Concentrated Citrus Poliphenols Extracts

Citrus fruits contain pretty high concentration of Flavanone glycosides Hesperidin and Naringin, plus smaller amount of many others flavonoids.
In well ripened fruits, flavonoids are mainly located in flavedo and in cores. Hesperidin is contained in sweet and bitter oranges, in lemons, in mandarins, in leaves, in twigs, nark and blossoms. Its biological purpose in citrus has not been completely identified, but taxonomic significance is evident. An orange contains, in average, 1,0 g of Hesperidin; processing wastes contain about  400 g/ton. As already mentioned, small size, immature  fruits are very rich in Hesperidin (about 10 % by weight on dry matters) and content decreases at full maturity  (< 0,5 % by weight on dry matters).
Traditional system for Hesperidin extraction involve peel treatment at pH 11,0 with calcium hydroxide. Use of lime rather than NaOH is necessary to allow release of bound water and prevent solubilization and hydrolysis of pectin avoiding large viscosity increases and slurry handling problems. After extraction, the alkaline slurry is clarified by pressing and filtration and the solution is adjusted to  pH 4,0 - 4,5 . This solution is held for 24 - 48 hours, allowing Hesperidin to crystallize, after which it is recovered by filtration. The product, crude Hesperidin (about 40 %) is dried and can be be further purified. Residue filtrate can be concentrated to increase concentration and is held for a second crystallization to improve recovery.
Hesperidin has beneficial effects on permeaility and fragility of capillaries and can be transformed in sweeteners and azoic pigments.
CITRECH has know-how to offer to interested citrus processors new technologies that afford to recover polyphenols in concentrated form.
As usual for CITRECH, technology is easily coupled into existing production lines; it is innovative and does not require any peels treatment like the traditional one.
With CITRECH technology You will be able to recover flavonoids both from endocarp and mesocarp (fruit's inside) and/or from esocarp (peel's outside) obtaining extracts which differs for chemical composition teflecting starting material. You'll will be entitled to choose if using solvents or not and products obtained can be thermally or membrane concentrated.
Please, see below for different possibilities:

I Polifenoli

Plants produce enormous amount of health-promoting compounds, including polyphenols, dietary fibers, vitamins, and minerals. Basically, food items of plant origin provide a high nutritive value, low caloric density, and low-energy constituents (e.g., minerals, vitamins, dietary fibers) as well as being rich sources of bioactive phytochemicals such as carotenoids, sterols, polyphenols, and glycosylates. Dietary polyphenols like flavonoids are strong antioxidants that act through interacting with reactive oxygen species (ROS) producing reactive compounds. For example, curcumin is a dietary polyphenol with strong antioxidant activity that have been used against a number of aging-related diseases like Alzeimer's disease for more than a century. Curcumin acts via chelating reactive metal ions like Fe2+, thus diminishes their oxidative power and the resulting OS due to free radicals. Ginkgo biloba extract has also been used to treat aging-related diseases. Ginkgo biloba extract contains various polyphenols, such as flavonoids, and terpenes, which interact with superoxide anion, hydroxyl, and peroxyl free radicals to quench the radical chain reactions in mitochondrial respiratory chain function. The important effect of tea polyphenols on cognitive function of elderly people is attributed to its flavonoid (catechin) content. Various studies have confirmed that vegetable and fruit juices available commercially also have high level of antioxidant polyphenolic compounds. This might be due to the high-pressure mechanical extraction during processing of vegetable and fruit juices that drives out a considerable amount of the antioxidant compounds from the peels and pulp in addition to the main fruit and vegetable fluids.
According to various in vitro, in vivo, and clinical studies, a stronger neuroprotective activity than antioxidant vitamins has been reported in polyphenols from grape, apple, and citrus fruit juices. Other antioxidant molecules like b-carotene, dietary polyphenols (flavonoids), and Se also reduce OS in neurons. The antioxidant, anti-inflammatory, and neuroprotective effect of vitamin D has also been widely reported. Due to the high nutritive values of plants and antioxidant potential, various polyphenolic compounds such as flavonoids, glycosidic derivatives of flavonoids, isoflavonoids, lignans and flavonolignans, stilbenoids, tannins, curcuminoids, coumarins and phloroglucinols, xanthones, and anthrones have been studied for their phytochemistry, classification, main representatives, biological activities, techniques of extraction and purification, techniques of identification and quantification, levels founds in foods, effects of food processing and pharmaceutical applications.

