus Melapona which can survive only in Central America or more specifically in Mexico. Therefore, all vanilla plants are nowadays hand-pollinated, also known as artificial pollination method.
When the flower is pollinated, the fruit (pod) is formed and these pods are commonly known as beans as they are cylindrical and pendular shaped. The pods can attain a length of about 10-15 cm and are green in colour when fresh and are harvested about eight to nine months after flowering. The pods are considered to be mature when the green colour changes to pale yellow.1 After that, the pods are cured, a process during which the aroma and the flavour of the vanilla pods are enhanced by a process of fermentation of the fleshy part of the pod. The curing process changes the colour of the pods to dark brown or black.
1.2 - Vanillin
A substance known as vanillin (C8H8O3) is what procures the taste and aroma of the pods along with nuances of several other compounds. However, since natural vanillin is scarce and is also very expensive, synthetic vanillin is synthesized from guaiacol; which is a naturally occurring phenolic compound derived from wood creosote or lignin; a wood constituent.
Natural vanillin is very expensive as compared to synthetic vanillin but the quality defined mainly by the taste and aroma of the natural vanillin is in no way comparable to that of the synthetic one.
1.3 ââ‚¬" Uses of vanilla
Vanilla is used a lot in bakery items, soft drinks, chocolate flavours, ice-creams and various other confectionaries. It is used to procure the aroma to the products. Vanilla is also used in personal care products such as perfumes, creams and soaps as well as detergents for the fragrance it procures.
1.1 Extraction of Secondary Metabolites: Methods
Extraction is the process in which pre-treated plant samples are used along with particular solvents so as to remove the metabolites found in the plant tissues. The solvents used are chosen in such a way that they extract the greater part of the metabolites which are themselves attracted to certain types of solvents. For example, the solvent hexane usually extracts flavonoids and alkaloids from plant tissues. These metabolites are dissolved in the solvent and pure extracts of those metabolites are obtained by removing the solvents from the extract-solvent mixture using a rotary evaporator.
The two more commonly used methods are decantation and the soxhlet extraction.
Decantation is a method used for the extraction of fixed oils. The clean plant material is first cut and then macerated so as to increase the extraction rate. It is then placed in a conical flask and submerged with the solvent(s). The flask is stoppered with a cotton plug and aluminium foil and is placed onto an orbital shaker for a minimum of 48hrs so as to extract the maximum amount of secondary metabolites. The mixture is then filtered and the filtrate is concentrated in a rotary evaporator to obtain a pure extract without the solvent. The extract is then poured in a dark bottle and kept for further analysis.
Soxhlet extraction is a method used to extract non-volatile oils by using different types of solvents ranging from polar solvents to non-polar ones. The plant sample is crushed in to a fine powder and then enveloped in a filter paper thimble. This thimble is placed in the soxhlet extractor. The solvent(s) to be sued for the extraction process is put in a round bottomed flask which is fitted with the soxhlet extractor. The solvent in the round-bottomed flask is heated electrically. When the solvent reaches its boiling point, it evaporates and the vapour goes up through the soxhlet extractor up to the condenser. There, it condenses back to liquid form and drips into the soxhlet extractor where the thimble containing the plant sample is found. Once the extractor is full, the solvent along with some extract from the plant sample drains back to the round-bottomed flask. The process is allowed to repeat until the colour of the solvent in the soxhlet extractor and in the round-bottomed flask is approximately the same. The mixture; that is the solvent and the oil extract, is then concentrated using a rotary evaporator.
1.2 Secondary Metabolites in Plants
Plant secondary metabolites are a diverse group of molecules that are involved in the adaptation of plants to their environment but are not part of the primary biochemical pathways of cell growth and reproduction(Harinder P.S.Makkar, P.Siddhuraju, & Klaus Becker 2007b). The production of these compounds is dependent on the surrounding environment of the plants and the conditions under which they are developing. These compounds are involved in defence against herbivores and pathogens, chemical inhabitation of competing plants species (allelopathy) and have many more functions depending on the situation in which they are being produced(Harinder P.S.Makkar, P.Siddhuraju, & Klaus Becker 2007b).
