The Effect of Different Extracts of Beetroots as Antioxidant and Anti-Anaemia On Phenylhydrazine-Induced Rats

Anaemia is a condition when the number of red blood cells or haemoglobin concentration in the body is lower than normal. Anaemia is a problem that can occur at any age. WHO estimates that 42% of children less than five years of age and 40% of pregnant women worldwide are anaemic. In Indonesia, anaemia's prevalence in women of reproductive age is 27.85% and in children under five years is 36.78% (World Health Organization, 2020). The main cause of anaemia is lack of nutrition (inadequate supply of iron), drug toxicity, blood loss, genetic, or pathological diseases (Chaddha & Mittal, 2016).

Anaemia due to iron deficiency has a significant impact on human health. However, this rarely directly causes death. Erythrocytes require large amounts of iron to produce heme and haemoglobin. Iron deficiency results in decreased formation of red blood cells, which causes anaemia. Based on the research by Jaiswal et al. (2014), the ethanol extract of beetroot contains high iron, folic acid, and vitamin C. These contents are presumed related to the extract hematinic effect on phenylhydrazine-induced rats. Beetroot also contains iron, magnesium, potassium, manganese, copper, sodium, calcium, zinc (Odoh & Okoro, 2013), and bioactive compounds such as betalains, alkaloids, flavonoids, terpenoids, steroids, glycosides, and saponins (Hadipour et al., 2020). These contents have benefits in improving the haematopoiesis process.

Flavonoids are polyphenolic secondary metabolic compounds. These compounds have a protective effect against membranes lipo-peroxidative damage caused by free radicals (Nijveldt et al., 2001). Flavonoid-rich extracts effectively overcome the effects of phenylhydrazine induced malondialdehyde (MDA) damage to red blood cells membrane glycerol back-bone and peroxidation of phospholipids (Ologundudu et al., 2009; Chaddha & Mittal, 2016). On the other hand, tannins were centred on tannic acid, and other hydrolysable tannins are responsible for reductions in feed intake related to the antinutritional effects. It could bind with protein and red blood cells (Chung et al., 1998), affecting haemoglobin levels. Tannins could also inhibit iron absorption. Therefore, most pregnant women who consume the source of tannins like tea go through anaemia (Shah et al., 2020), and it is recommended that tannin-rich foods are not consumed together with meals (Santos-Buelga & Scalbert, 2000). Alkaloids from Datura stramonium sp. can reduce levels of erythrocytes, haemoglobin, and haematocrit by interfering with the process of erythropoiesis and causing the process of destruction of blood cells (Benouadah et al., 2016). Quinoline group alkaloids are used as antimalarial drugs by providing a binding effect to heme, a product related to haemoglobin, making the heme-quinoline conjugate toxic and interferes with haemoglobin activity (Heinrich et al., 2012).

Extracting solvent is one of the factors that influence the extraction results. The solvent's polarity is an aspect that underlies the selection of the extracting solvent (Tiwari et al., 2011). The hydroalcoholic extract contains phenolics, flavonoids, alkaloids, carbohydrates, glycosides, and tannins in beetroot (Ahmad et al., 2013). Alkaloids in plant material can be removed by acidification using citric acid. The residue extracted with 70% ethanol still contains flavonoid compounds as in the crude extract (Widiyanti et al., 2016). Meanwhile, tannins are slightly soluble in dichloromethane. This study aims to evaluate the effects of anti-anaemia and antioxidants in different extracts include dichloromethane extract (DEB), hydroethanolic extract (HEB), and alkaloids-free hydroethanolic extract (AFHEB) of beetroots. The difference in chemical composition contained in each extract obtained is expected to indicate differences in anti-anaemia and antioxidant activities. Thus, this study can be used as a reference to optimize the development of beetroots extract as a supplement to overcome anaemia conditions.

Characteristics of the beetroot extracts include a percentage of yield, loss of drying, and ash content shown in Table 1. Based on Table 1, HEB contains more chemical compounds than other extracts (AFHEB > DEB).

Phytochemical screening aims to identify the presence of chemical substances in the sample qualitatively. Based on the results in Table 2, it can be concluded that phenolic, flavonoids, tannins, alkaloids, and saponin compounds are present in extracts. Meanwhile, saponins compounds are only found in HEB. HEB contains high levels of flavonoids and iron (Fe) compared to other extracts (AFHEB > DEB) (Table 3).

Based on Table 4, the erythrocyte, haemoglobin, and haematocrit levels of beetroot extract groups showed a significant difference compared to the negative control group (p < 0.05). HEB group showed the best result, where all the blood parameters comparable to normal control group. Beetroot extract also showed activity as an antioxidant, shown by reduced MDA levels (Figure 1). The best antioxidant activity also showed by HEB, comparable to the normal control (p = 0.352 > 0.05).

