Vanilla is a vine-like tropical orchid, from the Orchidaceous household. Among the orchids, apart from the cosmetic orchids ; grown for their flowers, vanilla is the lone genus of economic importance. Vanilla is chiefly cultivated for its sweet olfactory property and delicate spirits, much prized for assorted dishes all over the universe. It is known to be the 2nd most expensive flavorer spice after Crocus sativus in the universe.

Vanilla originates from the tropically-humid parts of Mexico and Central America, every bit good as the woods of South America..

There are about 110 species of vanilla which have been reported but among these, merely three species are cultivated for commercial usage. These are Vanilla planifolia ( Vanilla fragrans ) , Vanilla pompana and Vanilla tahitensis.

Cultivation of Vanilla pompana and Vanilla tahitensis are merely done on occasion as they yield inferior quality merchandises as compared to Vanilla planifolia.

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1.1 – Structure of Vanilla Plants

The vanilla workss largely grow on the borders of the tropical woods. Vanilla workss consist of long vines of about 35 meters, and they have alternate foliages spread along its length. The foliages are short, oblong and dark viridity in coloring material. Vanilla foliages are besides thick, leathery and fleshy.

The flowers of the works are green, white, pick or greenish-yellow. These flowers are intersexs ; intending that they possess both male and female variety meats. Therefore, pollenation of the flower simply requires the transportation of pollen from the anther to the stigma. However, the peculiar cannular form of the flower and the presence of rostellum which is a membrane dividing the stamen and the stigma in the flower, prevent the flower from being self-fertilized. Merely one specific pollinator is known to be for Vanilla planifolia and they are bees of the genus Melapona which can last merely in Central America or more specifically in Mexico. Therefore, all vanilla workss are nowadays hand-pollinated, besides known as unreal pollenation method.

When the flower is pollinated, the fruit ( cod ) is formed and these cods are normally known every bit beans as they are cylindrical and pendular shaped. The cods can achieve a length of about 10-15 centimeter and are green in coloring material when fresh and are harvested about eight to nine months after blooming. The cods are considered to be mature when the green coloring material alterations to blanch yellow.1 After that, the cods are cured, a procedure during which the olfactory property and the spirit of the vanilla cods are enhanced by a procedure of agitation of the heavy portion of the cod. The bring arounding procedure alterations the coloring material of the cods to dark brown or black.

1.2 – Vanillin

A substance known as vanillin ( C8H8O3 ) is what procures the gustatory sensation and olfactory property of the cods along with niceties of several other compounds. However, since natural vanillin is scarce and is besides really expensive, man-made vanillin is synthesized from guaiacol ; which is a of course happening phenolic compound derived from wood creosote or lignin ; a wood component.

Natural vanillin is really expensive as compared to man-made vanillin but the quality defined chiefly by the gustatory sensation and olfactory property of the natural vanillin is in no manner comparable to that of the man-made 1.

1.3 aa‚¬ ” Uses of vanilla

Vanilla is used a batch in bakeshop points, soft drinks, cocoa spirits, ice-creams and assorted other confectioneries. It is used to secure the olfactory property to the merchandises. Vanilla is besides used in personal attention merchandises such as aromas, picks and soaps every bit good as detergents for the aroma it procures.

LITERATURE REVIEW

1.1 Extraction of Secondary Metabolites: Methods

Extraction is the procedure in which pre-treated works samples are used along with peculiar dissolvers so as to take the metabolites found in the works tissues. The dissolvers used are chosen in such a manner that they extract the greater portion of the metabolites which are themselves attracted to certain types of dissolvers. For illustration, the solvent hexane normally extracts flavonoids and alkaloids from works tissues. These metabolites are dissolved in the dissolver and pure infusions of those metabolites are obtained by taking the dissolvers from the extract-solvent mixture utilizing a rotary evaporator.

The two more normally used methods are decantation and the soxhlet extraction.