The use of natural products (secondary metabolites), especially from plants, for healing, is as ancient and universal as medicine itself. Natural products played a prominent role in traditional medicine systems, such as Chinese, Ayurveda, and Egyptian. Nature has been a source of therapeutic agents for thousands of years, and an impressive number of modern drugs have been derived from natural sources, many based on their use in traditional medicine. According to the World Health Organisation (WHO), 75% of people still rely on plant-based traditional medicines for primary health care globally. Additionally, a number of top selling drugs have been developed from natural products or secondary metabolites; such as morphine from the plant Papaver somniferum. These secondary metabolites are also used in the natural pharmaceutical industry as well as in traditional medicine programs in the form of food supplements and nutraceuticals. (Satyajit D.Sarker, Zahid Latif, & Alexander I.Gray 2006)
The main secondary metabolites which are found in plants are coumarins, steroids, terpenes, anthraquinones, tannins, saponins, alkaloids, phenols, flavonols and leucoanthocyanins.
Coumarins are naturally occurring benzopyrene derivatives. It has been found in 150 plant species in more than 30 families (David Hoffman, FNIMH, & AHG 2003b). There are 3 major classes of coumarins:
Hydroxycoumarins; e.g. umbelliferone, esculetin
Furanocoumarins; e.g. angelicin
Pyranocoumarins; e.g. psoralen(David Hoffman, FNIMH, & AHG 2003b)
The most widespread plant coumarin is the parent compound, coumarin itself. It occurs in over twenty-seven plant families and is commonly found in many grasses and fodder crops. It is also present as the sweet smelling volatile compound which is often released from new mown hay(J.B.Harborne 1998b).
Coumarin is an important raw material in the fragrance industry and is widely used in hand soaps, detergents, lotions and perfumes. It is often associated in the perfume industry with herbaceous odours and is also used as an odour enhancer to achieve a long lasting effect when combined with natural essential oils such as lavender, citrus and rosemary. Coumarins are also used in the electroplating industry; more specifically in the automotive area (Paul M.Boisde & Walter C.Meuly 2000).
Terpenes or terpenoids are a large group of oily compounds composed of isoprene units (Caroline Bowsher, Martin Steer, & Alyson Tobin 2008c). They can be defined as a unique group of hydrocarbon-based natural products possessing a structure derived from isoprene (K.G.Ramawat, S.Dass, & Meetha Mathur 2009).
Steroids from part of the triterpene class and are in fact modified triterpenes. They are pharmacologically active and are very important as hormones, coenzymes and provitamins.
Anthraquinones make the largest group of naphthoquinones which are dark yellow pigments in the quinine family having a range of pharmacological properties. Anthraquinones are usually red or purple rather yellow in colour. They are widely distributed in several pant families, especially in the Fabaceae, Liliaceae, Polygonaceae and Rhamnaceae. The anthraquinones contain many important laxatives such as aloe-emodin from the Aloe species (David Hoffman, FNIMH, & AHG 2003b).
Tannins are polyphenolic compounds which are classified into two broad groups:
Hydrolysable tannins (Harinder P.S.Makkar, P.Siddhuraju, & Klaus Becker 2007b)
Condensed tannins also known as flavolans or proanthocyanidins occurs mostly in ferns and gymnosperms. They are also widespread in the angiosperms, especially in the woody plants (J.B.Harborne 1998a).
Hydrolysable tannins are mixed polymers of gallic acids (gallotannins) and ellagic acids (ellagitannins). They are more readily hydrolysed than the proanthocyanidins or condensed tannins (Caroline Bowsher, Martin Steer, & Alyson Tobin 2008b). Tannins have a variety of uses. They are mixed with iron salts and used in the manufacture of ink. They are also used in the process of tanning hides into leather.
Saponins are glycosidic triterpenoids widely found in the plant kingdom (Peter M.Bramley 1997). They represent the largest group of terpenoid poisons (Caroline Bowsher, Martin Steer, & Alyson Tobin 2008c). The steroid or triterpene (non polar) groups along with the polar groups (sugar) found in saponins provide the latter with strong surface-active properties. Those properties are responsible for the many adverse and beneficial aspects of saponins. The primary biological effect of saponins is the interaction with cellular and membrane components and saponins are thus able to haemolyse red blood cells (Harinder P.S.Makkar, P.Siddhuraju, & Klaus Becker 2007c). Saponins also have soap-like properties and are usually detected by their ability to cause foaming (J.B.Harborne 1998c). Saponins are known to occur in several plant species, including ivy (Hedera helix) and the horse chestnut tree (Aesculus hippocastanum) (Caroline Bowsher, Martin Steer, & Alyson Tobin 2008c).
