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The chemical composition and membrane stability activity of some herbs used in local therapy for anemia
Journal of Ethnopharmacology 102 (2005) 15–22
The chemical composition and membrane stability activity of some herbs used in local therapy for anemia O.S. Falade a , I.O. Otemuyiwa a , A. Oladipo b , O.O. Oyedapo c , B.A. Akinpelu c , S.R.A. Adewusi a,∗ b
a Department of Chemistry, Obafemi Awolowo University, Ile-Ife, Nigeria Centre for Energy Research and Development, Obafemi Awolowo University, Ile-Ife, Nigeria c Department of Biochemistry, Obafemi Awolowo University, Ile-Ife, Nigeria
Received 9 September 2004; received in revised form 22 April 2005; accepted 29 April 2005 Available online 21 July 2005
Abstract The chemical composition and the membrane stabilizing activity of the decoctions of the dry Hibiscus sabdariffa Linn. (Malvaceae) calyx (Sobo in a Nigerian language), Theobroma cacao Linn. (N.O. Sterculiaceae) root (cocoa) and the dry stalk of white and red cultivars of Sorghum bicolor (L.) Moench (Poaceae) were determined. These herbs are often used locally for the management of anemia. Ash content was from 3.5 to 7.2%, tannin 7–46 mg/kg catechin equivalent, ascorbic acid content 250–625 mg/kg and total anthocyanin 57–150 mg/kg cyanidin-3glucoside equivalent. Phytate was not detected in any sample but alkaloids and saponins were present except in Sobo. The decoctions contained 0.2–2.0% of the herbs’ total tannin content while the pH was 3.2–6.7. Iron and zinc content was highest in Sobo while Theobroma cacao root contained the highest calcium and magnesium. Solubility and mineral availability were highest in the decoction of Sobo and lowest but available in Theobroma cacao root decoction. The decoction of Theobroma cacao exhibited a high membrane stabilizing activity while those of Sorghum bicolor and Hibiscus sabdariffa were only average. The study indicated that the herbal decoctions could provide some minerals and also help to stabilize the red blood cells from stress injury. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Hibiscus sabdariffa; Sorghum; Cocoa; Tannin; Ascorbic acid; Membrane stabilizing activity
1. Introduction The use of traditional medicine has been recently encouraged by World Health Organization (WHO) and United Nation Children’s Fund (UNICEF) for its cultural role, greater availability and acceptability than the modern pharmaceutical agents (Mahu, 1988; Mabina and Pitose, 1997). In Nigeria, there is a general belief that certain herbs contain haematopoietic properties and could serve as “blood tonic”; the medicinal application of the infusions or decoctions of such herbs has gained wide popularity amongst the population. Such herbs include the stem, root and bark of Theobroma cacao Linn. (N.O. Sterculiaceae) (cocoa), the calyx of Hibis∗
Corresponding author. Tel.: +234 803 5528166. E-mail address: [email protected] (S.R.A. Adewusi).
0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.04.034
cus sabdariffa Linn. (Malvaceae) locally called “Sobo” and the stalk of Sorghum bicolor (L.) Moench (Poaceae). The therapeutic action of a plant depends on its chemical constituents. Plants have been reported to contain compounds such as tannin, saponin, phytate, fibre and phenolic compounds which play prominent roles in mineral nutriture as inhibitors. The presence of these compounds in tea, coffee and other beverages has been shown to inhibit iron absorption from composite meals (Gilooly et al., 1983; Brune et al., 1991; Hurrell et al., 1999). Other agents in plant products such as ascorbic and other organic acids have been known to enhance mineral solubility and uptake (Hallberg et al., 1986; Adewusi et al., 1999). The role of the decoctions from these herbs in enhancing iron availability from foodstuffs was highlighted in the first paper in this series (Otemuyiwa et al., 2005), to indicate the
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relevance of the herbal decoctions in the management of anemia in Nigeria. In addition, the possibility that the decoctions of these herbs could have other pharmacological effects such as the stabilization of erythrocyte membranes in vivo and in vitro was expressed as shown for the extracts of Zanthoxylum zanthoxyloides, Olax subscorpioides and Tetrapleura tetraptera (Oyedapo and Famurewa, 1995). The main objectives of the present study are to:
2.3.2. Determination of soluble minerals in herbs Ten grams each of washed and dried samples was boiled in 200 ml of distilled water for 30 min (as done traditionally). After boiling, the volume of the decoction was recorded and the solution was analyzed for soluble minerals (iron, zinc, calcium and magnesium) using the Atomic Absorption Spectrophotometer.
(a) evaluate the chemical contents of the common haematopoietic herbs used in Nigerian traditional medicine; (b) ascertain the level and effect of antinutritional factors as well as some of the enhancing agents on mineral availability in these herbs; (c) investigate the membrane stabilizing effect of the decoctions and thus supply some information on the potentials of these herbs for the management of anemia.
2.3.3. Determination of available minerals in herbs The available mineral content was determined by the method of Miller et al. (1981) with modifications. Titratable acidity was first determined on 50 ml of each herbal decoction as described earlier (Miller et al., 1981). Fifty millilitres of each decoction was measured into a 100 ml beaker; 10 ml of water, NaHCO3 corresponding to the titratable acidity (Miller et al., 1981) and a magnetic bar were placed in a dialysis tubing and put into the beaker of the decoction and incubated at 37 ◦ C for 2 h on a magnetic stirrer. The content of the dialysis tube was removed, the tube was then washed with distilled water and the volume of the dialysate noted and was then taken for mineral analysis using the Atomic Absorption Spectrophotometer.
2. Materials and methods 2.1. Herbal materials The dry fermented calyx of Hibiscus sabdariffa Linn. (Malvaceae) and the dry stalk of two cultivars of Sorghum bicolor (L.) Moench (Poaceae) coded red and white were bought at a local herb market in Ile-Ife, Nigeria, while the root of Theobroma cacao Linn. (N.O. Sterculiaceae) was collected from a cacao plantation on Obafemi Awolowo University Campus, Ile-Ife. Voucher samples were deposited in the herbarium of the Department of Botany, Obafemi Awolowo University, Ile-Ife: the root of Theobroma cacao—IFE 2282, Hibiscus sabdariffa calyx—IFE 2328, red colored stalk of Sorghum bicolor—IFE 10976 and the stalk of white Sorghum bicolor without a distinct coloration—IFE 10975. 2.2. Methods The herbs were washed with distilled water, dried at 50 ◦ C overnight, milled in a ball grinder (Siebtechnik TS 250) and then sieved with No. 72 mesh size (Griffin and George Ltd., London). The samples were stored in plastic containers with screw cap and kept in the freezer until used. 2.3. Analytical techniques The ash content of each sample was determined by the Association of Official Analytical Chemists (AOAC, 1984) method by heating each sample in a muffle furnace at 550 ◦ C until white ash was obtained. 2.3.1. Mineral content determination The ash from the procedure above was dissolved in 2 ml concentrated HNO3 and made up to mark in a 50 ml volumetric flask. The mineral content from the resulting solution was determined using the Atomic Absorption Spectrophotometer (Alpha 4 model, Fisons Chem-Tech, Analytical, UK).
