INSTRUMNETAL METHODS FOR THE QUANTIFICATION OF CAPSAICINOIDS.INTRODUCTION
Capsiacinoids are natural alkaloid compounds found in pepper fruits (Capsium spp.) which are responsible for the burning sensation experienced when chewed. They are typical of hot pepper fruits. They also cause the nose and eyes to run and induce perspiration. Biosynthesis of capsiacinoids occurs in the placenta and accumulate in vacoules by epidermal cells and are then excreted on the internal pericarp surface and seeds. They are mainly found in the placenta of peppers. Parrish 1996)
Quality of capsicium is dependent on colour, flavour and pungency (govidarajan 1986). The pungency of capsicium fruits which is due to the amount of capsaicinoids present result from the direct effect of capsiacinoids on the pain receptors in the mouth and throat. (Krajewska & Powers 1988). Capsaicinoids are products of the condensation of vanillylamine and fatty acids of varying chain lenths. This condensation reaction is compelled by the enzyme capsaicin synthase (CS) which acts pricisely on the chain length of the fatty acid. This reaction requires ATP, coenzyme A (CoA), Mg2+, the vanillylamine portion formed from phenylalainie and the fatty acid which is formed from valine or leucine(Maria de Loudes). The constitution of the lateral chain length ( 9 – 11 carbon atoms), number and position of double bonds are responsible for the structural differences amongst capsaicinoids.
There are different types of capsaicinoids present in capsicum but the most common types include; Capsaicin, nordihydrocapsaicin, dihydrocapsaicin, homocapsaicin, homodihydrocapsaicin, nornordihydrocapsaicin and also nonivamide which is often referred to as synthetic capsaicin (Cordell). The concentration of capsaicinoids varies with specie of pepperCapsaicin and dihydrocapsaicin account for more than 90% of pungency present n capsicum. (Kosuge & Murata. 1970). Capsaicin is the most abundant and pungent type of capsaicinoid.
The pungency of capsaicin has restricted its use in clinical trials even with the encouraging results of medical studies which shows potential favorable effects in treatment of many diseases such as (Maria de Loudes) arthritis, cystitis. The extraction, synthesis and classification of its non spicy analogues for clinical purpose are ongoing.
The aim of this…….. is to look at all possible methods for quantifying capasiacinoids present within the capsicum species
SPECIES OF HOT PEPPERS (CAPSICUM SPP)
Some of the most common species of Chile peppers includes;
* Capsicum chinense: includes the hottest peppers such as habaneros and scotch bonnets
* Capsicum frutescens: includes the tabasco and cayenne peppers ,
* Capsicum annuum: includes common varieties such as, jalapenos, bell peppers and paprika
* Capsicum pubescens: includes the rocoto peppers from South American
* Capsicum baccatum: includes the chiltepin
USES/IMPORTANCE OF CAPSAICINOID
Capsaicinoids are known for their neurological, pharmaceutical and dietary effect. They show positive effect on carbohydrate metabolism and gastrointestinal digestion even at low concentrations (govindarajan & Sathyanarayana 1991) they affect pain receptors, central and peripheral heat detectors and pulmonary aortas (Saria et al 1981)
A mixture of capsaicinoids are used by pharmaceutical companies to produce creams/ gel formulations used for topical application as pain relivers for psoriasis , pre and post surgical pain. It is believed to act selsctively on the nerve fibers; A-delta and C- fibers that are responsible for pain signals, prompting the depolorization of C- fibers by opening cation channels accessible to sodium and calcium ions. Capsaicin effect is restricted to area of application due to its high metabolism into inactive compounds. Capsaicinoids are also being studied as efficient therapeutic remedy for nerve fiber disorders, pain related with arthritis, diabetes, neuropathy and HIV. (Robbins)
Ingredients derived from Capsicum such as capsicum annuum extracts are used as flaovoring agents, fragrance components in hair and skin care products. Capsicumannuum fruit bextract is used as antidandruff, antifungal , antifmicrobial agent( Cosmetic Info. Capsicum annuum Extract. (n.d).
METHODS OF ANALYSIS OF CAPSAICINOIDS
SCOVILLE ORGANOLEPTIC TEST
The scoville organoleptic test was the first reliable and most common sensory method used for the measurement of pungency in capsium products. It was created by an American pharmacist; Willbur Scoville in 1912. This test involved an alcohol extraction of capsacinoids from a known quantity of pepper. The extracted sample is diluted with a solution of sugar in water until pungency is no longer detected by a panel of five tasters. The degree of dilution is referred to as Scoville Heat Unit (Scoville, 1912). Peppers such as the sweet pepper which contains no capsainoid would read as zero on the scoville scale while those such as Habaneros known as the hottest chili pepper would have a rating of 200,000 or more meaning that their extracts would have to be diluted over 200,000 times before the pungent taste is vague.
Major problem of this test is that it is biased as it relies on individual taste. Therefore more reliable method for testing and quantifying pungency amongst different pepper species has been developed.