Alkaloids are a diverse group of chemicals that are mostly synthesised from amino acid precursors (Caroline Bowsher, Martin Steer, & Alyson Tobin 2008a) and may be by-products of nitrogen metabolism in plants (A C Dutta 1964). Around 5500 alkaloids are known and they comprise the largest single class of secondary plant substances (Harinder P.S.Makkar, P.Siddhuraju, & Klaus Becker 2007a).
Alkaloids serve a range of functions in plants as poisons, feeding deterrents, antimicrobial defences and germination inhibitors (Caroline Bowsher, Martin Steer, & Alyson Tobin 2008a). The important families in which alkaloids occur are Apocynaceae, Papaveraceae, Fabaceae, Ranunculaceae, Rubiaceae, Rutaceae and Solanaceae as well as in some lower plants and fungi. They may be present systematically in the whole plants and may accumulate in large amounts in specific organs such as roots (aconite in belladonna), stem bark (cinchona in pomegranate) and seeds (nux vomica in Areca)(K.G.Ramawat, S.Dass, & Meetha Mathur 2009).
Alkaloids are nearly always poisonous but when used in appropriate doses, many of them possess medicinal properties. For example:
Quinine obtained from the bark of Cinchona officinalis is an effective antimalarial drug.
The alkaloids vincristine and vinblastine extracted from Catharanthus roseus, have anticancer properties and are particularly effective against childhood leukemia and Hodgkinââ‚¬â„¢s disease.
Morphine and codeine from Papaver somniferum are very effective pain-kilers.
Sanguinarine which has antibacterial and antiplaque properties is obtained from Eschscholzia californica and is used in toothpastes (Caroline Bowsher, Martin Steer, & Alyson Tobin 2008a).
Phenols are found in the class of phenolics compounds and is the simplest form of a plant phenolics compound (Caroline Bowsher, Martin Steer, & Alyson Tobin 2008b). Phenolics acids are ubiquitous in plants but free phenols are rare in occurrence (David Hoffman, FNIMH, & AHG 2003a). Hydroquinone is probably the most widely distributed among the different plants whereas the others such as catechol, orcinol, phloroglucinol and pyrogallol have been reported to occur in only a few sources (J.B.Harborne 1998a).
Phenols such as arbutin possess antimicrobial properties and salicylates are anti-inflammatory in nature. Guaiacol is used as an expectorant in veterinary practices and in humans; it is applied externally to treat eczema and other skin diseases (David Hoffman, FNIMH, & AHG 2003a).
Anthocyanins and Flavonols
The anthocyanins are the most important and widespread group of colouring matters in plants. They are responsible for nearly all the pink, scarlet, red, mauve, violet and blue colours in the petals, leaves and fruits of higher plants. The anthocyanins are chemically based on cyanidin (J.B.Harborne 1998a) and are soluble in water and alcohol (A C Dutta 1964).
Flavonols are vey widely distributed in plants both as co-pigments to anthocyanins in petals and also in leaves of higher plants. They occur most frequently in glycosidic combination. The most common flavonols which occur in plants are kaempferol, quercetin and myricetin (J.B.Harborne 1998a).
2.1 Sample collection:
The plant samples of Vanilla species were taken at St Aubin Sugar Estate which is found in the south of Mauritius. The samples were collected in the morning at around 9.00am. The samples consisted of:
4 green pods
4 cured pods
Approximately 100 green leaves
Approximately 100 yellow leaves which are a result of nitrogen deficiency in the plants.
The vanilla plants were cultivated under controlled conditions in a greenhouse. (Fig 1)
The environmental conditions were noted. The conditions were maintained humid for the optimum growth of the plants.
The plant leaves were cut randomly from the several vanilla plants found in the greenhouse and bagged for further use.
At the university, the samples were wrapped in a humidified newspaper (Fig 2) and kept in the refrigerator at 4oC.