Decantation

Decantation is a method used for the extraction of fixed oils. The clean works stuff is first cut and so macerated so as to increase the extraction rate. It is so placed in a conelike flask and submerged with the dissolver ( s ) . The flask is stoppered with a cotton stopper and aluminum foil and is placed onto an orbital shaker for a lower limit of 48hrs so as to pull out the maximal sum of secondary metabolites. The mixture is so filtered and the filtrate is concentrated in a rotary evaporator to obtain a pure infusion without the dissolver. The infusion is so poured in a dark bottle and maintain for farther analysis.

Soxhlet Extraction

Soxhlet extraction is a method used to pull out non-volatile oils by utilizing different types of dissolvers runing from polar dissolvers to non-polar 1s. The works sample is crushed in to a all right pulverization and so enveloped in a filter paper thimble. This thimble is placed in the soxhlet extractor. The dissolver ( s ) to be sued for the extraction procedure is put in a unit of ammunition bottomed flask which is fitted with the soxhlet extractor. The dissolver in the round-bottomed flask is heated electrically. When the dissolver reaches its boiling point, it evaporates and the vapor goes up through the soxhlet extractor up to the capacitor. There, it condenses back to liquid signifier and trickles into the soxhlet extractor where the thimble incorporating the works sample is found. Once the extractor is full, the dissolver along with some infusion from the works sample drains back to the round-bottomed flask. The procedure is allowed to reiterate until the coloring material of the dissolver in the soxhlet extractor and in the round-bottomed flask is about the same. The mixture ; that is the dissolver and the oil infusion, is so concentrated utilizing a rotary evaporator.

1.2 Secondary Metabolites in Plants

Plant secondary metabolites are a diverse group of molecules that are involved in the version of workss to their environment but are non portion of the primary biochemical tracts of cell growing and reproduction ( Harinder P.S.Makkar, P.Siddhuraju, & A ; Klaus Becker 2007b ) . The production of these compounds is dependent on the environing environment of the workss and the conditions under which they are developing. These compounds are involved in defense mechanism against herbivores and pathogens, chemical inhabitancy of viing workss species ( allelopathy ) and have many more maps depending on the state of affairs in which they are being produced ( Harinder P.S.Makkar, P.Siddhuraju, & A ; Klaus Becker 2007b ) .

The usage of natural merchandises ( secondary metabolites ) , particularly from workss, for healing, is as antediluvian and cosmopolitan as medical specialty itself. Natural merchandises played a outstanding function in traditional medical specialty systems, such as Chinese, Ayurveda, and Egyptian. Nature has been a beginning of curative agents for 1000s of old ages, and an impressive figure of modern drugs have been derived from natural beginnings, many based on their usage in traditional medical specialty. Harmonizing to the World Health Organisation ( WHO ) , 75 % of people still rely on plant-based traditional medical specialties for primary wellness attention globally. Additionally, a figure of top merchandising drugs have been developed from natural merchandises or secondary metabolites ; such as morphia from the works Papaver somniferum. These secondary metabolites are besides used in the natural pharmaceutical industry every bit good as in traditional medical specialty plans in the signifier of nutrient addendums and nutraceuticals. ( Satyajit D.Sarker, Zahid Latif, & A ; Alexander I.Gray 2006 )

The chief secondary metabolites which are found in workss are coumarins, steroids, terpenes, anthraquinones, tannic acids, saponins, alkaloids, phenols, flavonols and leucoanthocyanins.

Coumarins

Coumarins are of course happening benzopyrene derived functions. It has been found in 150 works species in more than 30 households ( David Hoffman, FNIMH, & A ; AHG 2003b ) . There are 3 major categories of coumarins:

Hydroxycoumarins ; e.g. umbelliferone, esculetin

Furanocoumarins ; e.g. angelicin

Pyranocoumarins ; e.g. psoralen ( David Hoffman, FNIMH, & A ; AHG 2003b )

The most widespread works coumarin is the parent compound, coumarin itself. It occurs in over 27 works households and is normally found in many grasses and fresh fish harvests. It is besides present as the sweet smelling volatile compound which is frequently released from new mown hay ( J.B.Harborne 1998b ) .