2.3.4. The effect of colored compounds on soluble and available minerals One hundred millilitres of the decoction was decolorized using 0.5 g acid washed Norit (activated charcoal) and made up to 100 ml with distilled water. A portion of the decolorized decoction was analyzed for soluble minerals while another portion was analyzed for mineral availability as described above (Miller et al., 1981). 2.3.5. The tannin content The tannin content in the samples was determined by the modified vanillin–hydrochloric acid (MV–HCl) method (Price et al., 1978) using catechin as a standard. 2.3.6. Phytate content Phytate content of the samples was determined by the anion exchange method (Harland and Oberleas, 1986) using KH2 PO4 solution (80 g/ml) as the standard. 2.3.7. The ascorbic acid The ascorbic acid content was determined using the method of Roe and Kuether (1943) with slight modifications. The milled sample (2.0 g) was extracted with 25 ml of 0.1% (v/v) oxalic acid, the residue washed twice with 10 ml of the extraction solvent, centrifuged and the supernatant was made up to 50 ml in a standard flask with the extraction solvent. Two millilitres of each filtrate was placed in two test tubes, a drop of 10% (v/v) thiourea solution and 0.5 ml of 2% (w/v) of 2,4dinitrophenyl hydrazine reagent were added. The test tubes were placed in a water bath at 37 ◦ C for 3 h and then placed in a beaker of ice water; 2.5 ml of 98% (v/v) H2 SO4 was added in drops from the burette within 1.0 min. The absorbance was
O.S. Falade et al. / Journal of Ethnopharmacology 102 (2005) 15–22
taken after 30 min at 520 nm using the Spectrophotometer and estimated against a standard ascorbic acid curve. 2.3.8. Total anthocyanin Total anthocyanin in Hibiscus sabdariffa and Sorghum bicolor was determined by the method of Abdel-Aal and Huel (1999) and the result calculated as cyanidin-3-glucoside equivalent. 2.3.9. The qualitative screening for alkaloids and saponins The samples were screened for the presence of alkaloids and saponins by the method of Odebiyi and Sofowora (1978). The presence of alkaloids was tested by Dragendorff’s reagents, 10% (w/v) tannic acid and saturated picric acid solution while saponin was tested by the frothing method. 2.3.10. Preparation of erythrocyte Bovine erythrocyte was prepared as previously described (Sadique et al., 1989; Oyedapo and Famurewa, 1995). Fresh blood samples were collected into an anticoagulant containing trisodium citrate (3.8%, w/v) and mixed thoroughly. The blood samples were then centrifuged at 3000 rpm for 5 min at room temperature. The supernatant was decanted and the packed red blood cells were washed in fresh isosaline solution followed by centrifugation. The washing process was repeated until the supernatant was clear. Then, 2% (v/v) erythrocyte suspension was prepared in normal saline solution. 2.3.11. Preparation of drugs and decoctions Standard drugs, Indomethacin and Ibuprofen, were prepared in iso-saline solution to a final concentration of 2.5 mg/ml. Ten grams of each herb was boiled in distilled water for 30 min, decanted, cooled and evaporated in vacuo at 40 ◦ C. The residue was weighed and re-dissolved in isosaline. 2.3.12. Assay of membrane stabilizing activity This was carried out as earlier described (Odebiyi and Sofowora, 1978; Sadique et al., 1989) using extract concentrations that provided maximum activities as follows: 3 mg/ml (Sobo); 5 mg/ml (red Sorghum bicolor); 1 mg/ml
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(white Sorghum bicolor); 1 mg/ml (Theobroma cacao); the standard drugs, 2.5 mg/ml. In essence, the assay mixture consisted of 2 ml of 0.25% (w/v) sodium chloride, 1.0 ml of 0.15 M sodium phosphate buffer (pH 7.4), 0.5 ml of 2% (v/v) erythrocyte suspension, 0–1.0 ml of drugs or decoctions and the final mixture was made up to 4.5 ml with iso-saline. Drugs were omitted in the first control while the drug control lacked the erythrocyte suspension. The reaction mixtures were incubated at 56 ◦ C for 30 min, cooled under running water and centrifuged at 5000 rpm. The supernatant was collected and the color of the released haemoglobin read at 560 nm on the Spectrophotometer. The percentage membrane stability was estimated from the expression: 100 − (absorbance drugtest − absorbance drugcontrol ) × 100 absorbance drugcontrol where the control represents 100% lysis or zero membrane stability.
3. Results 3.1. Chemical compositions The chemical compositions of the herbs are presented in Table 1. The analysis of whole samples revealed that the percentage ash content in the stalk of white sorghum was significantly higher (p < 0.05) than that of the red variety. The tannin content in herbs was high in Theobroma cacao root, low in Hibiscus sabdariffa calyx and moderate in both stalks of white and red sorghum. Phytate was not detected in any of the samples analyzed. The ascorbic acid content was highest in Hibiscus sabdariffa but undetectable in the Theobroma cacao root. The stalk of white sorghum contained the highest level of anthocyanin, closely followed by the red sorghum stalk while it was not detectable in Theobroma cacao root. Tannin was present in negligible quantity in all the herbal decoctions and totally unextractable in Sobo (Table 1). The pH of the decoction was lowest in Sobo and highest in the decoction of the white sorghum stalk. The phytochemical screening (Table 2) showed that alkaloids and saponins were present in all samples except Sobo.
Table 1 Chemical composition of the herbs (on dry weight basis) and the decoctions Sample
Hibiscus sabdariffa Sorghum bicolor (red) Sorghum bicolor (white) Theobroma cacao root
Herbs
Decoctions
Ash content (%)
Ascorbic acid (mg/kg)
Total anthocyanin (mg/kg)
Tannin (g/kg)
7.2 ± 0.2a 3.5 ± 0.1c 6.5 ± 0.3b 7.2 ± 0.3a
625 ± 0a 375 ± 0b 250 ± 1c N.D.
57 ± 0c 135 ± 0b 150 ± 1a N.D.
7.0 11.0 11.0 46.0
± ± ± ±
0. 9 c 0.6 b 0.2b 0.1a
Tannin (mg/dm3 )
Tannin (%)
pH
N.D. 0.03 ± 0.02 0.20 ± 0.00 0.11 ± 0.06
– 0.3 2.0 0.2
3.2 ± 0.1 5.5 ± 0.4 6.7 ± 0.1 6.5 ± 0.6
Mean ± standard deviation of triplicate analysis. Values in the same column with the same superscript letters (a–c) are not significantly different at p < 0.05. N.D.: not detected.