Figure 1: Vanilla plants grown in greenhouse at St Aubin Sugar Estate
The experiments started two days after the collection on Monday 20th of September 2010.
Figure 2: The collected samples wrapped in humidified newspaper
2.2 Extraction process
Brief Overview of the solvent system used:
Extraction by decantation process:
The plant samples (green and yellow leaves, green and cured pods) were cut up in little pieces so as to increase the rate of extraction of the metabolites from the plant materials. The seeds being already powder-like were used as such.
The samples were then individually weighed.
These samples were then soaked in different organic solvents; namely dichloromethane, methanol and a mixture of both at a ratio of 1:1.
The conical flasks were then stoppered using cotton plugs and aluminium foil. The flasks were also wrapped in paper to prevent light from reaching the plant-solvent mixture inside.
All the flasks were then placed on an orbital shaker for a minimum period of 48h. The constant shaking also increases the rate of extraction of the metabolites.
Afterwards, the different decanting mixtures were filtered under vacuum to separate the plant material (leaves, pods or seeds) from the mixture of solvent and secondary metabolites.
The filtrates were then concentrated using the rotary evaporator to separate the plant extracts from the solvent(s).
The weights of the extracts were taken.
The extracts and the solvents were stored separately in dark bottles at room temperature for further use.
Table 1 below include details on the mass of samples used and volume of solvent used for the extraction process using decantation. Table 2 includes details on the concentration of the extracts obtained.
Table 1: Mass of samples and volume of solvent(s) used for Vanilla extraction by decantation
Plant Parts Used
Mass Of Sample/g
Yellow leaves (N deficient)
Vanilla Green Seeds
Vanilla Green Pods
Vanilla Cured Pods
Vanilla Cured Seeds
Table 2: Separation and concentration of the extracts obtained by decantation method
Plant Part Used
Mass of Empty Flask/g
Mass of Empty Flask + Extract/g
Mass of extract only/g
Vanilla Green Leaves
Dichloromethane + Methanol
Vanilla Yellow Leaves
Dichloromethane + Methanol
Vanilla Green Pods
Dichloromethane + Methanol
Vanilla Green Seeds
Dichloromethane + Methanol
Vanilla Cured Seeds
Dichloromethane + Methanol
Vanilla Cured Pods
Dichloromethane + Methanol
Extraction using soxhlet apparatus
The sample has been previously oven-dried at about 70oC and then grinded in a blender until a fine powder was obtained.
Approximately 5g of the sample was weighed and then packed and tied in a filter paper. The small packet was then placed in the soxhlet apparatus.
The soxhlet extractor was then placed onto a round bottom flask.
Approximately 250ml of the solvent was then poured into the extractor and allowed to drain into the round bottom flask. 50ml of the solvent was then added in the extractor. In all, 300ml of solvent was used for the extraction.
The reflux condenser was placed on top of the soxhlet extractor and the round bottom flask containing the solvent is heated by electrical means until the solvent starts to boil.
The extraction is allowed to continue for about 30 mins and then the solvent is removed from the round bottom flask.
The solvent is stored in a dark-coloured bottle and sealed for further use.
2.3 Phytochemical Screening:
Test tube tests were used to detect the presence of coumarins, tannins, saponins, anthraquinones, leucoanthocyains and flavonols. Thin-layer chromatography techniques were used to detect the presence of phenols, alkaloids and steroids/terpenes.
The test for each phytochemical is detailed below.
Test for coumarins (Crowden, 1969)
To a small amount of the crude plant extract, some concentrated ammonia solution is added. Some of this solution is smeared onto a microscope slide and it is viewed under long wave UV light (366 nm). A green fluorescence indicates the presence of coumarins in the extract being tested.
Test for tannins (Hampton-Hoch, 1933)
A small amount of the crude plant extract is washed with petroleum ether. The mixture is then filtered. To the filtrate, an equal amount of freshly prepared ferric chloride and potassium hexacyanoferrate (III) is added drop wise. If hydrolysable tannins are present a blue precipitate will be obtained. However, if a green colour is obtained, then condensed tannins are present in the extract.
Test for saponins (Harborne, 1973)
Water is added to about 0.5g of some the plant material (previously dried and crushed) and the wole solution is kept at 100oC for approximately 5 minutes. Then the solution is cooled and shaken vigorously. If there is the formation of froth above the solution lasting for about 30 minute, then it means that saponins are present in the extract.
