As per the research project, review of literature is divided into three parts.

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1 Literature Review: As per the research project, review of literature is divided into three parts. 1. Extraction of phenolic compounds 2. Validation and identification of phenolic compounds 3. Use of phenolic compounds in adulteration. 1. Extraction of phenolic compounds Extraction of phenolic compounds from plant, fruits, vegetables and other products depend upon the physical and chemical properties of them. Here reviews of previous work related to the extraction of phenolic compounds are described as per the fruits or plant parts. Jerneja et al. (2009) used methanol and ethanol for the extraction of phenols from green walnut fruits. The total phenolics and some individual phenolics, such as gallic, chlorogenic, ellagic, sinapic and protocatechuic acid, (+)-catechin and juglone were detected by total phenolic content (TPC) and Total flavonoid content (TFC). They used methanol and ethanol as a extraction solvent and it was seen that methanol gives good recovery compare to ethanol. Uma et al. (2010) optimizeed yield of total phenols in henna leaves (Lawsonia inermis) Response surface methodology (RSM) was used with central composite rotatable design (CCRD) and finally found optimized condition was in acetone at concentration of % with extraction time min and temperature was o C. Jenesen et al. (2007) used ethanol as an extraction solvent for red grapes. In this work, they homogenized grape juice with 0.1 M hydrochloric acid in ethanol at 40 o C and allowed to neutralize grape juice with acid. This method showed recovery 82 % for TPC and 92 % for anthocyanins with deviation of only 6 % and 4 % for TPC and anthocyanins respectively. Haseeb et al. (2006) tried mixture of Ethanol and water in ratio of (1:1) for extraction of grape seed. Different protocol they used for batter recovery such as solid to liquid ratio they set from g/ml, number of extraction stages (Single, double and triple) and membrane pore size (0.22 and 0.45 µm), in these experiment they found highest recovery based on total weight of grape seed weight was 11.4 %.

2 Nicolas et al. (2005) analysed soluble phenolics in extracting solvent methanol at 70%. For detection they used HPLC with PDA detector. Soluble phenolics were Flavanoids, Quinones and phenyl propanoids. Weirong et al. (2010) used 80 % ethanol with temperature of 90 o C and ratio from material to solvent was 25:1 for batter extraction of flavonoids from Opuntia milpa alta skin. In this maximum yield was 5.55mg/g of dry weight. For extraction of flavonoids from citrus peel like lime, orange and tangerine advanced method ultrasound assisted extraction method was also used. In this TPC were ± 1.90, ± 0.75 and ± 4.01 mg GAE (gallic acid equivalents)/g, for lime, orange and tangerine, respectively which were obtained from different cultivators. Katie et al. (2010) worked for Extraction of Strawberry it was freeze dried and grinded to powder and extracted with acetone: water in ratio of (70:30) v/v. Detection of Anthocyanins, derivatives of HHDP, ellagic acid and flavonols were done by LCMSMS with the flow rate of 0.4 ml/min and 0.1 % formic acid in water and acetonitrile was used as a mobile phase. Hung-ju et al. (2012) worked on Chinese herb taraxacum formosanum kitam was extracted in 50 % ethanol and 29 phenolic and flavonoid compounds were separated on column within 68 min and analyzed by LCMS. Mantas et al. (2010) used same method of 50 % ethanol for dill, Onion, parsley and celery but they detected phenolic and flavonoid compounds by spetrophotometric and chromatographic methods. Highest content among them was found in above-ground part of dill and celery and lowest content was found in celery roots. Vranova (2005) developed method for isoflavones some part of which are phenolics, method for extraction was developed by on HPLC by using n-hexane and 80 % ethyl acetate by solid phase extraction method. Van-den et al. (2010) analysed vegetables like potatoes (purple fleshed) were extracted by thermal process in which freeze dried powder and steamed potatoes were extracted with acidified methanol using advanced solvent extractor Dionex ASE 200. Anthocyanins and anthocyanidins were observed by HPLC-DAD/ESI-MS. From above review it can be conclude that Ethanol and acidic methanol are good extracting solvents for phenolic compounds.