Figure 1

The selection of the suitable organic solvent affects the success in solid-liquid extraction. It is usually done based on the dielectric constant (ε) characteristic of the extracting solvent, which is closely related to its polarity index (Katritzky et al., 2004). Water has a dielectric constant of 78.30, while ethanol is 24.30 (Khoddami et al., 2013) and dichloromethane is 9.1 (Saeker et al., 2006). The polarity index values for water, ethanol, and dichloromethane were 9.1, 5.2, and 3.7, respectively (Snyder, 1974). It shows that water is more polar than the other two solvents (water> ethanol> dichloromethane). The principle of ‘like dissolved like’ is defined as the condition where a phytochemical substance is dissolved in a similar polarity solvent (Wakeel et al., 2019). In other words, a polar solvent will be able to dissolve a polar substance, and a nonpolar solvent can dissolve a nonpolar substance. Some solvents might have similar polarity index values but could attract compounds of different quantities (Khoddami et al., 2013). For example, in the use of absolute ethanol extracting solvents or ethanol-aqueous (with variations of ethanol concentration in them). This difference can occur due to the differences in each solvent's ability to form chemical bonds with the bioactive metabolites contained in the plant matrix (Huaman-Castilla et al., 2019). Other things that can also affect plant chemical constituents’ acquisition are the bioactive compounds’ chemical structure, the extraction time, and the temperature used (Khoddami et al., 2013). The difference in the type and polarity of the extracting solvent can cause differences in the quality, quantity, toxicity, bioactivity, and safety of the extract produced (Eloff, 1998).

In this study, the extracted beetroot activity was tested using a variety of solvents as anti-anaemia and antioxidants. A previous study by Jaiswal et al. (2014) reported that ethanol extract of beetroot (200 mg.Kg⁻¹ BW) extracted in a Soxhlet apparatus effectively increases the levels of haemoglobin and erythrocytes. Vitamins (such as Vitamin C and folic acid) and minerals (one of them is iron) in beetroots are active ingredients responsible for these activities. Since some of the compounds, such as tannins and alkaloids could interfere with the hematopoietic activity, in this research, we try to optimize the result by evaluating the anti-anaemic activity in dichloromethane extract (DEB), hydroethanolic extract (HEB), and alkaloids-free hydroethanolic extract (AFHEB) of beetroots.

The yields of different extracts of beetroots increased in the following order: DEB < AFHEB < HEB. The highest yield was found in HEB (34.52%) followed by AFHEB (24.55%) > DEB (1.92%). It shows that polar compounds dominate in beetroots. It might also contain non-secondary metabolite polar compounds that also dissolve during the extraction process, such as carbohydrates (which are composed of fibres and sugar) (Neha et al., 2018), proteins, essential and non-essential amino acids, and other compounds (Hadipour et al., 2020). Beetroot contains bioactive compounds such as phenolics (epicatechin, catechin hydrate, vanillic acid, p-coumaric acid, protocatechuic, caffeic acid, syringic acid, proline, dehydro vomifoliol, 4-hydroxybenzoic acid, chlorogenic acid, and ferulic acid, etc.), flavonoids (betagarin, betavulgarin, cochliophilin A, dihydroisorhamnetinas, 2,5-dihydroxy 6,7-methylenedioxyisoflavone, 3,5 dihydroxy-6,7-methylenedioxyflavanone, 5-hydroxy-6,7 methylenedioxyflavone, rutin, quercetin, and 40-hydroxy-5-methoxy-6,7-methylenedioxy flavanone), saponins (betavulgarosides (I, II, III, IV, V, VI, VII, VIII, IX, X), hederagenin, akebonoic acid and gypsogenin), betalains (betacyanin and betaxanthin), alkaloids (calystegine B1, calystegine B2, calystegine C1, calystegine B3 and ipomine), triterpenoids/steroids (beta-amyrin acetate, boehmerylacetate and friedelin), volatile compounds (pyridine and 4-picolene), and so on (Hadipour et al., 2020).