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Table 2 Results of phytochemical screening for alkaloids and saponins Sorghum bicolor (red)
Sorghum bicolor (white)
Theobroma cacao root
Hibiscus sabdariffa calyx
Picric acida Wagners reagenta Tannic acida Mayer’s reagenta Dragendorff’s reagenta
+ve +ve +ve +ve +ve
+ve +ve +ve +ve +ve
+ve +ve +ve +ve +ve
−ve −ve −ve −ve
Saponin Froth test Lieberman–Burchard test
−ve +ve
−ve +ve
+ve +ve
−ve −ve
Sample/reagents Alkaloida
a
The reagents were used to test for the presence of alkaloids.
3.2. Mineral content of the herbs The mineral content of the herbs presented in Table 3 indicated that total iron ranged between 60 and 177 mg/kg. The soluble iron concentration in the herbs was lowest in Theobroma cacao root decoction while the highest concentration was recorded for Sobo. Availability of iron was highest in Sobo and lowest in Theobroma cacao root but the highest percentage available iron was recorded for red sorghum stalk followed closely by Theobroma cacao root despite the low iron solubility recorded in the decoctions. The result presented in Table 3 also indicated that the total zinc in the herbs ranged from the low level (3 mg/kg) in red sorghum stalk to the highest level (28 mg/g) in Sobo. The soluble zinc concentration in the decoction was highest in Sobo and lowest in Theobroma cacao. Despite the fact that the zinc content of the Theobroma cacao root was the least soluble, its avail-
ability was virtually total. Analysis of the calcium content of the herbs (Table 3) indicated that Theobroma cacao root contained the highest level of total calcium (11,650 mg/kg) followed by Sobo (9700 mg/kg). The total calcium content of the stalk of the red and white Sorghum bicolor was 1300 and 1840 mg/kg, respectively. The highest soluble calcium was again found in the decoction of Sobo and lowest in Theobroma cacao. The available calcium and percentage availability were highest in Sobo and least in red Sorghum bicolor. The total magnesium content of the herbs was lowest (1750 mg/kg) in the stalk of white Sorghum bicolor and highest in Theobroma cacao (7500 mg/kg). The highest content of soluble magnesium, available and percentage available magnesium were recorded in Sobo while Theobroma cacao root recorded the lowest soluble magnesium. Sorghum bicolor red stalk recorded the least value for both available and percentage available magnesium.
Table 3 Mineral content of the herbs Sample
Total mineral (mg/kg)
Soluble mineral (mg/dm3 )
Soluble mineral (%)x
Available mineral (mg/dm)
Available mineral (%)y
Iron Hibiscus sabdariffa Sorghum bicolor (red) Sorghum bicolor (white) Theobroma cacao root
177 ± 60 ± 79 ± 144 ±
1a 8d 7a 30b
39.0 2.3 1.5 0.4
± ± ± ±
0.3a 0.1b 0.3bc 0.0c
22 4 2 0.3
4.80 0.80 0.30 0.13
± ± ± ±
0.10a 0.10b 0.10c 0.01d
12 35 20 33
Zinc Hibiscus sabdariffa Sorghum bicolor (red) Sorghum bicolor (white) Theobroma cacao root
28 ± 3± 8± 20 ±
2a 0d 1c 2b
10.0 1.0 2.0 0.5
± ± ± ±
1.2a 0.1c 0.1b 0.1c
36 33 25 3
1.30 0.26 0.26 0.46
± ± ± ±
0.10a 0.10c 0.10c 0.10b
13 26 13 92
Calcium Hibiscus sabdariffa Sorghum bicolor (red) Sorghum bicolor (white) Theobroma cacao root
9700 ± 1300 ± 1840 ± 11650 ±
900b 230d 137c 349a
3527 ± 208 ± 226 ± 109 ±
216a 41b 10b 7.5c
36 16 12 1
466 ± 3.2 ± 5.6 ± 4.0 ±
11a 0.4c 0.1b 0.4bc
13 2 3 4
Magnesium Hibiscus sabdariffa Sorghum bicolor (red) Sorghum bicolor (white) Theobroma cacao root
2150 ± 1850 ± 1750 ± 7500 ±
40b 180c 76d 713a
1341 ± 1086 ± 776 ± 515 ±
28a 28a 13c 3c
62 58 44 7
232 ± 13 ± 40 ± 20 ±
6a 4d 3b 1c
24 1.0 5 4
Mean ± standard deviation of triplicate analysis. Values in the same column with the same superscript letters (a–d) are not significantly different at p < 0.05. x Soluble mineral (%) is expressed as fraction of the total mineral in the herbs. y Available mineral (%) is expressed as a fraction of soluble mineral in the decoction.
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3.3. Effect of colored compounds on mineral solubility and availability The result of the effect of colored compounds on mineral (Fe, Zn, Ca and Mg) solubility and availability presented in Table 4 indicated that soluble iron content of the decolorized decoction of Sobo was significantly lower (t9.9 , p < 0.01) than the colored decoction but there was no significant difference in the available iron content from both decoctions. There was no significant difference (t4.3 , p < 0.05) in the soluble and available iron from decoctions of both white and red sorghum stalk. The result also indicated that there was no significant difference between the solubility and availability of zinc and calcium (t4.3+, p < 0.05) in colored and decolorized decoctions though the soluble magnesium from Hibiscus sabdariffa and red Sorghum bicolor decoctions was significantly (t4.3 , p < 0.05) higher than from the decolorized.
4. Discussion The ash content ranged from 3.5 to 7.2% with the least percentage ash recorded for the red Sorghum bicolor stalk while the highest was recorded for both Sobo and Theobroma cacao (Table 1). The range of 6.5–19.4% had been reported for some Nigerian vegetables (Awoyinka et al., 1995). The pH value (Table 1) indicated that Sobo decoction was acidic (pH 3.2), which is a consequence of the fermentation of the Hibiscus sabdariffa calyx before sale in Nigeria. The solubility of minerals such as iron and calcium increases in acidic solution, thereby promoting their availability (Champagne and Rao, 1985; Adewusi et al., 1999). pH also plays an important role in the binding of minerals by both dietary fibre and
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phytate (Brune et al., 1992; Vauquero, 1993). Even in the absence of dietary fibres, iron had been found to precipitate progressively as the pH increases (Reinhold et al., 1982). It is therefore not surprising that the low pH of the decoction of Sobo and the stalk of the red Sorghum bicolor would explain in part the increased solubility and availability of the minerals analyzed in this study. The ascorbic acid content (Table 1) was highest in Sobo (625 mg/kg) and may contribute to the low pH (3.2) of this decoction. The level of ascorbic acid in the stalk of red Sorghum bicolor (375 mg/kg) was higher than the white variety (250 mg/kg); this seemed to affect the pH of the decoctions accordingly. Ascorbic acid is heat labile and since these samples have been subjected to various degrees of heat treatment, the ascorbic acid of the decoction could have been reduced. It has been demonstrated that ascorbic acid is one of the ligands that promote absorption of iron from meals (Hallberg et al., 1986; Oladipo et al., 2004) and could therefore account in part for the availability of iron and other minerals induced by the decoctions. Phytate, which chelates minerals (Cheryan, 1980; Adewusi and Falade, 1996), was not detected in any of the herbs analyzed (Table 1). The tannin content in whole samples was lower than the range of 27–92 g/kg catechin equivalent reported for tea (green and black) and coffee powder (Brune et al., 1991) and lower still in all the decoctions and below detection level in the Sobo decoction. The absence of tannin in the decoction of Sobo and its presence below lethal level in other decoctions indicated that tannin from these herbs is not likely to play any significant role in mineral availability of composite meals. The range reported here for the total anthocyanin was lower than the range recorded in barley and wheat bran but
Table 4 Effects of colored compounds on mineral solubility and availability from the herbs Sample
Soluble mineral (mg/dm3 )
Available mineral (mg/dm3 )
COL.