Coumarin is an of import natural stuff in the aroma industry and is widely used in manus soaps, detergents, lotions and aromas. It is frequently associated in the aroma industry with herbaceous smells and is besides used as an odour foil to accomplish a long lasting consequence when combined with natural indispensable oils such as lavender, citrous fruit and rosemary. Coumarins are besides used in the electroplating industry ; more specifically in the automotive country ( Paul M.Boisde & A ; Walter C.Meuly 2000 ) .

Steroids/Terpenes

Terpenes or terpenoids are a big group of oily compounds composed of isoprene units ( Caroline Bowsher, Martin Steer, & A ; Alyson Tobin 2008c ) . They can be defined as a alone group of hydrocarbon-based natural merchandises possessing a construction derived from isoprene ( K.G.Ramawat, S.Dass, & A ; Meetha Mathur 2009 ) .

Steroids from portion of the triterpene category and are in fact modified triterpenes. They are pharmacologically active and are really of import as endocrines, coenzymes and provitamins.

Anthraquinones

Anthraquinones make the largest group of vitamin Ks which are dark xanthous pigments in the quinine household holding a scope of pharmacological belongingss. Anthraquinones are normally ruddy or violet instead xanthous in coloring material. They are widely distributed in several pant households, particularly in the Fabaceae, Liliaceae, Polygonaceae and Rhamnaceae. The anthraquinones contain many of import laxatives such as aloe-emodin from the Aloe species ( David Hoffman, FNIMH, & A ; AHG 2003b ) .

Tannins

Tannins are polyphenolic compounds which are classified into two wide groups:

Condensed tannic acids

Hydrolysable tannic acids ( Harinder P.S.Makkar, P.Siddhuraju, & A ; Klaus Becker 2007b )

Condensed tannic acids besides known as flavolans or proanthocyanidins occurs largely in ferns and gymnosperms. They are besides widespread in the flowering plants, particularly in the woody workss ( J.B.Harborne 1998a ) .

Hydrolysable tannic acids are assorted polymers of Gallic acids ( gallotannins ) and ellagic acids ( ellagitannins ) . They are more readily hydrolysed than the proanthocyanidins or condensed tannic acids ( Caroline Bowsher, Martin Steer, & A ; Alyson Tobin 2008b ) . Tannins have a assortment of utilizations. They are assorted with Fe salts and used in the industry of ink. They are besides used in the procedure of tanning fells into leather.

Saponins

Saponins are glycosidic triterpenoids widely found in the works land ( Peter M.Bramley 1997 ) . They represent the largest group of terpenoid toxicants ( Caroline Bowsher, Martin Steer, & A ; 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 belongingss. Those belongingss are responsible for the many inauspicious and good facets of saponins. The primary biological consequence of saponins is the interaction with cellular and membrane constituents and saponins are therefore able to haemolyse ruddy blood cells ( Harinder P.S.Makkar, P.Siddhuraju, & A ; Klaus Becker 2007c ) . Saponins besides have soap-like belongingss and are normally detected by their ability to do foaming ( J.B.Harborne 1998c ) . Saponins are known to happen in several works species, including Hedera helix ( Hedera spiral ) and the Equus caballus chestnut tree ( Aesculus hippocastanum ) ( Caroline Bowsher, Martin Steer, & A ; Alyson Tobin 2008c ) .

Alkaloids

Alkaloids are a diverse group of chemicals that are largely synthesised from amino acid precursors ( Caroline Bowsher, Martin Steer, & A ; Alyson Tobin 2008a ) and may be byproducts of nitrogen metamorphosis in workss ( A C Dutta 1964 ) . Around 5500 alkaloids are known and they comprise the largest individual category of secondary works substances ( Harinder P.S.Makkar, P.Siddhuraju, & A ; Klaus Becker 2007a ) .

