Vanillin (4-hydroxy-3-methoxybenzaldehyde) is an important flavoring agent mostly used in beverages, pharmaceutical industries, food products, etc. Naturally present as a vanillin glucoside in vanilla pods and used as an intermediate in the synthesis of some drugs. Vanillin possesses antimicrobial and antioxidant properties.
In the past, production of vanillin is very expensive and a very long process. Vanillin was obtained from the oxidation of lignin, or from ferulic acid pathway or some other pathways. The chemical production of vanillin from various methods had been described.
The method used in this research is High selectivity in the oxidation of Mandelic acid derivatives and in O-Methylation of Protocatechualdehyde. The starting material used here was catechol and nowadays this method is used for the industrial production of vanillin. The method used was tiresome but produces vanillin in good yield.
The analysis of obtained vanillin was done by using Thin Layer Chromatography and Infrared Spectroscopy. Infrared spectrum of obtained product and reference spectrum of vanillin were compared. The similarity of melting points of the obtained product and vanillin from literatures shows that the product obtained can be confirmed as vanillin.
Introduction
Vanillin (4-hydroxy-3-methoxybanzaldehyde) is a main part of natural vanilla. It is a major flavouring agent used widely in the food and dairy products, beverages and pharmaceutical industries etc. It is an organic aromatic compound which contains three functional groups (aldehyde, phenol and ether). Vanillin is obtained from the beans or pods of Vanilla plant (Vanilla planifolia). Its origin is from the subtropical forests of Mexico and Central America. Mayan and Aztec civilizations are the first who discover the properties of vanilla. It was first extracted by Nicholas Theodore Gobley in 1858.1
Vanilla Planifolia Vanilla pods Vanilla beans
In freshly harvested vanilla pods vanillin is binds with the β-D-glycoside.
Today vanillin is used in the preparation of many pharmaceutical preparations like Papaverine, Levodopa, Levomethyldopa and antimicrobial agent Trimethoprim and also for the production of herbicides and antifoaming agents. Vanillin also has properties like antioxidants and anti-tumor. Due to its aromatic properties it is widely used in the air fresheners, perfumes, incense and candle.1
Vanillin is extracted from the vanilla beans but due to its low natural production and high demand it is prepared by various synthetic methods like chemical synthesis, enzymatic synthesis etc.1
Physiochemical properties of Vanillin:
Vanillin is a white crystalline powder which has a melting point about 820C. The purity is generally above 99.0% w/w on dried basis. Vanillin has a characteristic pleasant smell and taste for which it is widely used in the world. The boiling point of vanillin is about 1540C and its sublimation temperature is reported to be 700C. Vanillin starts to decompose at 1600C. Vanillin has a vapour pressure of 0.0022 hPa at 250C and 0.0017 hPa at 650C and saturated air has a concentration of 0.00029 % at 250C, corresponding to 18.0 mg/m3. The vapour density of vanillin is found to be 5.3 at 250C. Its apparent specific gravity is 0.6 kg/dm3. Specific gravity of vanillin is reported to be 1056 kg/m3 at 200C. Vanillin is soluble in water and its solubility increases with increasing temperature. Its solubility in water at 25°C was reported to be 10g/L. Vanillin was reported to be readily soluble in alcohol (ethanol). Also vanillin shows slight solubility in ethyl acetone, methanol, and diethyl ether. The Octanol/ Water partition coefficient was found to be 1.21 which indicates that vanillin is unlikely to bio accumulate. The pH of vanillin in water is 4.3. The phenol group of vanillin has a pKa value of 7.38. With increasing pH the molecule will lose a proton, become negatively charged and more soluble in water. Vanillin dissolves in dilute solution of alkali hydroxides.16
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Production of vanillin:
The production of vanillin is a very long and expensive process which involves large number of steps as well. The pollination of flowers has to be done manually as there is lack of natural pollinators. The flowers have to be pollinated within 24 hours to bear fruits. The vanilla beans require 10- 12 months to mature from the time of pollination. The matured vanilla beans are yellowish green, and are bitter in taste. The matured beans lack the characteristic vanilla flavour which only develops upon curing, which involves three steps.