Spectrophotometry is a technique used to quantitatively measure the transmission or reflection abilities of a material as a function of its wavelength and which is then recorded as optical density or absorbance. It involves the use of spectrophotometers which is a device used to measure the intensity as a function wavelength of the light source light. Its light source includes ultra-violet, visible and infrared light and its wavelength ranges from 200nm to 2500nm. (Rendina)This is an important tool for determining the presence and amount of an analyte within a sample. (Skoog et al 7th ed)
The analysis of capsaicin content by spectrophotometry conducted by Tirimanna in 1972 was based on the colour reaction of capsaicin with molybdophosphoric acid and tungstophosphoric acid reagents with reference to a solution of standard capsaicin content. The extraction procedure used is based on North’s method using Thin Layer Chromatography to monitor capsaicin presence within extracts before they are disposed. The molybdophosphoric acid and tungstophosphoric acid reagents are then added to 5ml of the extracted test solution, mixed and filtered. The optical density of the solution was taken at wavelength of 735nm. Its major advantage is that absorption measurements of capsaicinoids can be quantitatively carried out without the equilibrium of the sample being disturbed and also interfering compounds are eliminated during extraction process. (Tirimanna).
In an experimental study carried out by Irena et al, the high correlation factor (0.93) acquired from the comparison of the determination of capsaicinoids extracted by TLC using spectrophotometry and High Performance Liquid Chromatography showed that both methods are capable of quantifying capsaicinoids and can be used in laboratories that are not so well equipped. (Perucka)
Another technique quiet similar to spectrophotometry is colorimetry which basically uses the same principle as a spectrophotometer. Most colorimetric methods suffer from instability of colour developed and cannot be used for extracts containing a large quantity of interfering matters. (Bajaj)
The increased interest in Capsaicinoids and capsaicin has led to the development and use of chromatography which is a more efficient and rapid technique compared to others like the Scoville Organoleptic test.
THIN LAYER CHROMATOGRAPHY
Thin Layer Chromatography involves the use of a glass plate coated with thin layer of absorbent material with finely divided particles like silica gel as its stationary phase. The sample is placed on the edge of the plate and placed into the mobile phase (developing solvent) where it is the separated as the developing solvent transverses across the length of the plate.
The use of TLC for the detection of capsaicin present in extracts from chilli has been carried out and it was observed that the lowest sensitivity limit for capsaicin is about 0.5I?g although there is the problem of reduced efficiency with multiple extractions (Tirimanna). At this low concentration, capsaicin was observed as a yellow colour instead of its normal pink madder lake color when sprayed with diazotized sulphanilic acid (Tirimanna). Another spray reagent which can also be used is iron III chloride-potassium ferricyanide which forms a Berlin blue colour (Spanyar)
Extraction procedure based on that of North’s involved the use of peroxide free diethyl ether and purified kerosene simultaneously. The kerosene-sample fraction was then re-extracted with about 20ml portions of water-acetone solution and then capsaicin was identified with TLC using glass plates coated with silica gel. Thin Layer Chromatography is also used to monitor the presence of capsaicin during extraction. The TLC plates were dried for an hour and stimulated at 100oC. The plates were sprayed with diazotized sulphanilic acid after being air dried, resulting in the production of a pink madder lake color.
Another method of extraction for samples containing capsaicin at levels below 10mg/100g of material, involved dissolving the residue from the extraction with diethyl ether in ethanol and mixing with light petroleum to remove coloring materials. The solution is then dried by evaporation then residue is extracted with ether. Spanyar et al. wrote that the best sample separation was achieved using chloroform-ethanol (99+1) solvent system. With the amount of preparative/extractions steps taken, there is the possibility of decomposition or failure of quantitative extraction of capsaicin especially with materials containing low amounts of capsaicin. (Spanyar). TLC is a reliable, easy and inexpensive method for not only purifying extracts but also quantitatively identifying and determining total capsaicinoids content (Jentzsch et al 1977). It can also be used by laboratories that are not so well equipped.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
HPLC according to Collin et al is a very efficient and precise method for quantifying capsaicinoids. This method enables the separation and quantification of the different natural capsaicinoids present. The use of different detectors such as fluorescence has made HPLC one of the most reliable methods for analyzing capsaicinoids. It’s highly sensitive as it has been applied to detect capsaicinoids in extracted sample solutions with low concentration of about 1 – 100ng/ml (Lu 1997)
According to Poyrazoglu et al, extracted sample is prepared simply by adding ethanol to a known quantity of ground pepper and refluxed at boiling point for 2.5 hours. The mixture is cooled, filtered and residue is washed with ethanol and filtered again. Sodium sulphate (anhydrous) is added to the filtrate and filtered. Acetonitrile -water (40:60 v/v) at pH 3 was used as the mobile phase with a UV-VIS photodiode array detector at 280nm. The peaks were highly resolved and were compared against their standard peak areas to calculate content of the different capsaicinoids in dry weight of pepper.
There are basically two types of Gas Chromatography; Gas-Solid Chromatography (GLC) and Gas-Liquid Chromatography which is also known as Gas Chromatography (GC). Due to tailing of peaks and semi retention of polar or active analytes, the use GLC is limited.
GC is based on the partitioning equilibrium of sample analytes between a mobile phase that is gaseous (like hydrogen and helium) and a stationary phase comprising of liquid that is inert on a solid surface. A major advantage of GC is that the mobile phase is chemically inert and therefore does not react with sample molecules. (Skoog). It separates compounds based on their level of volatility. It is always carried out in a column (packed or capillary)
Wooderck et al separated and quantified capsaicinoids eluting well resolved peaks at reasonable retention times using capillary Gas chromatography; capaicinoids were extracted with acetone and liquid-liquid extraction technique was used to purify sample to prevent interference or pollution of the column which was a polar silica column bonded with cyanopropylphenyldimethylsiloxane. Samples were injected with hydrogen as mobile phase.
Most GC quantification methods require the derivatization of samples to increase the volatility of capsaicinoids.