Natural Products Chemistry & Research

Flavonoids or bioflavonoids from the Latin word flavus meaning yellow, their color in nature are a class of plant secondary metabolites. Flavonoids were referred to as Vitamin P [1]. Probably because of the effect they had on the permeability of vascular capillaries, but the term has since fallen out of use [2]. Flavonoids are widely distributed in plants, fulfilling many functions. Flavonoids are the most important plant pigments for flower coloration, producing yellow or red/blue pigmentation in petals designed to attract pollinator animals [3].

The unique nutrient richness of every whole, natural food can be showcased in a variety of ways. But there is no better way to highlight the unique nutrient richness of foods than to focus on their flavonoid content! Flavonoids are a quite remarkable group of phytonutrients that fall into the chemical category of polyphenols. They're perhaps most famous for their rich diversity of color-providing pigments. The name of these phytonutrients actually derives from their color-related chemistry. As a group, however, flavonoids are highly bioactive and play a wide variety of different roles in the health of plants, animals, and human health.

The flavonoid nutrient family is one of the largest nutrient families known to scientists. Over 6,000 unique flavonoids have been identified in research studies, and many of these flavonoids are found in plants that are routinely enjoyed in delicious cuisines throughout the world. In terms of nutrient richness, we get far more flavonoids from plant foods than from animal foods, and in particular, vegetables and fruits can be especially nutrient-rich in this type of phytonutrient.

Flavonoids are best known for their antioxidant and antiinflammatory health benefits as well as the support of the cardiovascular and nervous systems. Because they also help support detoxification of potentially tissue-damaging molecules, their intake has often, although not always, been associated with decreased risk of certain types of cancers, including lung and breast cancer [4].

Flavonoids are widely distributed in plants, fulfilling many functions. Flavonoids are the most important plant pigments for flower coloration, producing yellow or red/blue pigmentation in petals designed to attract pollinator animals. In higher plants, flavonoids are involved in UV filtration, symbiotic nitrogen fixation and floral pigmentation.

They may also act as chemical messengers, physiological regulators, and cell cycle inhibitors. Flavonoids secreted by the root of their host plant help Rhizobia in the infection stage of their symbiotic relationship with legumes like peas and beans. In addition, some flavonoids have inhibitory activity against organisms that cause plant diseases [3].

Flavonoids (specifically flavanoids such as the catechins) are "the most common group of polyphenolic compounds in the human diet and are found ubiquitously in plants". The widespread distribution of flavonoids, their variety and their relatively low toxicity compared to other active plant compounds (for instance alkaloids) mean that many animals, including humans, ingest significant quantities in their diet. Foods with a high flavonoid content include fruits and vegetable. E.g., parsley, onions, blueberries and other berries, black tea, green tea and bananas, all citrus fruits, red wine and dark chocolate (with a cocoa content of 70% or greater) [5].

A variety of potential health benefits including reduced risk for coronary artery disease and cancer are thought to be associated with dietary flavonoids [6]. These effects of flavonoids are generally attributed to their ability to scavenge reactive oxygen and nitrogen species (O2– • OH, NO•, RO•, ROO•) and reduce oxidative stress, which may contribute to the progression of many diseases. For these reasons flavonoid-rich diet, supplements and cosmetics are widely recommended for improving health status and prevention of chronic diseases [7].

Equipments/apparatus

Fourier Transform Infrared (FT-IR), UV/Visible Spectrometer, laboratory glass ware, analytical balance, Pestle and Mortar, Sieve, Whatman filter paper.

Collection and preparation of plant materials

Fresh parts of five herbal plants leaves Vernonia baldwinii, Moringa oleifera, Telfairia occidentalis, Ocimum gratissimum and Cassia tora were collected from different areas in Jalingo and Ardo-kola Local Government Area of Taraba State in the North Eastern region of Nigeria. The plant materials were authenticated at the Department of Chemistry Modibbo Adama University of Technology Yola Adamawa State.