1) Killing, the green beans are treated with variety of methods such as scalded with hot water, exposed to sun, wilted in the oven, scarred, treated with ethylene gas, or frozen to disrupt tissue integrity. The second method is the cheapest but most labour- intensive. In this step tissues completely lose their integrity, but still contains high amount of moisture which has to be removed by the sweating process. This step runs for 7-10 days, during moisture content of the beans reduced to 60- 70 %. After losing the moisture the beans turn dark brown in colour and start to develop their characteristic vanilla flavour. To reduce microbial spoilage and to concentrate the flavour, the moisture content were further reduced to 25- 30 %. After this step, the beans are stored in closed containers to reach their highest flavour content and then their conditioning is done either by hot water treatment or by sun drying.5
2) Process of curing and drying together requires 4-5 months. The cured beans pods may be covered with tiny crystals of vanillin. This coating is known as givre, which sometimes used as criterion for quality assessment.6
3) During the fermentation process, vanillin is released from its non-volatile glucoside by the action of vanilla α-glucosidase on vanillin glucoside.3 Vanillin alone is not present in the extract of V. Plantifolia, some related phenylpropanoid (C3-C6) compounds [mainly p-hydroxybenzaldehyde(8.6%), vanillic acid (4.3%), p-hydroxybenzyl methyl ether (0.9%) ] are also present which gives the unique flavour to natural vanilla.7,8,9
However, vanillin has also been found to be present in traces amount in plants like tobacco, fruits and fruit products like orange, grapefruit, and tangerine. In mango, vanillin is present both as ‘free vanillin’ and ‘vanillyl glucoside’. It is also reported to be present in elderberry juice, blueberries, orange juice, strawberries, passion fruit juice, leeches, and wines. It has also been found in diverse food products such as popcorn, extruded oat flour, apple cider brandy, commercial liquid smoke flavourings, mushrooms and chocolate.3, 10
Vanillin is added in the concentrations ranging from 1 to 26 milimolar in the food products which depend upon the nature of the product. Vanillin has a low flavor threshold value of 20µg/L in water at 20 °C.11Although more than 12000 tonnes of vanillin are produced each year only 1% of it comes from the natural sources and the rest are synthesized by chemical synthesis.12 Moreover, the high demand for vanillin and the fact that the vanillin derived from plants is relatively expensive ($1200- 1400 per kilo compared to synthetically produced which is <$15 per kilo), because of causes such as scare availability of vanilla pods, climate associated fluctuations of the harvest yields, labour-intensive cultivation, pollination, harvesting and curing of pods.13
These reasons has led to the exploration of other biotechnological routes such as the microbial production from phenolic stilbenes, lignin, eugenol, and ferulic acid.8, 11 Vanillin can be obtained from chemical synthesis, biotransformation or from degradation of waste sulphite liquors.11
What is Vanilla extract?
It is full of the sapid and odorous principles of the matured pods, and is one of the mostly used flavouring aromas. The aroma of vanilla pods developed during the fermentation process after picking. Because of the cleavage of glycosidic bonds, vanillin aromatic acids, aldehyde and alcohols are liberated. Essences are prepared by the direct extraction of the pods with aqueous ethanol or by diluting the concentrated extract.
The high cost and demand of vanilla extract lead to adulteration and blending. Extracts are adulterated by addition of flavours like natural vanillin obtained from lignin, or more tasting ethyl vanillin. Sometimes coumarin also added. It is therefore essential to control the quality of the vanillin extracts to declare them as authentic. Several methods are used for the authentication of vanillin extracts such as AOAC methods which apply GC, TLC and PC methods. But some of this methods are time consuming and laborious. As natural vanillin contains more deuterium and carbon-13 as compared to synthetic vanillin, one way could be the determination of stable isotope ratio of vanillin by mass spectroscopy. One alternative method for this is the determination of stable isotope ratio of other compounds (4- hydroxybenzaldehyde, 4-hydroxybenzoicacid) or the measurement of the site-specific natural isotope ratio of deuterium/hydrogen by nuclear magnetic resonance (SNIF-NMR).
Because vanilla extract are very complex in their composition, the analysis of components occurring in authentic vanilla extract at certain concentrations, is another way to characterize vanilla extract. Various chromatographic methods are used for the separation of vanilla extracts such as Gas Chromatography, Gas Liquid Chromatography and High Performance Liquid Chromatography.14
General method for isolation of vanillin from extract:
Vanilla extract containing approximately 0.1 g of vanillin were diluted with water or methanol/water (1:1 v/v) depending on their alcohol and sugar content, respectively. This is then extracted three times with 25 ml of diethyl ether. The combined organic extracts were concentrated on a rotating evaporator to a viscous liquid. The residue was taken up in ethanol and diluted with water to an alcohol content of less than 30%. Final volume was measured to be 7 ml. The solution was cleared by filtering through a paper filter.15
Chemical methods of synthesis of vanillin
In 1876 Reimer-Tiemann method for the extraction of vanillin was used from guaiacol with an alkaline solution of chloroform.1
In early 20th century chemists found another source of vanillin and that was lignin and after 1920s most of vanillin was obtained from lignin waste.