Tiwari et al. (2011) explained that 70% ethanol as an extraction solvent was able to dissolve phenolic compounds (include flavonoids) better than absolute ethanol. Ethanol dissolves polyphenolic compounds, tannins, flavonoids (flavonols), triterpenoids, steroids, and alkaloids. In comparison, DEB showed the least number of phytochemicals. Dichloromethane (CH₂Cl₂) is a solvent with a medium polarity level. The polarity of dichloromethane due to its electronegative Cl atoms (Houghton & Raman, 1998). Dichloromethane is specially used for the selective extraction of only terpenoids (Tiwari et al., 2011) but is still able to dissolve alkaloids and aglycones (include flavonoids) (Houghton & Raman, 1998). Dichloromethane has a low ability to dissolve tannin compounds. Therefore, in the phytochemical screening evaluation (Table 1), tannins were not detected in DEB. Meanwhile, the AFHEB contains tannin but no alkaloids. Both total flavonoid and iron levels in the AFHEB are lower than in the HEB (Table 3). In this research, the procedure for removing alkaloids from the dried powder of beetroot not only results in the loss of alkaloids but also other compounds including flavonoids, iron or might be the pigment compounds of beetroot. Betalains are ‘chromoalkaloids’ found in plants of the order Caryophyllales (except in the Caryophyllaceae and Molluginaceae families) (Wink, 2010). Betalains are a natural nitrogen-containing pigment compounds that have two structural groups, viz. the red-violet betacyanins (betanin, prebetanin, isobetanin and neobetanin) and the yellow to orange betaxanthins (vulgaxanthin-1, vulaxanthin-2, and indicaxanthin) (Hadipour et al., 2020). These compounds are indole-derived (Ninfali et al., 2017; de Oliveira et al., 2020) that can be water or alcohol-extracted and stable at pH from 2 to 6 (Azeredo, 2009). Halwani et al. (2018) reported that red pigment compounds (betanine and vulgaxanthin-1) were found in high amounts in water extract and citric acid extract of beetroot in low pH conditions. The presence of this compound can result in a false positive reaction in alkaloid identification using Dragendorff and Mayer precipitated reagents. It is known that these two reagents can also react with nitrogen-containing compounds other than alkaloids (Evans, 2009). We recommend that when identifying alkaloids with these reagents, the test solution must be free from compounds with nitrogen atoms (such as proteins, amino acids, or betalains).

The results showed that phenylhydrazine induction for three days in the negative control group reduced the number of erythrocytes by 32.4%. Phenylhydrazine induces oxyhaemoglobin into methaemoglobin and produces hydrogen peroxide. This hydrogen peroxide causes lipid peroxidation in the red blood cell membrane resulting in lysis of the blood cells, leads to the condition known as haemolytic anaemia (Singh et al., 2014). In this study, the increase in Fe levels and antioxidant activity was in line with the increase in anti-anaemia activity. The DEB and AFHEB showed less potential activities antioxidant (Figure 1.) and lower Fe content than the HEB. The same trend was shown in the anti-anaemia activity (Table 4). Iron levels influence haemoglobin production in the body. Iron is an essential component in the formation of heme molecules. The body needs nearly 30 mg of iron each day in the erythropoiesis process (the formation of erythrocytes in bone marrow), and only 1–2 mg can be absorbed (Lesjak & Srai, 2019). As the flavonoid-rich plant, administration of beetroot prevents further oxidation on the red blood cells, therefore prevent further damage to the cells (Chaddha & Mittal, 2016). The pigment in beetroot also acts as an antioxidant. Research showed that beetroot betalains could reduce MDA levels in male mice (Clifford et al., 2015). Red pigment betacyanin is also a powerful antioxidant and protects against several types of cancer (Neha et al., 2018). Betacyanins have better antiradical activity in vitro due to their hydroxyl group position than betaxanthin (Azeredo, 2009).

Polyphenols (including phenolic acids, flavonoids, lignans, and stilbenes) are a source of natural antioxidants that can be obtained from medicinal plants. Flavonoids (including flavonols, flavanones, catechins, flavones, anthocyanidins, and isoflavonoids) are found in vegetables and fruits. Flavonoids have also been studied to improve haematological parameters, increase iron levels in the spleen tissue, and ferroportin expression in iron deficiency anaemia (Mazhar et al., 2017). In addition, secondary metabolites such as saponins, phenolics, and glycosides might also be responsible for the acclaimed anti-anaemic potential of plants used in traditional medicine (Gbadamosi et al., 2012).

This research shows that the content of tannins and alkaloids in the beetroot ethanol extract does not affect its activity as anti-anaemia. However, it is crucial to quantitatively measure the presence of tannins and alkaloids in each extract to support definite conclusions regarding their connection to anti-anaemia activity in beetroots.

The hydroethanolic extract of beetroot has good anti-anaemia and antioxidant activities. Based on this study, the chemical constituents of the extract plays an important role in its pharmacological activity. The appropriate extraction methods of beetroot can produce an extract as a source of raw materials with maximum chemical content to achieve the intended pharmacological activity.