(fig. 2)
Polyphenols are a group of secondary plant metabolites comprising more than 9000 compounds identified till date. Based on the number of phenolic rings, they are categorized into different classes, namely, are flavonoids, lignans, and stilbenoids. On the other hand, flavonoids have seven subclasses, which represents 60% of dietary polyphenols, including flavones, flavonols, flavanones, isoflavones, flavanols, anthocyanins, and chalcones. Flavonoids are biological active polyphenols with potential antioxidant and immunomodulatory bioactivities. Various epidemiological and animal model studies indicated that dietary flavonoids can reduce the incidence of age-related diseases. Structurally flavonoids are biosynthesized from aromatic rings of the phenyl- and malonyl-coenzyme A. Their classification thus depends on the position of different functional groups on ring C and the position of ring B as shown in above figure. In ring C of flavanones and flavanols, the bond between second and the third atoms is a single bond where as it is an aromatic or a double bond in the case of flavones, flavonols, isoflavones, and anthocyanidins. With the exception of isoflavones, the attachment of ring B to the ring C is carried out at position 2.
Flavonoids mainly occur as glycosides instead of aglycones. Chalcones are the precursors of flavonoids composed of two benzene rings connected by a three-carbon a,b-unsaturated carbonyl structure. On the other hand, dihydrochalcones, which are a small group of flavonoids, have an open C-ring structure. The dihydrochalcones have a limited dietary significance and they are abundantly found in apples (Malus domestica) and rooibos tea. The other groups of flavonoids with a double bond at C-2 are the flavones, which include chrysin, apigenin, rutin, and the isoflavones genistin, genistein, daidzin, and daidzein. Apart from lacking C-3 oxygen flavones, apigenin, luteolin, wogonin, and baicalein bears structural resemblance to flavonols.
The content of flavones in celery (Apium graveolens) and parsley (Petroselinum hortense) is reported considerably high. Besides, other plants of the citrus family are rich in polymethoxylated flavones like nobiletin and tangeretin.
Isoflavones are a group of phytoestrogens that have structural and/or functional similarity to 17-estradiol. Therefore, they are known to have potent estrogenic or antiestrogenic actions. This is a group of flavonoids with the B-ring attached at C-3 instead of C-2. They exist abundantly in legumes with significant quantities of daidzein and genistein. In soybean, isoflavones occur mainly as 7-O-(6"-O-malonyl)- glucosides, 7-O-(6"-acetyl)-glucosides, 7-O-glucosides, and aglycones.
The oxidation at the C-3 position of the flavonoid structure gives flavonols such as kaempferol, isorhamnetin, myricetin, and quercetin. Flavonols are found abundantly in plants mainly as glycosides with conjugate bonds at 5, 7, 3', 4', and 5' positions in their structure.
Yellow and red onions (Allium cepa) are potential sources of flavonols with significantly high amount of quercetin-4’-O-glucoside and quercetin-3,4'-O-diglucoside.
Flavanones are flavonoids formed as a result of closure of the C-ring that produces chromone unit and they have a C-4 carbonyl group. The most common flavanones include naringin, naringenin, taxifolin, eriodictyol, and hesperidin. Flavanones occur in abundant amount in flavedo of citrus fruits as hydroxyl, glycosylated, and O-methylated derivatives where the most common one is hesperetin-7-O-rutinoside (hesperidin). The flavanone naringin is a glycoside containing of naringenin, an aglycone, and neohesperidose on the hydroxyl group at C-7. Naringin has a bitter taste because of its glucose moiety, but its 1,3-diphenylpropan-l-one derivative produced by treating naringin with a strong base is about 300-1800 times sweeter than sugar.
The flavan-3-ols such as catechin, epicatechin, and epigallocatechin gallate, and the anthocyanidins include apigenidin and cyanidin are formed through the reduction of the flavonols by reductases. The flavan-3-ols are subclass of flavonoids, which consists of the monomers, oligomers, and polymeric proanthocyanidins (condensed tannins). At positions C-2 and C-3 of the monomeric flavan-3-ols, there are two chiral centers that result in four isomeric structures.
Two of these isomeric flavan-3-ols, (+)-catechin and (—)-epicatechin, are abundant, whereas the others are found rarely in nature. For instance, flavan-3-ol monomers such as (-)-epigallocatechin, (-)-epigallocatechin-3-O-gallate, and (-)-epicatechin-3-O-gallate are available in green tea (Camellia sinensis) in very high quantity. Moreover, there are over 500 anthocyanin derivatives identified in plants with a varying degree of hydroxylation and/or methoxylation.