The fresh plant materials were collected, and the voucher specimen were numbered 1-5 and kept in Chemistry Researsh Laboratory of Modibbo Adama University of Technology Yola Adamawa State. The part of the plant materials collected were freed from twigs and extraneous matter. Soil grit, Sand and dirty were removed by sifting. In other to remove the remnants of adhering foreign matter, the samples were rapidly and thoroughly washed under tap water and rinsed with distilled water and then shade dried at room temperature for 15 days. After drying, the plant materials were ground to fine powder and transferred into airtight containers with proper lebeling for future use.

Flavonoid extraction

About 5 g of the dried sample was extracted with ethanol 70% for 60 min. at 60°C. Extracts were filtered in vacuum using whatman filter paper. Aqueous as well as hydrochloric extracts were evaporated to dryness. The dried weigh was measured. The yield was defined:

Crude extract/Plant material weight × 100

Synthesis of metal-flavonoid complexes

solution of Copper chloride, Zinc sulphate, Nickel chloride each (0.0249 g, 1.25 × 10^-4 mol) was measured and (2 cm³) distilled water was added and the solution was slowly added drop wise to a solution of flavonoid (0.146 g, 2.5 × 10^-4 mol) in methanol (10 cm³). The mixture was stirred for 30 min at room temperature. The complex was filtered in a vacuum system, washed with water and dried by lyophilization. A pure green solid was obtained and weighed.

Flavonoid extracts

The result for the extraction of flavonoid in Moringa oleifera, Cassia tora, Ocimum gratissimum, Vernonia and Telfairia occidentalis leaves is presented in Table 1. The result shows that Vernonia leaves produced highest percentage yield of flavonoid extracts while Moringa oleifera leaves produced the lowest percentage yield of the flavonoid extracts. The flavonoid produced in Moringa oleifera leaves is ˂Cassia tora leaves>Ocimum gratissimum leaves˂Vernonia leaves>Telfairia occidentalis .

Table 1: Flavonoids extracts in gram and percentage yield in the plant leaves.

The synthesized metal-flavonoid complexes

The percentage yield for the complexes in Table 2 shows that Znflavonoid complex in Ocimum gratissimum has the highest percentage yield while Zn-flavonoid complex in Moringa oleifera contained the lowest percentage yield. The percentage yield of the metal-flavonoid complexes were higher when compared with the result of Regina [8].

Table 2: Percentage yield of metal-flavonoid complexes from flavonoid extracts of Moringa oleifera, Cassia tora, Ocimum gratissimum, Vernonia baldwinii and Telfairia occidentalis leaves.

The physical properties of the complexes

In this study, the investigation of physical properties of complexes in Table 3 indicates that the highest pH value of the complexes was found in Zn-flavonoid complex of Vernonia baldwinii while the lowest pH value was found in Zn-flavonoid complex of Cassia tora . According to these results, the complexes were formed at slightly acidic condition between the pH value 3.51 to 4.65. The optimal pH for complex formation, although strongly dependent on the features of the metal ion, is around pH 6. Complex formation at pH values lower than 3.0 is difficult because the flavonoids are predominantly present in their undissociated form. Although higher pH values favour deprotonation of flavonoids and consequently, more complex species at higher pH values metal ions are often involved in side reaction (hydrolysis) and hydroxo-complexes are formed [9-12]. In general the conductivity values of Ni-flavonoid complexes ˂Cu-flavonoid complexes ˂Znflavonoid complexes. The lowest conductivity of all the complexes was obtained from Zn-flavonoid complexes as a result of its largest surface area, weak bonding and being far away from the nucleus. Therefore, Ni-flavonoid complexes had higher conductivity because of their small surface area and are closer to the nucleus and having stronger bonding than Cu-flavonoid complexes and Zn-flavonoid complexes. The highest melting point of all the complexes was obtained from Znflavonoid complex of Ocimum gratissimum while the lowest melting point was obtained from Ni-flavonoid complex of Moringa oleifera . The results shows that Ni-flavonoid complex of Moringa olifera had shorter time to be melted than all the complexes and weak bonding exist in the complex but Zn-flavonoid complex of Ocimum gratissimum had strong bonding and take longer time to be melted. The melting point of the complexes were low when compared with the result of Regina [13] as a result of different in samples and environmental condition. The pH values of the complexes agreed with the pH value stated by Duśan and Vesna [14] for metal-flavonoid complexes.