DECOL.
DF (%)
COL.
Iron Hibiscus sabdariffa Sorghum bicolor (red) Sorghum bicolor (white)
39.0 ± 0.3a 2.3 ± 0.1 1.5 ± 0.3
30.6 ± 0.5b 2.2 ± 0.2 1.3 ± 0.1
22 4 13
4.8 ± 0.1 0.8 ± 0.1 0.3 ± 0.1
Zinc Hibiscus sabdariffa Sorghum bicolor (red) Sorghum bicolor (white)
10.0 ± 0.2 1.0 ± 0.1 2.0 ± 0.6
8.5 ± 1.7 1.0 ± 0.2 1.5 ± 0.3
15 – 25
Calcium Hibiscus sabdariffa Sorghum bicolour (red) Sorghum bicolor (white)
3527 ± 21 208 ± 41 226 ± 0
3505 ± 63 189 ± 0 224 ± 1
Magnesium Hibiscus sabdariffa Sorghum bicolor (red) Sorghum bicolor (white)
1341 ± 28a 1086 ± 48a 776 ± 13
1160 ± 11b 1005 ± 7b 777 ± 36
0.6 9 0.8 13 7 –
DECOL.
Available (%) DF (%)
COL.
DECOL.
4.3 ± 0.1 0.6 ± 0.0 0.2 ± 0.0
10.4 22.5 26.6
12 34 20
14 28 31
1.3 ± 0.1 0.3 ± 0.1 0.3 ± 0.1
1.3 ± 0.1 0.3 ± 0.1 0.2 ± 0.0
4 0 15
13 26 13
15 26 15
466 ± 11 3.2 ± 0.4 5.6 ± 0.1
495 ± 5.6 3.2 ± 0.1 4.2 ± 0.8
+6 1 25
13 1.5 2.4
14 1.6 2.8
323 ± 6 13 ± 4 40 ± 3
344 ± 4 12.4 ± 1 35 ± 6
+6 5 13
24 1.2 5.2
29 1.2 4.5
Mean ± standard deviation of triplicate analysis. Values with superscript letters (a and b) are significantly different at (t9.9 , p < 0.01), other values are not significantly different. DF (%) is expressed as percentage difference in available and soluble mineral in colored and decolorized decoction. Available mineral (%) is expressed as a fraction of soluble mineral in colored and decolorized decoctions. COL.: colored decoction. DECOL.: decolorized decoction.
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higher than 22.5 mg/kg reported for wheat flour (Abdel-Aal and Huel, 1999). Anthocyanins have been reported to form complexes with metal ions, especially iron, magnesium, zinc and calcium, and this could affect their availability, but the present study has revealed that the presence of these water soluble pigments did not seem to affect mineral availability from the herbs. The content of iron in the herbs (Table 3) indicated that Sobo had the highest total iron while the least was recorded for the red sorghum stalk. The values recorded for the herbs were higher than 28 mg/kg total iron reported for cowpea leaves (Potter, 1983) and the range reported for green beans (Martinez et al., 1998). Iron was found to be more soluble and available from the decoction of Sobo probably with a low pH value, which is an indication of a high level of organic acids resulting from the fermentation of the calyx (Adewusi et al., 1999). Theobroma cacao root recorded the least soluble and available iron despite its high total iron content probably due to the high level of fibre, most especially cellulose, which has been reported to bind minerals (Reinhold et al., 1982). The high pH of its decoction (pH 6.5) could also contribute to the low solubility of its iron as indicated earlier (Clydesdale and Camire, 1983). The result also showed that iron was more soluble and available from the stalk of red Sorghum bicolor compared to the white cultivar possibly reflecting the difference in their pH (6.7 and 5.5 for white and red cultivars, respectively) and tannin (0.20 and 0.03 mg/dm3 for white and red cultivars, respectively). Decolorization did not seem to have a significant role in mineral availability (Table 4) since there was no significant difference (p < 0.05) between available iron from the decolorized and colored decoctions. The total zinc content recorded for Sobo compared favourably with 24 mg/kg total zinc reported for amaranthus (Adewusi et al., 1999). The highest soluble and available zinc was also recorded for Sobo while the least was recorded for Theobroma cacao. Theobroma cacao sample contained the highest tannin content (Table 1), which has been reported to inhibit the speciation of zinc and may thus be responsible for low zinc availability from the decoction. It is interesting to note that the decoction of Theobroma cacao root which recorded the least solubility and availability for zinc showed the highest percentage zinc availability (92%). The implication is that the little amount of zinc that was soluble was readily available. This decoction needs to be further investigated. The observed total calcium in this study was higher than the 7792–9135 mg/kg values reported earlier for amaranthus (Adewusi et al., 1999). The highest calcium solubility and availability was recorded for Sobo as reported earlier for iron and possibly for the same reasons (Adewusi et al., 1999). Other decoctions recorded less than 5% available calcium, which may be a consequence of their tannin and fibre content and more importantly the high pH of the decoctions in which calcium is expected to precipitate (Clydesdale and Camire, 1983). As observed in iron, colored compounds (Table 4) did
Fig. 1. Membrane stabilizing activity of the decoctions of Hibiscus sabdariffa calyx, white Sorghum bicolor and red Sorghum bicolor stalk.
not seem to have any significant effect on the availability of calcium. The highest solubility and availability of magnesium was recorded in the Sobo decoction. Magnesium forms water soluble complexes which could be responsible for its high solubility as well as availability of this mineral in Sobo. Colored compounds do not seem to have any significant role in magnesium availability from these decoctions as observed in iron and calcium. Theobroma cacao recorded the highest total magnesium but its solubility as well as availability was the lowest. This is expected since the roots’ main constituent, cellulose, is well known to bind minerals. The result of the bovine erythrocyte membrane stabilizing activity of the decoctions indicated that Hibiscus sabdariffa and red and white Sorghum bicolor stalks exhibited moderate membrane stabilizing activity (Fig. 1) which could be biphasic especially at high concentration of the extracts. The extract of the Theobroma cacao root exhibited a very high membrane stabilizing activity comparable to the membrane stabilization activity of the Olax subscorpioides (87.5%), Aspilia Africana (85%) and Fagara zanthoxyloides (83.8%) (Oyedapo and Famurewa, 1995; Oyedapo et al., 1997) extracts against heat
Fig. 2. Membrane stabilizing activity of the decoctions of Theobroma cacao root and the standard drugs—Ibuprofen and Indomethacin.