The classic method for the production of vanillin is by isomerisation of eugenol into isoeuginol and oxidation of isoeuginol to vanillin.1
Vanillin’s molecular formula is C8H8O3 and molecular weight is 152.1 g/mol having functional groups like ether, aldehyde and phenol. Vanillin is white to slightly yellowish in colour in the form of crystalline powder or needles, which is slightly soluble in water, freely soluble in alcohol. It has a melting point of 81-84oC2 and boiling point at 285oC and its density is 1.056 g/cm3.2
But today vanillin is synthesized by reaction of guaiacol with glyoxylic acid to obtain catechol and than vanillin. This is how we are producing a lot of vanillin today.1
In laboratories vanillin is produced from 4-hydroxybenzaldehyde.1
Biosynthetic method of natural vanillin production:
Several biosynthetic routes have been proposed for the biosynthesis of natural vanillin. In 1965, the first biosynthetic route was proposed by Zenk. He proposed a biosynthetic pathway in which vanillin was derived from ferulic acid. In this route Zenk described that firstly β- oxidation of ferulic acid leads to formation of vanilloyl-CoA which was then reduced to vanillin or alternatively can be deacylated to vanillic acid.
The formation of vanillin from ferulic acid by Zenk.
The second biosynthetic route was proposed by Funk and Brodelius in 1990 and 1992 based on the results of feeding radio labelled compounds to vanilla tissue cultures. They proposed a very complex process in which caffeic acid was first methylated at 4- position to iso-ferulic acid which was then further methylated at 3-position to produce 3,4- dimethoxycinnamic acid. It was then demethylated at the 4- position. After which glucosylation leads to the production of vanillic acid this was then reduced to form vanillin.3, 5
The formation of vanillic acid from isoferulic acid
Kanisawa et al. in 1994 proposed a very complex route which came from measurements of the levels of simple phenolic compounds and their glucosides during the time course of development of vanilla pods.5
Vanillin production by Kanisawa et al.
A very interesting mechanism was derived for formation of vanillin by Overhage et al. in 1999. In this route he described the formation of ferulic acid from eugenol and the formation of vanillin from ferulic acid.3, 5
Vanillin Production by Overhage et al.
Biotechnological methods of the production of vanillin
Enzymatic methods:
Vanillin can be obtained from vanilla pods by using preparations containing β-glucosidase which helps to release vanillin from pods. This method can be used as an alternative to conventional curing. Enzymes can also be used to generate vanillin from other plant material by biotransformation. For example: Soybean lipoxygenase produces vanillin from esters of coniferyl alcohol. Van den Heuvel et al. used Penicillium flavoenzyme to produce vanillin by biotransformation of vanillylamine and of cresol.7
Cell or tissue culture:
This technique has been used for years to produce vanillin. The benefit of this technique is that with the production of vanillin some of the different compounds can be prepared which are present in the vanilla pods. For example: vanilla cells and organs and cells of Capsicum frutescens have been successfully cultured and found to accumulate vanillin and other metabolites. But the disadvantage is that the production of vanillin is always low with these techniques. Some measures had been taken to increase the yield of vanillin by these routes. Like feeding of putative precursors, the use of hormones or elicitors, use of adsorbent such as charcoal, and adjustment of environmental culture conditions. But none of these measures had been able to produce vanillin in good yield.7
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Genetic engineering for vanillin in plants:
By use of genetic engineering a new way of vanillin production from plants had been proposed by Brodelius and Xue. They proposed to introduce an enzyme or pathway to generate vanillin from a mainstream intermediate of the plant phenylpropanoid pathway. By isolation of HCHL (4-hydroxycinnamoyl-CoA hydratase/lyase) enzyme, had raised some possibilities for vanillin production. For example: feruloyl-CoA, which is an intermediate in plant monolignol pathway, could be converted to vanillin and acetyl-CoA in a single step. Any vanillin obtained via genetic modification would not be in a free state but would be converted to its β-D-glucoside. However, once the vanillin is formed it will be very difficult to prevent it from oxidation and reduction. It requires the down regulation of the enzyme activities responsible which would had to be identified and their genes isolated. It may be possible that the enzymes will perform some other vital functions which will interfere with vanillin production.7
Pharmacokinetics of Vanillin:
Vanillin can be given orally, or by subcutaneous, intraperitoneal, and intravenous route. Metabolism of vanillin yields mainly vanillic acid and glucuronide and sulfate conjugates which are excreted in urine along with other metabolites. 94% percent of an oral dose of 100 mg/kg vanillin was excreted as urinary metabolites and unchanged vanillin within 48 hours of treatment.