Alkaloids serve a scope of maps in workss as toxicants, feeding hindrances, antimicrobic defense mechanisms and sprouting inhibitors ( Caroline Bowsher, Martin Steer, & A ; Alyson Tobin 2008a ) . The of import households in which alkaloids occur are Apocynaceae, Papaveraceae, Fabaceae, Ranunculaceae, Rubiaceae, Rutaceae and Solanaceae every bit good as in some lower workss and Fungis. They may be present consistently in the whole workss and may roll up in big sums in specific variety meats such as roots ( aconite in deadly nightshade ) , root bark ( Peruvian bark in Punica granatum ) and seeds ( nux vomica in Areca ) ( K.G.Ramawat, S.Dass, & A ; Meetha Mathur 2009 ) .

Alkaloids are about ever toxicant but when used in appropriate doses, many of them possess medicative belongingss. For illustration:

Quinine obtained from the bark of Cinchona officinalis is an effectual antimalarial drug.

The alkaloids Oncovin and Velban extracted from Catharanthus roseus, have anticancer belongingss and are peculiarly effectual against childhood leukaemia and Hodgkinaa‚¬a„?s disease.

Morphine and codeine from Papaver somniferum are really effectual pain-kilers.

Sanguinarine which has bactericide and antiplaque belongingss is obtained from Eschscholzia californica and is used in toothpastes ( Caroline Bowsher, Martin Steer, & A ; Alyson Tobin 2008a ) .

Phenols

Phenols are found in the category of phenoplasts compounds and is the simplest signifier of a works phenoplasts compound ( Caroline Bowsher, Martin Steer, & A ; Alyson Tobin 2008b ) . Phenolic resins acids are omnipresent in workss but free phenols are rare in happening ( David Hoffman, FNIMH, & A ; AHG 2003a ) . Hydroquinone is likely the most widely distributed among the different workss whereas the others such as catechol, orcinol, phloroglucinol and pyrogallic acid have been reported to happen in merely a few beginnings ( J.B.Harborne 1998a ) .

Phenols such as arbutin possess antimicrobic belongingss and salicylates are anti-inflammatory in nature. Guaiacol is used as an expectorator in veterinary patterns and in worlds ; it is applied externally to handle eczema and other tegument diseases ( David Hoffman, FNIMH, & A ; AHG 2003a ) .

The soxhlet extractor was so placed onto a unit of ammunition underside flask.

Approximately 250ml of the dissolver was so poured into the extractor and allowed to run out into the unit of ammunition underside flask. 50ml of the dissolver was so added in the extractor. In all, 300ml of dissolver was used for the extraction.

The reflux capacitor was placed on top of the soxhlet extractor and the unit of ammunition underside flask incorporating the dissolver is heated by electrical agencies until the dissolver starts to boil.

The extraction is allowed to go on for approximately 30 mins and so the dissolver is removed from the unit of ammunition underside flask.

The dissolver is stored in a dark-colored bottle and sealed for farther usage.

2.3 Phytochemical Screening:

Test tubing trials were used to observe the presence of coumarins, tannic acids, saponins, anthraquinones, leucoanthocyains and flavonols. Thin-layer chromatography techniques were used to observe the presence of phenols, alkaloids and steroids/terpenes.

The trial for each phytochemical is detailed below.

Trial for coumarins ( Crowden, 1969 )

To a little sum of the petroleum works infusion, some concentrated ammonia solution is added. Some of this solution is smeared onto a microscope slide and it is viewed under long moving ridge UV visible radiation ( 366 nanometer ) . A green fluorescence indicates the presence of coumarins in the infusion being tested.

Trial for tannic acids ( Hampton-Hoch, 1933 )

A little sum of the petroleum works infusion is washed with crude oil quintessence. The mixture is so filtered. To the filtrate, an equal sum of newly prepared ferrous chloride and K hexacyanoferrate ( III ) is added bead wise. If hydrolysable tannic acids are present a bluish precipitate will be obtained. However, if a green coloring material is obtained, so condensed tannic acids are present in the infusion.