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and hypotonic induced stress and even marginally exceeded the activity of the standard drug Ibuprofen (Fig. 2). The haematopoietic activity of the decoction of Theobroma cacao root in vivo would therefore seem to be through a combination of two systems via the enhancement of mineral availability from foods as well as its membrane stabilizing activity
5. Conclusion The data reported here showed that decoctions of the herbs are capable of providing an alternative source of minerals as well as enhancing mineral availability from other food sources through the low pH of the decoctions. This could be especially true of the decoction from the fermented Hibiscus sabdariffa calyx which is now a popular drink called “Sobo” in Nigeria. The presence of ascorbic acid in the decoctions may be another mechanism whereby the herbs enhance mineral availability. There is a high positive correlation factor between the ascorbic acid content (Table 1) and the percent soluble minerals presented in Table 3: iron (r = 0.88), zinc (r = 0.93), calcium (r = 0.98) and magnesium (r = 0.92). This seems to implicate ascorbic acid in the speciation of the minerals in the herbs and hence their availability. This could prevent mineral deficiency diseases such as anemia. These findings give credence to the claim that the herbs could be effective in ameliorating iron deficiency anemia. Theobroma cacao root extract possesses the additional property of a high level of membrane stabilizing activity which could stabilize red blood cells from stress injury. This result will serve as a base data for nutritionists, traditional and orthodox medical practitioners who may be looking for an alternative source of iron for the treatment of anemia and some other mineral deficiency diseases. During the course of this study, a new herbal preparation “JUBI” incorporating Sorghum bicolor stalk has been introduced into the market in Nigeria as a support for the “management of moderate and severe anemia”. It was also reported that Hibiscus sabdariffa is a well known medicinal plant in Thailand (Chewonarin, 1999).
References Abdel-Aal, E.S.M., Huel, P., 1999. A rapid method for quantifying total anthocyanin in blue aleron and purple pericarp wheat. Cereal Chemistry 76, 350–354. Adewusi, S.R.A., Falade, O.S., 1996. The effects of cooking on extractable tannin, phytate, sugars and mineral solubility in some improved Nigerian legume seeds. Food Science and Technology International. 2, 231–240. Adewusi, S.R.A., Ojumu, T.V., Falade, O.S., 1999. The effect of processing on total organic acid content and mineral availability of simulated cassava-vegetable diets. Plant Foods for Human Nutrition 40, 367–380. AOAC, 1984. Official Methods of Analysis. Association of Official Analytical Chemists, Washington, DC.
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Awoyinka, A.F., Abegunde, V.L., Adewusi, S.R.A., 1995. Nutrient content of young cassava leaves and assessment of their acceptance as a green vegetable in Nigeria. Plant Food for Human Nutrition 47, 21–28. Brune, M., Hallberg, L., Skanberg, A., 1991. Determination of iron binding phenolic groups in foods. Journal of Food Science 56, 128–131. Brune, M., Rossander, H., Hallberg, L., Gleerup, A., Sandberg, A., 1992. Iron absorption from bread in humans; inhibiting effect of cereal fibre, phytate and inositol phosphate. Journal of Nutrition 122, 442–449. Champagne, E.T., Rao, P., 1985. Solution behaviour of the mineral, protein, and phytic acid in rice bran with time, temperature and pH. Cereal Chemistry 62, 218–222. Cheryan, M., 1980. Phytic acid interaction in food systems. Critical reviews. Food Science and Nutrition 14, 297–302. Chewonarin, T., 1999. Effect of Roselle (Hibiscus sabdariffa Linn.) a Thai medicinal plant on mutagenicity of various known mutagens in Salmonella typhimurium. Food Chemistry and Toxicology 37, 591–601. Clydesdale, F.M., Camire, A.L., 1983. The effect of pH and heat on the binding of iron, calcium, magnesium and zinc and the loss of phytic acid in soy flour. Journal of Food Science 48, 1272–1283. Gilooly, M., Bothwell, T.H., Torrance, J.D., Macphail, A.P., Derman, D.P., Bewoda, W.R., Mills, W., Charlton, R.W., Meyer, F., 1983. The effect of organic acids, phytate and polyphenols on the absorption of iron from vegetables. British Journal of Nutrition 49, 331–342. Hallberg, L., Brune, M., Rossander, L., 1986. Effect of ascorbic acid on iron absorption from different types of meals. Human NutritionApplied Nutrition 40A, 97–113. Harland, B.F., Oberleas, D., 1986. Anion exchange method of determination of phytate in foods collaborative study. Journal of Association Official Analytical Chemistry 69, 667–670. Hurrell, R.F., Reddy, M., Cook, J.D., 1999. Inhibition of non-heme iron absorption in man by polyphenolic containing beverage. British Journal of Nutrition 81, 289–295. Mabina, M.H., Pitose, S.B., 1997. The use of traditional herbal medication during pregnancy. Tropical Doctor 27, 84–86. Mahu, J.L., 1988. Prevalence and etiology of anemia in reunion children. Nutrition Research 8, 733–744. Martinez, C., Ros, G., Periago, M., Ortino, J., Lopez, G., 1998. In vitro digestibility and mineral availability of Green Beans (Phaseolus vulgaris L.) as influenced by variety of pod size. Journal of the Science of Food and Agriculture 1, 41–42. Miller, D.D., Schricker, B.R., Rasmussen, R.R., Van campen, D., 1981. An in-vitro method for estimation of Iron availability from meals. American Journal of Clinical Nutrition 34, 2278–2257. Odebiyi, O.O., Sofowora, E.A., 1978. Phytochemical screening of Nigerian medicinal plants II. Lloydia, Ohio 41, 234– 246. Oladipo, A., Falade, O.S., Otemuyiwa, I.O., Adewusi, S.R.A., 2004. Ascorbic acid and mineral availability in two Nigerian plant foods. African Journal of Medicine and Medical Sciences 33, 171– 175. Otemuyiwa, I.O., Falade, O.S., Oladipo, A., Adewusi, S.R.A., 2005. Effect of some herb decoctions on in vitro mineral availability from two cultivars of cowpea (Vigna unguiculata L. Walp). Journal of Food, Agriculture and Environment 3, 327–332. Oyedapo, O.O., Akindele, V.R., Okunfolami, O.K., 1997. Effects of extracts of Olax subscorpioides and Aspilia africana on bovine red blood cells. Phytotherapy Research 11, 305–306. Oyedapo, O.O., Famurewa, A.J., 1995. Antiprotease and membrane stabilizing activity of extracts of Fagara zanthoxyloides, Olax subscorpioides and Tetrapleura tetraptera. International Journal of Pharmacognosy 33, 65–69. Potter, N.N., 1983. Nutrient content of raw and cooked cowpea leaves. Journal of Food Science 48, 1252–1254. Price, M.L., Van Scoyoc, S., Butter, L.G., 1978. A critical evaluation of the vanillin reaction as an assay for tannin in sorghum grains. Journal of Agricultural and Food Chemistry 26, 1214–1216.
