Flavonoids: isolation, characterization, and health benefits

The unique and vast pharmacological activities of flavonoids have made them of research interest. This led to the use of various techniques to isolate and characterize them, intending to determine their potential health benefits. The medicinal values of plant-based flavonoids that this literature review tends to summarize the pharmacological activities of these secondary metabolites from 22 selected plant families. The pharmacological shreds of evidence reported in the literature have proven that flavonoids have shown anti-cancer, anti-microbial, anti-oxidant, anti-inflammatory, anti-fungal, anti-ulcer, and anti-edematogenic activities. Out of these, 30% showed to have anti-oxidant activity, key in protecting the body against free radicals. Besides, 18% of the references showed anti-microbial and anti-cancer activities. Further literature reports indicated that flavonoids from these families exhibited anti-inflammatory and anti-edematogenic (9%), anti-viral and anti-ulcer (5%), anti-fungal, anti-nociceptive, and anti-histamine (2%). The pharmacological activities of flavonoids from the various sources reviewed in this study show that the secondary metabolites could provide a scaffold for the development of potent anti-cancer drugs in the future.


Background
Flavonoids are phytochemicals responsible for the various colors in the seeds, flowers, fruits, leaves, and bark [1]. Flavonoids are a large class of natural aromatic compounds as there are reported to be the most common plants' phenolics [2,3]. Over the years, flavonoids have represented a vast percentage of phytochemicals from natural sources. It has been reported that more than 10, 000 different classes of flavonoids have been found in kingdom Plantae [4][5][6]. Flavonoids are secondary metabolites found in organs of these plants with different functions [7,8]. Also, they have been reported from sources such as vegetables, wine, fruits, and beverages (tea) [9].
The chemical structures of flavonoids consist of C6-C3-C6 [10] rings which correspond to two aromatic rings A and B linked by three carbon atoms, which may lead to the formation of a third ring (C). Variations in this basic structure give the various subclasses of flavonoidal compounds. These are flavanones, isoflavones, flavones, flavanols (catechins), chalcones, flavonols, and anthocyanins [11][12][13][14]. The flavonoids present in the diet help in the prevention of cardiovascular disease [15]. The biological and oxidative properties of flavonoids are responsible for their anti-allergic, cardioprotective, antidiabetic, anti-inflammatory, anti-oxidative activity, and free radical scavenging capacity [15,16]. Also, flavonoids have been reported to exhibit anti-cancer activity [5]. Studies of flavonoids revealed that they are free radical scavengers and reducing agents [17]. Recent researches have focused on the health benefits of these secondary metabolites because of their preventive activity against diseases and anti-oxidative activity, anti-cancer activities, anti-viral activities, and anti-inflammatory [18,19]. Aside from the antioxidant activity of flavonoids, chelating properties [20], their usage as anti-aging substances [21], capillary permeability, and inflammatory response [22], anti-bacterial and therapeutic [23], gastro-protective, and anti-diabetic activity [3] of these phenolics have been reported.
The protective effects such as the anti-inflammatory, anti-oxidant, anti-viral, and anti-tumor activity of flavonoids from natural sources are well documented [24]. The anti-carcinogenic activity of flavonoids has been linked to their anti-oxidant properties [25] which is due to the hydroxyl groups on the structure of the flavonoids [26]. Because of the importance of these phytochemicals, this review summarizes the isolation, characterization, and health benefits of these flavonoids taking into consideration those phytochemicals responsible for these activities. The health benefits reviewed were anti-cancer, anti-microbial, anti-oxidant, anti-inflammatory, antifungal, anti-ulcer, and anti-edematogenic activities.
2 Main text

Methods
The search was done by using keywords such as flavonoids on "science direct," "google scholar," "Scopus" database, and many journal sites. Journals employed in the search are Elsevier and Springer. Other search engines used as well as papers published between 2006 and 2019.

Flavonoids biosynthetic pathway
Flavonoids or stilbenes biosynthetic pathway ( Fig. 1) follow the extension of 4-hydroxycinnamoyl-CoA with three malonyl-CoA units, in which the poly-β-keto chain folded in different ways, via Aldo or Claisen reactions. Stilbene synthase and chalcone synthase (enzymes) couple the three malonyl-CoA with cinnamoyl-CoA unit to give chalcones or stilbene. Chalcones are precursors for the vast range of flavonoids and their derivatives found in plants. The nucleophilic attack (Michael type) of a phenolic group on α, β-unsaturated ketone forms a six-membered heterocyclic ring such as naringenin. This isomerization reaction in an acid condition favors the flavanone while in basic condition, the chalcone [27]. Flavanones then give rise to variants of flavonoids such as flavonols, flavones, anthocyanidins, and catechins as shown in Fig. 1

Sources and classification of flavonoids
The various sources of flavonoids have been reported [28] and are given in Table 1 while their classification [29] is shown in Table 2 showing the subclasses. Similarly, Fig. 2 showed the basic skeleton of flavonoids and their various classes.

Isolation of flavonoids
The isolation of flavonoids involves various techniques available to natural products researchers that have simplified their isolation from crude extracts. These techniques are column chromatography (CC), high-performance liquid chromatography (HPLC), high-speed counter-current chromatography (HSCCC), counter-current chromatography (CCC), open centrifugal preparative thin layer chromatography (CPTLC), preparative thin-layer chromatography (PTLC), medium pressure liquid chromatography (MPLC), and high-pressure preparative liquid chromatography (HPLC) [29,30]. The details of the extraction, steps, and the solvents used for the structural elucidation and characterization of the flavonoids summarized in this review are shown in Table 5.