Trial for saponins ( Harborne, 1973 )

Water is added to about 0.5g of some the works stuff ( antecedently dried and crushed ) and the wole solution is kept at 100oC for about 5 proceedingss. Then the solution is cooled and shaken smartly. If there is the formation of foams above the solution lasting for approximately 30 minute, so it means that saponins are present in the infusion.

Trial for anthraquinones ( Harborne, 1973 )

Some of the concentrated works infusion is dissolved in warm distilled H2O. The solution is so filtered and the filtrate is extracted with benzine. Ammonia solution is added to the infusion and the mixture is shaken. If a ruddy coloring material develops in the lower aqueous bed of the mixture, so it can be concluded that anthraquinones are present in the infusion.

Trial for leucoanthocyanins and flavonols ( Harborne et al. , 1975 )

The petroleum infusion is washed with crude oil quintessence until all of the pigments present are extracted. Ethanol is so added to the washed stuff and the whole mixture is so filtered. Concentrated hydrochloric acid is added to the filtrate. The filtrate is so separated in half in separate trial tubing. One of the tubing incorporating the mixture of the filtrate with concentrated hydrochloric acid is placed in a hot H2O bath and allowed to stand for approximately 30 proceedingss. If a ruddy color is observed in the trial tubing, so it indicates a positive trial for the presence of flavonols. Magnesium turnings are added to the other trial tubing and allowed to stand for 10 proceedingss. A ruddy color in this trial tubing indicates the presence of leucoanthocyanins in the infusion.

Trial for alkaloids, phenols and steroids/terpenes utilizing thin-layer chromatography.

Alkaloids, phenols and steroids/terpenes are tested for utilizing the thin-layer chromatography technique. Specific solvent systems are prepared for each phytochemical by blending different dissolvers together. A bead of the infusion to be tested is placed on a TLC home base. The home base is so placed in a beaker incorporating the mixture of dissolvers. These solvent systems ; due to their mutual opposition, will enable the compounds in the infusion being tested to divide. The TLC home base is allowed to stand in the solvent mixture until the dissolvers have separated the compounds. After that, the home base is dried and so sprayed with a specific staining reagent. Just like the dissolvers systems, the staining reagent varies depending on the phytochemical the infusion is being tested for.

The infusions obtained by decantation were found to incorporate largely coumarins, tannic acids, terpenes, phenols and alkaloids. They besides have hints of flavonols and leucoanthocyanins were found in hints merely in the green vanilla cods. Leucoanthocyanins were absent in the green seeds, foliages and besides in the nitrogen-deficient foliages. Anthraquinones and saponins were non present at all in the healthy and nitrogen-deficient foliages and neither in the seeds and in the cods.

Alkaloids are known to possess antimicrobic belongingss every bit good as analgetic belongingss. Since the vanilla leaves, cods and seeds contain alkaloids, vanilla may be presumed to possess the above mentioned belongingss. Similarly, since phenols, steroids, terpenes, tannic acids and coumarins are present in the vanilla leaves, seeds and cods, vanilla can be assumed to possess the belongingss conferred by these phytochemicals to workss.

The healthy foliages and the N deficient leaves showed no difference in their phytochemical fundamental law. Since, the N lack was fixed really quickly in the nursery by supplementing the workss with fertilizers ; the lack did non hold any permanent consequence on the plantaa‚¬a„?s metamorphosis.

The vanilla green cods contain trace sums of coumarins whereas the seeds were positive for the presence of coumarins. The cods besides contain more condensed tannic acids than the seeds which have more hydrolysable tannic acids. Phenols and alkaloids were every bit present in both seeds and cods and there were steroids and terpenes in the vanilla seeds and hints of them in the vanilla cods. Flavomols while wholly absent in the seeds, were present in the cods. The cods besides contained hints of leucoanthocyanins which may be the ground for their deep brown coloring materials but the phytochemical was wholly absent in the seeds.

Based on the preliminary consequences obtained so far, vanilla works, like many other orchids, shows a great potency for medicative belongingss. Therefore, alternatively of merely its seeds being used in commercialism, its foliages or some other portion may be used for its medicative belongingss.

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