Test for anthraquinones (Harborne, 1973)
Some of the concentrated plant extract is dissolved in warm distilled water. The solution is then filtered and the filtrate is extracted with benzene. Ammonia solution is added to the extract and the mixture is shaken. If a red colour develops in the lower aqueous layer of the mixture, then it can be concluded that anthraquinones are present in the extract.
Test for leucoanthocyanins and flavonols (Harborne et al., 1975)
The crude extract is washed with petroleum ether until all of the pigments present are extracted. Ethanol is then added to the washed material and the whole mixture is then filtered. Concentrated hydrochloric acid is added to the filtrate. The filtrate is then separated in half in separate test tubes. One of the tube containing the mixture of the filtrate with concentrated hydrochloric acid is placed in a hot water bath and allowed to stand for about 30 minutes. If a red colouration is observed in the test tube, then it indicates a positive test for the presence of flavonols. Magnesium turnings are added to the other test tube and allowed to stand for ten minutes. A red colouration in this test tube indicates the presence of leucoanthocyanins in the extract.
Test for alkaloids, phenols and steroids/terpenes using thin-layer chromatography.
Alkaloids, phenols and steroids/terpenes are tested for using the thin-layer chromatography technique. Specific solvent systems are prepared for each phytochemical by mixing different solvents together. A drop of the extract to be tested is placed on a TLC plate. The plate is then placed in a beaker containing the mixture of solvents. These solvent systems; due to their polarity, will enable the compounds in the extract being tested to separate. The TLC plate is allowed to stand in the solvent mixture until the solvents have separated the compounds. After that, the plate is dried and then sprayed with a specific staining reagent. Just like the solvents systems, the staining reagent varies depending on the phytochemical the extract is being tested for.
Table 3 below includes details on the solvent systems used ; the volume of solvents used to prepare each of them, the staining reagent used for the detection of each of the phytochemical mentioned above and the results expected for the presence of these phytochemicals to be confirmed.
Table 3: Solvents used to test for each phytochemical, staining reagent used and the expected results for the presence of alkaloids, phenols or steroids/terpenes.
Staining Reagent Used
Orange-brwn spots on a yellow background
Results and Discussion
Table 4 below shows the results for the phytochemical tests performed on the extracts obtained by decantation from the Vanilla plant samples.
The extracts obtained by decantation were found to contain mostly coumarins, tannins, terpenes, phenols and alkaloids. They also have traces of flavonols and leucoanthocyanins were found in traces only in the green vanilla pods. Leucoanthocyanins were absent in the green seeds, leaves and also in the nitrogen-deficient leaves. Anthraquinones and saponins were not present at all in the healthy and nitrogen-deficient leaves and neither in the seeds and in the pods.
Alkaloids are known to possess antimicrobial properties as well as analgesic properties. Since the vanilla leaves, pods and seeds contain alkaloids, vanilla may be presumed to possess the above mentioned properties. Similarly, since phenols, steroids, terpenes, tannins and coumarins are present in the vanilla leaves, seeds and pods, vanilla can be assumed to possess the properties conferred by these phytochemicals to plants.
The healthy leaves and the nitrogen deficient leaves showed no difference in their phytochemical constitution. Since, the nitrogen deficiency was fixed very rapidly in the greenhouse by supplementing the plants with fertilisers; the deficiency did not have any lasting effect on the plantââ‚¬â„¢s metabolism.
The vanilla green pods contain trace amounts of coumarins whereas the seeds were positive for the presence of coumarins. The pods also contain more condensed tannins than the seeds which have more hydrolysable tannins. Phenols and alkaloids were equally present in both seeds and pods and there were steroids and terpenes in the vanilla seeds and traces of them in the vanilla pods. Flavomols while completely absent in the seeds, were present in the pods. The pods also contained traces of leucoanthocyanins which may be the reason for their deep brown colour but the phytochemical was completely absent in the seeds.
Based on the preliminary results obtained so far, vanilla plant, like many other orchids, shows a great potential for medicinal properties. Thus, instead of only its seeds being used in commerce, its leaves or some other part may be used for its medicinal properties.