Absorption: No data for absorption of vanillin is found in humans. The acute toxicity of vanillin in animals exposed orally and by intraperitoneal, subcutaneous, and intravenous injections indicates that it is absorbed by those routes. Systemic effects observed in animals following oral sub chronic treatment with vanillin further indicates that gastrointestinal absorption occurs.
Distribution: No information was found regarding the distribution of vanillin.
Metabolism: Metabolism of vanillin in humans yields vanillic acid. 100mg/kg of vanillin given to rats yields mainly glucuronide and sulfate conjugates in urine within 24 hours of treatment after 48 hours, 94% of vanillin dose was accounted for: 7 % as vanillin, 19% as vanillyl alcohol, 47% as vanillic acid, 19% as vanilloylglycine, 8% as catechol, 2% as 4-methylcatechol, 0.5% as guaiacol, and 0.6% as 4-methylguaiacol. Vanillin administered intraperitoneally to rats gave rise to several urinary metabolites, consisting mainly of vanillic acid in both free and conjugated forms, and smaller amounts of conjugated vanillin, conjugated vanillyl alcohol, and catechol.
Excretion: Vanillin given to rats either orally or intraperitoneally excretes vanillin or its metabolites in urine. 94% of an oral dose of 100 mg/kg was excreted as urinary metabolites and unchanged vanillin within 48 hours of treatment.17
Uses of Vanillin
Vanillin is used as flavoring agent in the food industry, usually in chocolate and ice- cream industry. 75% of vanillin produced is used in these two chocolate and ice-cream industries and rest of 25% is used in confectionaries and baking industry.18 Also vanillin has been found to preserve food as it is an antioxidant and antibacterial properties. Researches also shows that cattle who eat vanilla flavored food gain more weight because they eat more.18
Vanillin as an intermediate product in the synthesis of various drugs like Levodopa, L-Methyldopa, and Trimethoprim etc.18
Levodopa
L-Methyldopa
Trimethoheprim
Levodopa is mainly used in the treatment of Parkinson’s disease, a slowly progressive disease which alters the cells of substantial nigra. These cells produce a chemical called dopamine. Dopamine is given to the patient in the form of Levodopa because dopamine is not able to cross blood brain barrier.18
L-Methyldopa is used in the treatment of the hypertension and has a vasodilator effect.18
Trimethoprim is an antibiotic comes under the class of chemotherapeutic agents and it is mainly used in the treatment of the urinary tract infections.18
Anticarcinogenic properties
Karran and Durant carried out studies that prove that vanillin has Anticarcinogenic effects. These studies proves that vanillin belongs to the DNA-PK inhibitor family.19
Anticlastonegic properties
Studies carried out by Keshava et al shows that vanillin has an ability to reduce chromosomal damage caused by X-rays and UV light.20
Antimicrobial Properties
Another study carried out by Fitzgerald et al proves the strong Antimicrobial properties of the vanillin. It was discovered that during fermentation vanillin is bioconverted into vanillyl alcohol and vanillic acid but it was found that neither of these metabolites were antagonist to yeast growth.21
Antitumor Properties
Antitumor property of vanillin is proved by a study carried out by Lirdprapamongko et al on a mouse model shows that vanillin is a novel inhibitor of class 1 P13K enzymes, presence of an aldehyde group in the vanillin structure is important for the inhibition. Vanillin also shows suppression of HGF-induced cancer cell migration and to reduce angiogenesis in-vivo.22, 23
Vanillin Prodrug (MX-1520)
A paper by Zhang et al published in The British Journal of Hematology shows that a vanillin prodrug had successfully been made which reduces red blood cell sickling in rats with sickle cell mutation. Vanillin bonds to the sickle hemoglobin and inhibits cells sickling in-vitro but in case of in-vivo vanillin is degraded in the digestive tract before being functional in the body. To prevent this degradation MX-1520 was prepared as a prodrug of vanillin which shows 30 times more bioavailability then vanillin.24
Synthesis
Vanillin can be synthesized by various methods. In this project vanillin was synthesized by High selectivity in the oxidation of Mandelic acid derivatives and in O-Methylation of Protocatechualdehyde.25
Chemicals used: catechol, aluminium oxide, glyoxylic acid, sodium hydroxide solutions, ethyl acetate, copper chloride dihydrate, dichloro methane, diethyl ether, chloroform, methanol, hydrochloric acid and sodium sulphate.