Table 3: Some investicated physical properties for metal-flavonoid complexes of Moringa oleifera, Cassia tora, Ocimum gratissimum, Vernonoia and Telfairia occidentalis plant leaves extracts.

The pH, Conductivity and Melting point obtained from metalflavonoid complexes of Moringa oleifera, Cassia tora, Ocimum gratissimum, Vernonoia and Telfairia occidentalis plant leaves extract as shown in Tables 4-8 and graphical represention shown in Figures 1a-10.

Table 4: Assignment of FTIR Absorption for Cu-flavonoid complex, Zn-flavonoid complex, Ni-flavonoid complex and flavonoid extracts from Moringa oleifera dried plant leaves.

Table 5: Assignment of FTIR Absorption for Cu-flavonoid complex, Zn-flavonoid complex, Ni-flavonoid complex and flavonoid extracts from Cassia tora dried plant leaves extracts.

Table 6: Assignment of FTIR Absorption for Cu-flavonoid complex, Zn-flavonoid complex, Ni-flavonoid complex and flavonoid extracts from Ocimum gratissimum dried plant leaves.

Table 7: Assignment of FTIR Absorption for Cu-flavonoid complex, Zn-flavonoid complex, Ni-flavonoid complex and flavonoid extracts from Vernonia baldwinii dried plant leaves.

Table 8: Assignment of FTIR Absorption for Cu-flavonoid complex, Zn-flavonoid complex, Ni-flavonoid complex and flavonoid extracts from Telfairia occidentalis dried plant leaves.

Figure 1a: FTIR Absorption Spectra for Cu-flavonoid complex from Moringa oleifera leaves.

Figure 1b: FTIR Absorption Spectra for Zn-flavonoid complex from Moringa oleifera leaves.

Figure 1c: FTIR Absorption Spectra for Ni-flavonoid complex from Moringa oleifera leaves.

Figure 2: FTIR Absorption Spectra for flavonoid extracts from Moringa oleifera leaves.

Figure 3a: FTIR Absorption Spectra for Cu-flavonoid complex from Cassia tora leaves.

Figure 3b: FTIR Absorption Spectra for Zn-flavonoid complex from Cassia tora leaves.

Figure 3c: FTIR Absorption Spectra for Ni-flavonoid complex from Cassia tora leaves.

Figure 4: FTIR Absorption Spectra for flavonoid extracts from Cassia tora leaves.

Figure 5a: FTIR Absorption Spectra for Cu-flavonoid complex from Ocimum gratissimum leaves.

Figure 5b: FTIR Absorption Spectra for Zn-flavonoid complex from Ocimum gratissimum leaves.

Figure 5c: FTIR Absorption Spectra for Zn-flavonoid complex from Ocimum gratissimum leaves.

Figure 6: FTIR Absorption Spectra for flavonoid extracts from Ocimum gratissimum leaves.

Figure 7a: FTIR Absorption Spectra for Cu-flavonoid complex from Vernonia baldwinii leaves.

Figure 7b: FTIR Absorption Spectra for Zn-flavonoid complex from Vernonia baldwinii leaves.

Figure 7c: FTIR Absorption Spectra for Ni-flavonoid complex from Vernonia baldwinii leaves.

Figure 8: FTIR Absorption Spectra for flavonoid extracts from Vernonia baldwinii leaves.

Figure 9a: FTIR Absorption Spectra for Cu-flavonoid complex from Telfairia occidentalis leaves.

Figure 9b: FTIR Absorption Spectra for Zn-flavonoid complex from Telfairia occidentalis leaves.