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Reinhold, J.G., Garcia, S.L., Garzan, P., 1982. Binding of iron by fibre in wheat and maize. American Journal of Clinical Nutrition 34, 1384–1391. Roe, J.H., Kuether, G.A., 1943. The determination of ascorbic acid in whole blood and urine through 2,4-di nitro phenyl hydrazine. Journal of Biological Chemistry 47, 399–410.
Sadique, J., Al-Raobah, N.A., Baghaith, M.F., El-Gindy, A.R., 1989. The bioactivity of certain medicinal plants on the stabilization of red blood cell membrane system. Fitoterapia LX, 525–532. Vauquero, M.P., 1993. Effect of phytate on the in-vitro solubility of Al3+ , Ca2+ , Hg2+ , Pb2+ , as a function of pH at 37 ◦ C. Journal of the Science of Food and Agriculture 64, 94–99.
The chemical composition and membrane stability activity of some herbs used in local therapy for anemia O.S. Falade a , I.O. Otemuyiwa a , A. Oladipo b , O.O. Oyedapo c , B.A. Akinpelu c , S.R.A. Adewusi a,∗ b
a Department of Chemistry, Obafemi Awolowo University, Ile-Ife, Nigeria Centre for Energy Research and Development, Obafemi Awolowo University, Ile-Ife, Nigeria c Department of Biochemistry, Obafemi Awolowo University, Ile-Ife, Nigeria
Received 9 September 2004; received in revised form 22 April 2005; accepted 29 April 2005 Available online 21 July 2005
Abstract The chemical composition and the membrane stabilizing activity of the decoctions of the dry Hibiscus sabdariffa Linn. (Malvaceae) calyx (Sobo in a Nigerian language), Theobroma cacao Linn. (N.O. Sterculiaceae) root (cocoa) and the dry stalk of white and red cultivars of Sorghum bicolor (L.) Moench (Poaceae) were determined. These herbs are often used locally for the management of anemia. Ash content was from 3.5 to 7.2%, tannin 7–46 mg/kg catechin equivalent, ascorbic acid content 250–625 mg/kg and total anthocyanin 57–150 mg/kg cyanidin-3glucoside equivalent. Phytate was not detected in any sample but alkaloids and saponins were present except in Sobo. The decoctions contained 0.2–2.0% of the herbs’ total tannin content while the pH was 3.2–6.7. Iron and zinc content was highest in Sobo while Theobroma cacao root contained the highest calcium and magnesium. Solubility and mineral availability were highest in the decoction of Sobo and lowest but available in Theobroma cacao root decoction. The decoction of Theobroma cacao exhibited a high membrane stabilizing activity while those of Sorghum bicolor and Hibiscus sabdariffa were only average. The study indicated that the herbal decoctions could provide some minerals and also help to stabilize the red blood cells from stress injury. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Hibiscus sabdariffa; Sorghum; Cocoa; Tannin; Ascorbic acid; Membrane stabilizing activity
1. Introduction The use of traditional medicine has been recently encouraged by World Health Organization (WHO) and United Nation Children’s Fund (UNICEF) for its cultural role, greater availability and acceptability than the modern pharmaceutical agents (Mahu, 1988; Mabina and Pitose, 1997). In Nigeria, there is a general belief that certain herbs contain haematopoietic properties and could serve as “blood tonic”; the medicinal application of the infusions or decoctions of such herbs has gained wide popularity amongst the population. Such herbs include the stem, root and bark of Theobroma cacao Linn. (N.O. Sterculiaceae) (cocoa), the calyx of Hibis∗
Corresponding author. Tel.: +234 803 5528166. E-mail address: [email protected] (S.R.A. Adewusi).
0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.04.034
cus sabdariffa Linn. (Malvaceae) locally called “Sobo” and the stalk of Sorghum bicolor (L.) Moench (Poaceae). The therapeutic action of a plant depends on its chemical constituents. Plants have been reported to contain compounds such as tannin, saponin, phytate, fibre and phenolic compounds which play prominent roles in mineral nutriture as inhibitors. The presence of these compounds in tea, coffee and other beverages has been shown to inhibit iron absorption from composite meals (Gilooly et al., 1983; Brune et al., 1991; Hurrell et al., 1999). Other agents in plant products such as ascorbic and other organic acids have been known to enhance mineral solubility and uptake (Hallberg et al., 1986; Adewusi et al., 1999). The role of the decoctions from these herbs in enhancing iron availability from foodstuffs was highlighted in the first paper in this series (Otemuyiwa et al., 2005), to indicate the
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relevance of the herbal decoctions in the management of anemia in Nigeria. In addition, the possibility that the decoctions of these herbs could have other pharmacological effects such as the stabilization of erythrocyte membranes in vivo and in vitro was expressed as shown for the extracts of Zanthoxylum zanthoxyloides, Olax subscorpioides and Tetrapleura tetraptera (Oyedapo and Famurewa, 1995). The main objectives of the present study are to:
2.3.2. Determination of soluble minerals in herbs Ten grams each of washed and dried samples was boiled in 200 ml of distilled water for 30 min (as done traditionally). After boiling, the volume of the decoction was recorded and the solution was analyzed for soluble minerals (iron, zinc, calcium and magnesium) using the Atomic Absorption Spectrophotometer.
(a) evaluate the chemical contents of the common haematopoietic herbs used in Nigerian traditional medicine; (b) ascertain the level and effect of antinutritional factors as well as some of the enhancing agents on mineral availability in these herbs; (c) investigate the membrane stabilizing effect of the decoctions and thus supply some information on the potentials of these herbs for the management of anemia.
2.3.3. Determination of available minerals in herbs The available mineral content was determined by the method of Miller et al. (1981) with modifications. Titratable acidity was first determined on 50 ml of each herbal decoction as described earlier (Miller et al., 1981). Fifty millilitres of each decoction was measured into a 100 ml beaker; 10 ml of water, NaHCO3 corresponding to the titratable acidity (Miller et al., 1981) and a magnetic bar were placed in a dialysis tubing and put into the beaker of the decoction and incubated at 37 ◦ C for 2 h on a magnetic stirrer. The content of the dialysis tube was removed, the tube was then washed with distilled water and the volume of the dialysate noted and was then taken for mineral analysis using the Atomic Absorption Spectrophotometer.