Anthocyanins possess radical scavenging property in relation to the hydroxyl backbone in their structure. Anthocyanidins are well known for their characteristic colors in fruits and flowers. The formation of various colors ranging from blue and purple to orange and red is due to the presence of conjugates produced as a result of a reaction between anthocyanidin aglycones (aglycones are pelargonidin, cyanidin, delphinidin, peonidin, petunidin, and malvidin) and organic acids. These compounds mainly occur in epidermal cell layer of vegetative tissues at some developmental stage and serve as photo protectants and attractants for pollinators or seed-dispersing organisms. An anthocyanidin is a stable anthocyanin-containing aglycone at C-3 position, while the additions of other moieties like acyl and hydroxycinnamic acid to the backbone yield complex anthocyanins.
The structural activity relationship (SAR) of flavonoids depends, in general, on the nature of substitution on rings B and C. The SAR explains their radical-scavenging, metal-chelating, and other biological activities in relation to various moieties on the main structures. Particularly, an ortho-dihydroxyl substitution on ring B (catechol group) confers higher activity because of the electron delocalization that stabilizes the carboxyl moiety, which also serves as a binding site for trace metals. Furthermore, the antioxidant activity is enhanced as the number of OH groups increases especially at positions 3,4, and 5 of the ring B. In addition, presence of a conjugated 4-oxo or a 3-OH group along with a double bond between C-2 and C-3 position of the ring C further enhances the radical-scavenging activity of flavonoids. In contrast, the methoxy derivatives of flavonoids formed by substitution of OH groups of ring B have lower radical-scavenging potential.
Flavonoids are a class of polyphenols with low molecular weight and they have an important role in the cell wall synthesis. They have a wide range of pharmacological activities, including anticancer, and antidiabetic activities, neuroprotective role and reduce the risk of cardiovascular diseases (CVDs). The term "phytopharmaceutical" is coined to plant secondary metabolites possessing biological activities. Naringin is one of the "phytopharmaceutical" among many flavonoids, which have hypolipidemic, antiatherosclerotic, antidiabetic, neuroprotective, hepatoprotective, and anticancer properties. In regard to their antidiabetic activity, flavonoids had shown to act via different intracellular signaling mechanisms such as regulating insulin secretion, insulin signaling, carbohydrate digestion, and glucose uptake in insulin-sensitive tissues. Various epidemiological studies support the potential of flavonoids to reduce the risk of Cardio Vascular Diseases (CVDs). Especially their activity toward reducing oxidation of low-density lipoproteins (LDL) is attributed to prevent endothelial dysfunction, platelet aggregation and adhesion, and smooth muscle cell migration and proliferation. For instance, there is a growing interest in the role of chocolate and one of its bioactive metabolites, such as flavan-3-ol, has efficacy in the prevention and management of CVDs due to improving metabolic syndrome risk factors. A large number of observational studies have demonstrated a significant association between cocoa use, which minimized risk of CVDs and related mortality. Moreover, numerous other in vitro studies have exhibited the potential of flavan-3-ols activity toward reduction of CVDs risk via their effect on angiotensin-converting enzyme (ACE) activity, inflammation, platelet function, endothelial function, and glucose transport, whereas their effectiveness is still unclear in in vivo model. In addition, experimental studies on flavonoids suggested that their potential antiepileptic activity is modulated via g-aminobutyric acid type A (GABAA)-Cl-channel complex due to their structural resemblance to benzodiazepines. Such results from herbal remedies containing flavonoids with neuroactive properties play a profound evidence for their potential action against GABAA receptor-mediated pathologic conditions. Hence, flavone derivatives are considered interesting principal compounds in the discovery of selective and potent benzodiazepine-like. The neuroprotective effect of flavonoids was initially suggested to be through an indirect action as a result of their antioxidant and free-radical scavenging activities. However, it was later discovered that flavonoids exert a direct pharmacological action on enzymes, receptors, and signaling pathways. Currently, much attention is given to the biological and physiochemical activities of flavonoids against neurodegeneration associated with Parkinson's and Alzheimer's disease and improve cognitive function in central nervous system (CNS).
The antimicrobial potential of flavonoids is a widely reported biological activity especially in plants containing flavones and flavonones. Structurally diverse isolates of the root bark of Morus like morusin, kuwanon C, sanggenon B and D were shown to have potent and wide spectrum activity against a range of microbes. Flavonones, pinocembrin and cryptocaryone, from the leaves of Cryptocarya chinensis demonstrated significant activity against M. tuberculosis in in vitro model. Besides, biflavonoids from Garcinia livingstonei leaves such as amentoflavone and 4' monomethoxy-amentoflavone have also shown potential antibacterial activity against Mycobacterium smegmatis.