Figure 9c: FTIR Absorption Spectra for Ni-flavonoid complex from Telfairia occidentalis leaves.

Figure 10: FTIR Absorption Spectra for flavonoid extracts from Telfairia occidentalis leaves.

The FTIR results

Evidence of formation of complexes for the extracts

In the complexes spectra, the bands assigning to the carbonyl group are shifted to lower wave number in comparison with that of the free ligands as a result of its coordination [13].

The IR spectrum of the flavonoid shows a band at 1732 cm^-1 for C=O, which reduced to 1723 cm^-1, 1725 cm^-1 and 1725 cm^-1 for Cuflavonoid complex, Zn-flavonoid complex and Ni-flavonoid complex respectively in Moringa oleifera . In Cassia tora , the IR spectrum of flavonoid shows a band at 1727 cm^-1 for C=O, which shifted to 1641 cm^-1, 1723 cm^-1, 1724 cm^-1 for Cu-flavonoid complex, Zn-flavonoid complex and Ni-flavonoid complex respectively.

The IR spectrum of flavonoid shows a band 1733 cm^-1 for C=O, and shifted to 1685 cm^-1 for Cu-flavonoid complex, 1688 cm^-1 for Znflavonoid and 1640 cm^-1 for Ni-flavonoid complex in Ocimum gratissimum . The IR spectrum of flavonoid shows a band 1717 cm^-1 for C=O, and shifted to 1640 cm^-1 for Cu-flavonoid complex, 1712 cm^-1 for Zn-flavonoid and 1710 cm^-1 for Ni-flavonoid complex in Vernonia baldwinii while the IR spectrum of flavonoid shows a band 1723 cm^-1 and shifted to 1645 cm^-1 for Cu-flavonoid complex, 1718 cm^-1 for Zn-flavonoid and 1716 cm^-1 for Ni-flavonoid complex in Telfairia occidentalis. This results indicated that complexes were formed.

UV-Visible spectra of complexes

The result of the UV-Visible spectra of wavelengths 200 nm-900 nm for the different complexes of Cu-flavonoid complex, Zn-flavonoid complex and Ni-flavonoid complex from Moringa oleifera, Cassia tora, Ocimum gratissimum, Vernonia and Telfairia occidentalis are shown in Figures 11-15 and the results were in agreement with the results of Duśan and Vesna [14].

Figure 11: UV-Visible Absorption Spectra of synthesized Cu- Flavonoid Complex, Zn-flavonoid Complex and Ni-Flavonoid Complex from Moringa oleifera plant leaves extract.

Figure 12: UV-Visible Absorption Spectra of synthesized Cu- Flavonoid Complex, Zn-Flavonoid Complex and Ni-Flavonoid Complex from Cassia tora plant leaves extract.

Figure 13: UV-Visible Absorption Spectra of synthesized Cu- Flavonoid Complex, Zn-Flavonoi Complex and Ni-Flavonoid Complex from Ocimum gratissimum plant leaves extract.

Figure 14: UV-Visible Absorption Spectra of synthesized Cu- Flavonoid Complex, Zn-Flavonoid Complex and Ni-Flavonoid Complex from Vernonia baldwinii plant leaves extract.

Figure 15: UV-Visible Absorption Spectra of synthesized Cu- Flavonoid Complex, Zn-Flavonoid Complex and Ni-Flavonoid Complex from Telfairia occidentalis plant leaves extract.

In Figure 11 the maximum absorption peak of 10 was observed at 450 nm by Ni-flavonoid complex and followed by the peak of 4.309 observed at 250 nm for Cu-flavonoid complex and the Zn-flavonoid complex has minimum absorption peak and observed at 3.386 at 245 nm. The absorption ranged from 3.386-10 was demonstrated by the complexes.