2. Materials and methods 2.1. Herbal materials The dry fermented calyx of Hibiscus sabdariffa Linn. (Malvaceae) and the dry stalk of two cultivars of Sorghum bicolor (L.) Moench (Poaceae) coded red and white were bought at a local herb market in Ile-Ife, Nigeria, while the root of Theobroma cacao Linn. (N.O. Sterculiaceae) was collected from a cacao plantation on Obafemi Awolowo University Campus, Ile-Ife. Voucher samples were deposited in the herbarium of the Department of Botany, Obafemi Awolowo University, Ile-Ife: the root of Theobroma cacao—IFE 2282, Hibiscus sabdariffa calyx—IFE 2328, red colored stalk of Sorghum bicolor—IFE 10976 and the stalk of white Sorghum bicolor without a distinct coloration—IFE 10975. 2.2. Methods The herbs were washed with distilled water, dried at 50 ◦ C overnight, milled in a ball grinder (Siebtechnik TS 250) and then sieved with No. 72 mesh size (Griffin and George Ltd., London). The samples were stored in plastic containers with screw cap and kept in the freezer until used. 2.3. Analytical techniques The ash content of each sample was determined by the Association of Official Analytical Chemists (AOAC, 1984) method by heating each sample in a muffle furnace at 550 ◦ C until white ash was obtained. 2.3.1. Mineral content determination The ash from the procedure above was dissolved in 2 ml concentrated HNO3 and made up to mark in a 50 ml volumetric flask. The mineral content from the resulting solution was determined using the Atomic Absorption Spectrophotometer (Alpha 4 model, Fisons Chem-Tech, Analytical, UK).
2.3.4. The effect of colored compounds on soluble and available minerals One hundred millilitres of the decoction was decolorized using 0.5 g acid washed Norit (activated charcoal) and made up to 100 ml with distilled water. A portion of the decolorized decoction was analyzed for soluble minerals while another portion was analyzed for mineral availability as described above (Miller et al., 1981). 2.3.5. The tannin content The tannin content in the samples was determined by the modified vanillin–hydrochloric acid (MV–HCl) method (Price et al., 1978) using catechin as a standard. 2.3.6. Phytate content Phytate content of the samples was determined by the anion exchange method (Harland and Oberleas, 1986) using KH2 PO4 solution (80 g/ml) as the standard. 2.3.7. The ascorbic acid The ascorbic acid content was determined using the method of Roe and Kuether (1943) with slight modifications. The milled sample (2.0 g) was extracted with 25 ml of 0.1% (v/v) oxalic acid, the residue washed twice with 10 ml of the extraction solvent, centrifuged and the supernatant was made up to 50 ml in a standard flask with the extraction solvent. Two millilitres of each filtrate was placed in two test tubes, a drop of 10% (v/v) thiourea solution and 0.5 ml of 2% (w/v) of 2,4dinitrophenyl hydrazine reagent were added. The test tubes were placed in a water bath at 37 ◦ C for 3 h and then placed in a beaker of ice water; 2.5 ml of 98% (v/v) H2 SO4 was added in drops from the burette within 1.0 min. The absorbance was
O.S. Falade et al. / Journal of Ethnopharmacology 102 (2005) 15–22
taken after 30 min at 520 nm using the Spectrophotometer and estimated against a standard ascorbic acid curve. 2.3.8. Total anthocyanin Total anthocyanin in Hibiscus sabdariffa and Sorghum bicolor was determined by the method of Abdel-Aal and Huel (1999) and the result calculated as cyanidin-3-glucoside equivalent. 2.3.9. The qualitative screening for alkaloids and saponins The samples were screened for the presence of alkaloids and saponins by the method of Odebiyi and Sofowora (1978). The presence of alkaloids was tested by Dragendorff’s reagents, 10% (w/v) tannic acid and saturated picric acid solution while saponin was tested by the frothing method. 2.3.10. Preparation of erythrocyte Bovine erythrocyte was prepared as previously described (Sadique et al., 1989; Oyedapo and Famurewa, 1995). Fresh blood samples were collected into an anticoagulant containing trisodium citrate (3.8%, w/v) and mixed thoroughly. The blood samples were then centrifuged at 3000 rpm for 5 min at room temperature. The supernatant was decanted and the packed red blood cells were washed in fresh isosaline solution followed by centrifugation. The washing process was repeated until the supernatant was clear. Then, 2% (v/v) erythrocyte suspension was prepared in normal saline solution. 2.3.11. Preparation of drugs and decoctions Standard drugs, Indomethacin and Ibuprofen, were prepared in iso-saline solution to a final concentration of 2.5 mg/ml. Ten grams of each herb was boiled in distilled water for 30 min, decanted, cooled and evaporated in vacuo at 40 ◦ C. The residue was weighed and re-dissolved in isosaline. 2.3.12. Assay of membrane stabilizing activity This was carried out as earlier described (Odebiyi and Sofowora, 1978; Sadique et al., 1989) using extract concentrations that provided maximum activities as follows: 3 mg/ml (Sobo); 5 mg/ml (red Sorghum bicolor); 1 mg/ml
17
(white Sorghum bicolor); 1 mg/ml (Theobroma cacao); the standard drugs, 2.5 mg/ml. In essence, the assay mixture consisted of 2 ml of 0.25% (w/v) sodium chloride, 1.0 ml of 0.15 M sodium phosphate buffer (pH 7.4), 0.5 ml of 2% (v/v) erythrocyte suspension, 0–1.0 ml of drugs or decoctions and the final mixture was made up to 4.5 ml with iso-saline. Drugs were omitted in the first control while the drug control lacked the erythrocyte suspension. The reaction mixtures were incubated at 56 ◦ C for 30 min, cooled under running water and centrifuged at 5000 rpm. The supernatant was collected and the color of the released haemoglobin read at 560 nm on the Spectrophotometer. The percentage membrane stability was estimated from the expression: 100 − (absorbance drugtest − absorbance drugcontrol ) × 100 absorbance drugcontrol where the control represents 100% lysis or zero membrane stability.
3. Results 3.1. Chemical compositions The chemical compositions of the herbs are presented in Table 1. The analysis of whole samples revealed that the percentage ash content in the stalk of white sorghum was significantly higher (p < 0.05) than that of the red variety. The tannin content in herbs was high in Theobroma cacao root, low in Hibiscus sabdariffa calyx and moderate in both stalks of white and red sorghum. Phytate was not detected in any of the samples analyzed. The ascorbic acid content was highest in Hibiscus sabdariffa but undetectable in the Theobroma cacao root. The stalk of white sorghum contained the highest level of anthocyanin, closely followed by the red sorghum stalk while it was not detectable in Theobroma cacao root. Tannin was present in negligible quantity in all the herbal decoctions and totally unextractable in Sobo (Table 1). The pH of the decoction was lowest in Sobo and highest in the decoction of the white sorghum stalk. The phytochemical screening (Table 2) showed that alkaloids and saponins were present in all samples except Sobo.