Instruments and Glassware: Beakers, Conical flasks, pipettes, measuring cylinder, Watch glass, Separating funnels, Magnetic stirrer, Oil bath, and Thermometer.
Reaction for the synthesis of Vanillin:
Monomethylation of catechol (Preparation of 3, 4-dihydroxy mandelic acid).
5.00 g of catechol was dissolved in aqueous NaOH (3.21g in 55.0 ml of water) in a beaker and to it 2.04 g of Aluminium oxide was added. Then after 5 min. 7.10g glyoxylic acid was added and the reaction mixture was heated at 600C for 24 hours under vigorous stirring. Then the reaction was allowed to precipitate for 10 min. After precipitation was complete, the reaction was filtered to remove Al2O3. The obtained filter cake was washed with 20ml of NaOH solution. The basic washing water was combined with the water solution and this was acidified to pH 3- 4 with 6.0 ml of 37% HCl and extracted with ethyl acetate to recover the unreacted catechol. The aqueous solution was further acidified to pH 1-2 by 2 ml of concentrated HCl and extracted with ethyl acetate to obtained mandelic acid derivatives.25
Electrophilic Substitution of glyoxylic acid on guaiacol (Preparation of Protocatechualdehyde).
2g of 3, 4-dihydroxymandelic acid was dissolved in 140ml of ethyl acetate and separately 11.11g of Cucl2.2H2O was dissolved in 30 ml of water. These two solutions were mixed by vigorous stirring and heated for 5 hours under nitrogen atmosphere at 600C. After the 5 hour duration time the two phases separated in organic and aqueous phase. The solution was transferred to a separating funnel and the organic phase was removed. The analysis of product confirmed the conversion of mandelic acid derivatives to protocatechualdehyde. The yield of the product was calculated and analysis was done by taking TLC and IR.25
Catalytic Oxidative, Decarboxylation of VMA (Preparation of vanillin).
7.2 ml of protocatechualdehyde and 0.8 ml of NaOH were mixed and dissolved in 25 ml of water; 20 ml of dichloro methane was added to the above solution. To this solution 0.5 ml of diethyl ether was added dropwise under vigorous stirring during 1 hour at a temperature of 55-60oC. The reaction was run further for 4 hour at the same temperature. This solution was transferred to a separating funnel and two phases were separated. The aqueous phase was extracted with 2×25 ml of CH2Cl2. Then the organic layers were combined and dried with sodium sulphate. After that the solvent was removed under vacuum and the desired product was obtained. The yield of the product was calculated and analysis was done by TLC and IR.25
Discussion:
The results obtained from the IR spectrum of catechol were matched with the standards. It means that the catechol used was pure and of a good quality. The IR spectrums of catechol and 3, 4- dihydroxymandelic acid were matched and it can be seen that considerable changes has been occurred. A carboxylic group was attached at the expense of a hydrogen ion. The IR spectrum of catechol shows a weak broad band due to the intermolecular hydrogen bonded -OH in the region of 3004- 3500 cm-1. The fingerprint region of catechol was weak band at 1261.7 cm-1 which gets matched with the standards (Sang-Hee Jang et al, 2002). While the fingerprint region for the step 1 product i.e. 3, 4- dihydroxymandelic acid was found at 1715.6 cm-1 which shows a -C=O stretch of carboxylic acid.
The results of analysis of protocatechualdehyde nearly match with the standards. The Rf value of protocatechualdehyde was calculated to be 0.88 which nearly matches with the standard of 0.90. The fingerprint region for protocatechualdehyde was found to be at 1710.2 cm-1 which matches with the standard for -C=O stretch for aromatic aldehyde as shown by the IR correlation tables. This can confirm that the product obtained was protocatechualdehyde.
The standard Rf value of vanillin from literature is 0.35+/- 0.3. The calculate value of vanillin was found to be 0.28. The calculated value for vanillin nearly matches with that of standard. The yield of vanillin obtained is 66.20%. The melting point of obtained product is 83-85 0C, which matches with the standard melting point (81-840C) of vanillin in literatures. The infrared spectrum of vanillin shows a -C=O stretch for aromatic aldehyde and an aromatic -C-O stretch for an ether which are unique fingerprint regions for vanillin. These figures shows that the product obtained can be regarded as vanillin.
Conclusion: The results obtained from the thin layer chromatography and spectrums obtained from the Infrared spectroscopy nearly matches with the standard values from the literatures. Also the melting points of the obtained product and of vanillin cited from the literatures are similar. This can confirmed that the obtained product was vanillin. The yield of vanillin obtained was quite good.
Future work: In future there is a need to develop a method which should not be too long and should be simple for the synthesis of vanillin.
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