The result of the UV-Visible absorption spectra for Cu-flavonoid complex, Zn-flavonoid complex, and Ni-flavonoid complex from Cassia tora plant leaves extracts are shown in Figure 12. The maximum absorption peaks of 10 were observed at 220 nm, 390 nm, 410 nm, 420 nm, 430 nm and 440 nm by Zn-flavonoid complex and followed by Niflavonoid complex with the absorption peak of 4.381 at 445 nm and the least absorption peak was observed 3.438 at 245 nm for Cuflavonoid complex. The ranged was 3.438-10 and 245-445 nm.

Figure 13 shows the result for the UV-Visible absorption spectra of complexes in Ocimum gratissimum . The maximum absorption peak of 9.999, 9.999, 9.999, 9.999, 9.999, 9.999 and were observed at 505 nm, 500 nm, 490 nm, 485 nm, 480 nm and 475 nm respectively in Cuflavonoid complex. In Zn-flavonoid complex, the absorption peak 3.386 was observed at 245 nm while another absorption peak 10 were also observed at 510 nm, 505 nm, 495 nm, 490 nm, 485 nm, 475 nm and 450 nm in Ni-flavonoid complex.

The result for UV-Visible in Figure 14 maximum absorption spectra was observed 10 at 505 nm, 495 nm and 490 nm in Zn flavonoid complex and Cu-flavonoid complex and Ni-flavonoid complex absorption spectra were observed at 4.160 and 3.409 at 450 nm and 400 nm respectively in Vernonia baldwinii .

The result in Figure 15 for UV-Visible absorption spectra for complexes, Zn-flavonoid complex had the maximum absorption peak at 9.999 at 300 nm and 240 nm. The absorption peak of 4.47 was observed at 240 nm in Ni-flavonoid complex. The minimum spectrum was recorded at 3.247 at 240 nm in Cu-flavonoid complex. The ranged of the peak was 3.247-9.999 at the ranged of 240-300 nm.

UV-Visible spectra of flavonoid extracts

The UV-Visible absorption spectra for flavonoid extracts from the study plant leaves were also determined by UV-Visible spectroscopic and shown in Figures 16-20. In Moringa oleifera plant extract, the maximum absorption peak was 0.873 at 370 nm and the minimum absorption peak of 0.004 at 807 nm. The maximum peak of 0.982 at 286 nm and the minimum absorption peak of 0.022 at 829 nm were observed in Cassia tora plant leaves extracts. The maximum peak of 0.932 at 289 nm and the minimum absorption peak of 0.009 at 802 nm were observed in Ocimum gratissimum leaves extracts. In Vernonia baldwinii leaves extracts, the maximum absorption peak was 1.252 at 289 nm while the minimum absorption peak was 0.003 at 829 nm. The plant extracts of Telfairia occidentalis recorded the maximum absorption peak at 0.832 at 290 nm and the minimum absorption peak was 0.020 at 804 nm. These results agreed with the results of Duśan and Vesna [14].

Figure 16: UV-Visible Absorption Spectra for Moringa oleifera plant leaves extracts.

Figure 17: UV-Visible Absorption Spectra for Cassia tora plant leaves extracts.

Figure 18: UV-Visible Absorption Spectra for Ocimum gratissimum plant leaves extracts.

Figure 19: UV-Visible Absorption Spectra for Vernonia baldwinii plant leaves extracts.

Figure 20: UV-Visible Absorption Spectra for Telfairia occidentalis plant leaves extracts.

The study established that complexes were formed between metal and the flavonoid. The results showed a significant coordination for complexes as the values of the carbonyl functional group C=O of the free flavonoid shifted to lower peak in metal-flavonoid complexes. The complexes were formed at slightly acidic condition between the pH value 3.51 to 4.65. The study presents many applications, such as the possession of higher potencies toward superoxide than the parent flavonoids. Transition metals also enhance the anti-flammatory activities of flavonoids and their cytoprotective effect against oxidative injury in isolated cell. In addition, the study shows an outstanding impact as anti-oxidants thereby making them more effective in protecting red blood cells. This research will be very helpful in discovering new drugs and products for use in agriculture, health, medicine and pharmacy.