The other prominent biological activity that flavonoids possess is inhibitory effect on cancer cell growth in in vitro and in vivo study. Apigenin is a flavonoid with a strong radical scavenging activity and anti-inflammatory property. Moreover, a number of studies on apigenin action against several cancer cell lines demonstrated that it vigorously inhibits tumor cell invasion, metastasis, mitogen-activated protein kinases
(fig. 3)
(MAPKs), and downstream oncogenes. On the other hand, chrysin, which is structurally similar to apigenin, is a potent inhibitor of aromatase of human immunodeficiency virus (HIV) in latent infection model. It was also reported that chrysin with slight modification has anti-inflammatory and antioxidant activities in addition to its cancer chemo preventive potential in in-vitro model.
Currently, over 9000 different flavonoids are available with a varying substitution pattern on their ring C. These flavonoids include various subclasses such as chalcones, flavones, flavonols, flavanones, anthocyanins, and isoflavonoids. Most of these flavonoids have considerable commercial importance. For instance, the flavonoid resveratrol (a stilbene), which is used for its animal longevity effect, is now gaining a greater market as nutritional supplement in products.
On the other hand, the metabolic engineering of flavonoid pathways, which became outstanding research area in the past few decades, begins since 1987. Particularly, in the area of ornamental plant breeding, engineering of flavonoids plays a major role in generating uncommon variety of flower colors such as blue and yellow flowering cultivars. In this regard, the molecular methods in combination with classical methods have been used in metabolic engineering to cultivate flowers and obtain novel colors while keeping other desirable original traits of the plant. Furthermore, flavonoids are indicated as screening pigments against UV-B, phytoalexins (antipathogenic microorganisms), antifeedants, pollination agent attractants, and plant growth promoters.
The extraction, separation, and purification of polyphenols are a challenging process due to their chemical complexity and similar structural features. However, recent advancements in technologies and instrumentations have made them easier. Overall, the collected samples to be extracted undergo drying, freezing, or lyophilizing ahead of extraction to avoid degradation, which is particularly a problem during high- temperature drying. Besides, the sample should be kept in a closed and opaque container since the composition of polyphenolic compounds is mostly affected by exposure to light and oxygen. Once the sample is ready, different extraction methods ncluding liquid/liquid partitioning and solid/liquid extraction, can be employed immediately after preliminary sample pretreatments (filtration and centrifugation).
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Schematic diagram showing the process of extraction/ fractionation/ and purification strategies of flavonoids from biological fluids, plants, and food (including beverages). Abbreviations: SEE, solid-phase extraction; TLC, thin-layer chromatography; GC, gas chromatography; HPLC, high-performance liquid chromatography; LC- MS, tandem liquid chromatography-mass spectrometry; C£, capillary electrophoresis; NMR, nuclear magnetic resonance.

In the process of crude extraction of flavonoids, heating the sample-solvent mixture at about 60°C in ethanol/water mixture (3:7, v/v), which is mostly considered suitable solvent, is the first step. After heating the sample-solvent mixture for enough period of time, the extract will be cooled, filtered, and the excess solvent will be evaporated to obtain the final crude extract. However, the crude extract needs further purification in order to obtain a pure flavonoid. Thus, a solution of the crude extract will be prepared and poured to a column (400 x 2.5 cm i.d.) packed with pretreated macroporous absorber resin. Once the solution is absorbed, distilled water will be used to wash carbohydrates out of the column and then preferably 65% ethanol is poured into the column to elute flavonoids. The collected mixture will be concentrated by rotary evaporation apparatus and dried under vacuum. Since the obtained eluate of flavonoids could contain many derivatives, further chromatographic procedures such as HPLC, GC, and others can be employed to get component flavonoids.
Dietary supplements and nutraceuticals that contain important flavonoids are widely available in the market. However, they have not been extensively utilized to benefit world population. Being strong antioxidants in nature, flavonoids have a range of biological activities as well as wider application in food-processing industries. Moreover, their characteristic coloring ability, especially that of anthocyanins, has been of interest as dyes in textile, food-processing, and cosmetic industries. Generally, the current trends in flavonoids research indicate there is an emerging potential to drive a lead compound or a drug candidate that can be developed to a drug for treating infectious diseases, diabetes, cancer, Alzheimer's disease, and other age-related ailments. Therefore, further research is important to provide reliable data on the flavonoids and their metabolites that are active and their respective doses, their interaction with other drugs as well as their mechanisms of actions.