3 2. Validation and identification of phenolic compounds After extraction, identification and validation are important part of protocol. This can be done by specific instrument. Effective analysis completely depends on the instrument we use as every instrument has their limit of detection. Once method is developed it is necessary that we validate the method for constant and reliable results. Total phenol content is determined by using Folinciocaltue assay and spectrophotometrically at 765 nm by using the aluminium chloride colorimetric assay. Ana et al. (2009) and Marinova et al. (2005) worked on different fruits (banana, cherry and blue berries) and vegetables (green and red peppers and onion) by using TPC method and calculated the total phenolic content in fruits and vegetables by using gallic acid as a standard. This method gives total content of phenols. Ozsoy et al. (2008) in the same above way TFC of C. asiatica was determined. The method in that they have used 5% sodium nitrite solution and 10 % ammonium chloride coloring reagent after subsequent reaction they added 1 M sodium hydroxide solution and obtained reagent mixture was subjected to spectrophotometric analysis at 510 nm using UV light spectrophotometer. Tania et al. (2012) used vanillin method for the determination of total phenolic content. They worked on fruit of perkier aculeate which has high protein content and found TPC of 64.9 mg/100 gm of gallic acid equivalent. Trans-β-carotene was the main carotenoid followed by α- carotene. Aneta et al. (2007) worked on 32 species of extracts by using HPLC. They determined quercetin, apigenin, kaempferol, luteolin and isorhamnetin qualitatively and quantitatively. The antioxidant activity was determined by method DPPH, ABTS and ferric reducing power was described as a TEAC (Total equivalent antioxidant activity). Truong et al. (2007) used Some other phenolic compounds such as caffeic acid, chlorgenic acid, 4,5-di-O-caffeoylquinic acid, 3,5-di-O-cafferoylquinic acid and 3,4-di-O-

4 caffeoylquinic acid) were separated iso-cratically on HPLC in sweet potatoes. They used 25 min run compared to 120 min require for equilibration of gradient method. Hiroyuki et al. (2002) used little higher version of instrument called DAD (Diode array detector), in which they analysed samples with 100 pure standards. Here retention time and wavelength were used to identify the compound. For extraction same as above method 90 % methanol was used. Recovery was 68 to 92 % and reproducibility was in range of 1 to 9 %. While Guanghou et al. (2004) used higher version of analytical instrument for phenolic compound study called ESI-LCMSMS. In that they determined proanthocyanidins from pycnogenol. Others compound they analysed were dimmers, trimers, tetramers and pentamers of catechin or epicatechin. Stephen et al. (2006) in their research Other compounds such as Isoflavones, salvinorin A, synepherine isomers and their metabolites were analysed in serum, urine and aqueous humour by LCMSMS showed good sensitivity and specificity. Verica et al. (2009), furthermore fruits (apricort, idared, apple, madjarska, blueberry, bluecrop, kuno, orange, sour cherry, strawberry, Maya, peach, redhayen and marasca and vegetables (broccoli, belster-flower and stem, cauliflower, kale, Melissa, leek-leaf and Fayola) were analyzed for phenolic compounds. Jie et al. (2005), advanced technology Q TRAPTM instrument was used to reduce the analysis time of phenolic content in adinandra nitida leaves, it was performed with UV spectrometer as well ESI-MS with negative ion mode. For this analysis Hypersil C18 column was used. Jungmin et al. (2008) in analysis effect of ph was studied. Effect of ph on anathocyanins was observed on HPLC in which two different columns and mobile phase were used. Significant variation was seen in the retention time and peak area of anthocyanins at different ph. Xiuling et al. (2004), other than natural products, phenolic compounds like naringin, hesperidin and neohesperidin were also analyzed in rat serum. To do this they used validated