Techniques used to elucidate the structure of flavonoids
Natural products researchers use the following spectroscopic techniques to elucidate the structure of flavonoids. These are infrared spectroscopy (IR), nuclear magnetic resonance (NMR), ultra-violet spectrophotometry (UV), mass spectrometry (MS), and physical properties as electronic circular dichroism (ECD), melting point (m.pt), and specific rotation power ð½α T D ) for flavonoids with a stereocenter for ECD and [α]D. Flavonoids have unique chemical shifts. These characteristic chemical shifts make it easier to characterize them. The characteristic chemical shift values reported [29] for some flavonoid classes are given (Table 3) and the UV absorption ranges for these flavonoids are shown ( Table 4).
The isolation, characterization, and health benefits of these flavonoids are represented (Scheme 1).

Health benefits of flavonoids
Extensive biological studies of flavonoids have revealed their health benefits including disease prevention [5,72]. They have exhibited anti-oxidant, anti-inflammatory, antibacterial, and anti-viral activities [73], anti-oxidant [74], anti-allergic anti-carcinogenic properties [75]. The protective effects of flavonoids have been reported as they help to reduce oxidative stress in the body. The cholesterollowering activity, anti-cancer, anti-oxidant of myricetin, tricin, apigenin, luteolin, quercetin, and isorhamnetin has been reported [24]. The anti-viral, anti-bacterial, anticancer, cardioprotective, and anti-inflammatory activity [76], as chelating agents and, are strong topoisomerase inhibitors [24] anti-aggregational, anti-atherosclerotic, and detoxification activities [77] of various flavonoids have been reported. These biological activities depend to a larger extent on the hydroxyl group in the flavonoids [78]. Quercetin has reduced the risk of cancer, eye diseases, arthritis, and allergic disorders [9]. The decreased risk of cardiovascular disease by proanthocyanins and flavone-3-ols has been reported [23]. The techniques used for the isolation, characterization, and the health benefits of these flavonoids are as shown in Table 5.
The pharmacological activities of the phytochemical constituents from 22 plant families reported in the literature as reviewed in Fig. 3 showed the percentages of these activities. Out of the references cited, 30% of the flavonoids showed anti-oxidant activity. Because of this vast anti-oxidant activity, flavonoids reduce aging by protecting the body against free radicals oxidation [26,79].

Anti-oxidant activity
Anti-oxidants are compounds that slow or prevent oxidation in living cells. They act against the effects of free radicals. Flavonoids protect the body against reactive oxygen species. Chemically, flavonoids have hydroxyl groups and a highly conjugated π-electron system, which allows them to act as free radical scavengers [80]. Antioxidant activity of flavonoids [16,20,33,76,81], chelating properties [20], makes them acts as protective agents against free radicals [26,79]. In the body, anti-oxidants protect the human body from free radicals oxidation [17] thereby retarding the progress of many chronic diseases. Epicatechin, epigallocatechin, and gallocatechin have exhibited anti-oxidant activity [31].

Anti-cancer activity
The term cancer refers to a disease in which cells of a tissue undergo uncontrolled and often rapid proliferation [85]. This is also the loss of control of growth [86]. Alternative medicine has been used to treat cancer [87]

Aesculus indica
Sapindaceae Anti-oxidant activity [44] Ekalu and Habila Beni-Suef University Journal of Basic and Applied Sciences (2020) 9:45 and flavonoids, especially from plant sources, have not been left out. The pharmacological properties of flavonoids have made them useful alternatives to inhibit cell damage [73]. Flavonoids have been reported to be good anti-cancer therapeutics [88]. Quercetin particularly has been reported to be effective in the treatment of stomach, lung, prostate, and breast cancers [29]. Pradhan et al. 2015 reported the anti-tumor activity of quercetin without toxicity on the breast cancer cell, MCF-7 [89]. The anticancer activity of quercetin has been linked to the inhibition of the enzyme (DNA gyrase) [90]. Luteolin isolated from the leaves of Struchium sparganophora has caused cell death of melanoma and ovarian cancer cell lines [51]. Luteolin-7-O-β-glucopyranoside, formononetin-7-O-β-Dglucoside, and quercetin-3-O-β-d-glucuronide isolated from the leaves of Cassia tora linn were active against breast cancer (MCF7) [53]. Luteolin from the Vitex negundo Lin was an active anti-tumor agent [55]. In another study, the 2′, 5-dihydroxy-7-methoxyflavanone and 2′, 5-dihydroxy-7-methoxyflavone isolated from Andrographis glandulosa were active against HeLa, MIA PaCa, and U-8 [56]. Quercetin has been reported to induce

Anti-ulcer activity
An ulcer is a disease of the alimentary tract caused by an inflamed break in the mucus lining membrane [100]. The anti-ulcer activity of quercetin in animals has been reported [72]. The phytochemical investigation of the showed a powerful anti-ulcer [64]. The hesperidin isolated from Citrus sinensis showed anti-ulcer activity [65].

Conclusions
Twenty-two members of the different families containing flavonoids studied for their health benefits confirmed the medicinal importance of these phytochemicals from these sources. The pharmacological pieces of evidence reported in the literature has proven that these flavonoids have shown anti-cancer, anti-microbial, anti-oxidant, anti-inflammatory, anti-fungal, anti-ulcer, and anti-edematogenic activity. Out of the references cited, 30% focused on the anti-oxidant activity of flavonoids, key in protecting the body against free radicals and oxidative stress. Also, 18% of the references showed anti-microbial and anti-cancer activities. Further literature reports indicated that flavonoids from these families exhibited anti-inflammatory and anti-edematogenic (9%), antiviral and anti-ulcer (5%), anti-fungal, anti-nociceptive, and anti-histaminice (2%). The pharmacological activities of flavonoids from the various sources reviewed in this study show that the secondary metabolites could provide a scaffold for the development of potent anti-cancer drugs in the future.