5 LCMS method with solid-phase extraction to get clear matrix. For validation they considered following parameters recovery, linearity, accuracy and precision (intra- and inter-day variation). Unyong et al. (2011), Same phenolic compound (hesperidin) in rat liver microsomes were analysed by using ESI-LCMS in the presence of NADPH and UDP-glucoronic acid. Chemical structure of hesperidine was characterized on an ion trap mass spectrometer. In pomogranet anthocynins, flavonoids and ellagic acid were determined by HPLC-UV method. The main compound in anthocynins were delphinidin 3,5-diglucoside, delphinidin 3-glucoside and pelargonidin 3,5-diglucoside. Gelareh et al. (2009) used high performance liquid chromatography attached with UV detector for quantitative calculation of six anthocyanins, phytoestrogenic flavonoids and ellagic acid in pomegranate juice of eight Iranian cultivators. Total phenolic content was determined by folin Ciocalteu and DPPH. Fatima et al. (2002) developed a good resolution giving method for anthocyanins in strawberry in which they used low flow rate compatible for mass analysis. They used fraction chromatography to extract anthocyanins and analyzed obtained compound on liquid chromatography attached with mass spectrometer and photo diode array. Gupta et al. (2010) analyzed twelve phenolic compounds on HPLC by using two wavelength 280 and 360 nm for detection purpose. Symmetry C18 column with flow rate 1.0 ml/min was used, for batter separation methanol and acetic acid as a mobile phase at different concentration were used which took 22 to 25 min to elute out the phenolic compound while acetonitrile and phosphoric acid took 30 to 45 min for elution. Laurent et al. (2007) to know the pharmacodynamic and pharmacokinetic activity of isoflavones in prostate tissue, LCMS method was used. They used 12 men with benign prostatic hyperplasia received soy extract supplementation for 3 days before prostate surgery them they received blood and tissue samples and analyzed successfully on LCMS. Zoubida et al. (2007) used LCMS in negative mode with ESI source for the analysis of phenolic and flavonoids compounds in argan pulp. They identified sixteen flavanoid glycoside and flavanoid aglycons in argan pulp.

6 Pavel et al. (2005) used HPLC with multi-channel electrochemical coulometric detection for 32 phenolic compounds and flavonoids in beverages and plant extract using gradient analysis. This method was also suitable for beer, wine, tea and yacon extract. 3. Sources and role of phenolic compounds. like Phenolic compound mainly act as an antioxidant which is proved by many researchers Fatemeh et al. (2012) worked on banana s antioxidant activity at different stage, in different varieties and with different parts. To do this they used total phenolic content and total flavanoid contents and proved that banana has a good antioxidant activity at different level and in different parts Though, Massimo et al. (2007) and Lopez-yelez et al. (2010) said that they have many other health beneficiary pharmacological and therapeutic actions. They prevent many disease of human being. Claudine et al. (2005) measured pharmacokinetic study of 18 major phenolic compounds in a body. They monitored maximal plasma concentration-time curve, the elimination half-life and the relative urinary excretion. Grynkiewicz et al. (2005), worked on Soy-derived food products and nutraceuticals and found that isoflavone-rich material are beneficial for cardiovascular health and bone metabolism and they also contribute significantly in prevention of cancer. Michalaka (2006) found that phenolic compound in plant act as a metal chelator and oxygen scavenger when oxidative stress was applied. Such mechanism called homeostatic mechanisms it maintain the correct concentrations of essential metal ions in cellular compartments to minimize the damaging effects of an excess of nonessential ones.

7 Otto et al. (1999) observed that when microbial infection, chemical stress and UV light induce synthesis of phenolic compound in plant to prevent and combat with the stresses while insecticide and fungicide show lesser effect compare to previous one. Igual et al. (2010), stability of phenolic compound in grape fruit juice was evaluated. They used different storage condition like refrigerated and frozen. Finally concentration and antioxidant activity was found on spetrophotometrically. Many researchers have worked on Total phenolic intake to monitor the daily intake of it through fruits and vegetables. Pierre et al. (2006) were one of them who conducted research on fruits and vegetables in France and they found that highest concentration of TPC is in artichokes, parsley, and brussels sprouts (Fresh vegetables) and in fresh fruits were strawberries, litchi, and grapes. Silva et al. (2000) performed next project on 17 samples of commercially available jam in Portuguese by. They used RP HPLC-DAD for analysis of 8 phenolic compounds and several unidentified procyanidin polymers, in this project they also found that some jam samples were fraudulently adulterated with pear puree. Georg et al. (2012), the taste, aroma and colour of fruits, vegetable and plant parts are just because of phenolic compounds such observation was made them. In their research they found that pungency and bitterness of olive oil is because of phenolic compounds. For measurement of phenolic compound they used two analytical methods first one is based on the photometric measurement and second one is based on HPLC-MS. Michaelis et al. (2008) has proved that anthocyanins act as a ph indicator and it gives color to various fruits and vegetables. Phenolic compounds can be used as a finger print to determine the fraudulent and adulterated products. Elhuyar (2008) and Leo Nollet (2004) worked on few citrus fruit juices and developed the finger print on LCMS. As different fruit has different phenolic ratio and content so it helps in development of finger print of each fruit otherwise it becomes difficult to find sample adulterated with similar inferior class of fruits. They also worked on food adulteration by using LCMS.

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