Table 1 Chemical composition of the herbs (on dry weight basis) and the decoctions Sample
Hibiscus sabdariffa Sorghum bicolor (red) Sorghum bicolor (white) Theobroma cacao root
Herbs
Decoctions
Ash content (%)
Ascorbic acid (mg/kg)
Total anthocyanin (mg/kg)
Tannin (g/kg)
7.2 ± 0.2a 3.5 ± 0.1c 6.5 ± 0.3b 7.2 ± 0.3a
625 ± 0a 375 ± 0b 250 ± 1c N.D.
57 ± 0c 135 ± 0b 150 ± 1a N.D.
7.0 11.0 11.0 46.0
± ± ± ±
0. 9 c 0.6 b 0.2b 0.1a
Tannin (mg/dm3 )
Tannin (%)
pH
N.D. 0.03 ± 0.02 0.20 ± 0.00 0.11 ± 0.06
– 0.3 2.0 0.2
3.2 ± 0.1 5.5 ± 0.4 6.7 ± 0.1 6.5 ± 0.6
Mean ± standard deviation of triplicate analysis. Values in the same column with the same superscript letters (a–c) are not significantly different at p < 0.05. N.D.: not detected.
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Table 2 Results of phytochemical screening for alkaloids and saponins Sorghum bicolor (red)
Sorghum bicolor (white)
Theobroma cacao root
Hibiscus sabdariffa calyx
Picric acida Wagners reagenta Tannic acida Mayer’s reagenta Dragendorff’s reagenta
+ve +ve +ve +ve +ve
+ve +ve +ve +ve +ve
+ve +ve +ve +ve +ve
−ve −ve −ve −ve
Saponin Froth test Lieberman–Burchard test
−ve +ve
−ve +ve
+ve +ve
−ve −ve
Sample/reagents Alkaloida
a
The reagents were used to test for the presence of alkaloids.
3.2. Mineral content of the herbs The mineral content of the herbs presented in Table 3 indicated that total iron ranged between 60 and 177 mg/kg. The soluble iron concentration in the herbs was lowest in Theobroma cacao root decoction while the highest concentration was recorded for Sobo. Availability of iron was highest in Sobo and lowest in Theobroma cacao root but the highest percentage available iron was recorded for red sorghum stalk followed closely by Theobroma cacao root despite the low iron solubility recorded in the decoctions. The result presented in Table 3 also indicated that the total zinc in the herbs ranged from the low level (3 mg/kg) in red sorghum stalk to the highest level (28 mg/g) in Sobo. The soluble zinc concentration in the decoction was highest in Sobo and lowest in Theobroma cacao. Despite the fact that the zinc content of the Theobroma cacao root was the least soluble, its avail-
ability was virtually total. Analysis of the calcium content of the herbs (Table 3) indicated that Theobroma cacao root contained the highest level of total calcium (11,650 mg/kg) followed by Sobo (9700 mg/kg). The total calcium content of the stalk of the red and white Sorghum bicolor was 1300 and 1840 mg/kg, respectively. The highest soluble calcium was again found in the decoction of Sobo and lowest in Theobroma cacao. The available calcium and percentage availability were highest in Sobo and least in red Sorghum bicolor. The total magnesium content of the herbs was lowest (1750 mg/kg) in the stalk of white Sorghum bicolor and highest in Theobroma cacao (7500 mg/kg). The highest content of soluble magnesium, available and percentage available magnesium were recorded in Sobo while Theobroma cacao root recorded the lowest soluble magnesium. Sorghum bicolor red stalk recorded the least value for both available and percentage available magnesium.
Table 3 Mineral content of the herbs Sample
Total mineral (mg/kg)
Soluble mineral (mg/dm3 )
Soluble mineral (%)x
Available mineral (mg/dm)
Available mineral (%)y
Iron Hibiscus sabdariffa Sorghum bicolor (red) Sorghum bicolor (white) Theobroma cacao root
177 ± 60 ± 79 ± 144 ±
1a 8d 7a 30b
39.0 2.3 1.5 0.4
± ± ± ±
0.3a 0.1b 0.3bc 0.0c
22 4 2 0.3
4.80 0.80 0.30 0.13
± ± ± ±
0.10a 0.10b 0.10c 0.01d
12 35 20 33
Zinc Hibiscus sabdariffa Sorghum bicolor (red) Sorghum bicolor (white) Theobroma cacao root
28 ± 3± 8± 20 ±
2a 0d 1c 2b
10.0 1.0 2.0 0.5
± ± ± ±
1.2a 0.1c 0.1b 0.1c
36 33 25 3
1.30 0.26 0.26 0.46
± ± ± ±
0.10a 0.10c 0.10c 0.10b
13 26 13 92
Calcium Hibiscus sabdariffa Sorghum bicolor (red) Sorghum bicolor (white) Theobroma cacao root
9700 ± 1300 ± 1840 ± 11650 ±
900b 230d 137c 349a
3527 ± 208 ± 226 ± 109 ±
216a 41b 10b 7.5c
36 16 12 1
466 ± 3.2 ± 5.6 ± 4.0 ±
11a 0.4c 0.1b 0.4bc
13 2 3 4
Magnesium Hibiscus sabdariffa Sorghum bicolor (red) Sorghum bicolor (white) Theobroma cacao root
2150 ± 1850 ± 1750 ± 7500 ±
40b 180c 76d 713a
1341 ± 1086 ± 776 ± 515 ±
28a 28a 13c 3c
62 58 44 7
232 ± 13 ± 40 ± 20 ±
6a 4d 3b 1c
24 1.0 5 4
Mean ± standard deviation of triplicate analysis. Values in the same column with the same superscript letters (a–d) are not significantly different at p < 0.05. x Soluble mineral (%) is expressed as fraction of the total mineral in the herbs. y Available mineral (%) is expressed as a fraction of soluble mineral in the decoction.
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3.3. Effect of colored compounds on mineral solubility and availability The result of the effect of colored compounds on mineral (Fe, Zn, Ca and Mg) solubility and availability presented in Table 4 indicated that soluble iron content of the decolorized decoction of Sobo was significantly lower (t9.9 , p < 0.01) than the colored decoction but there was no significant difference in the available iron content from both decoctions. There was no significant difference (t4.3 , p < 0.05) in the soluble and available iron from decoctions of both white and red sorghum stalk. The result also indicated that there was no significant difference between the solubility and availability of zinc and calcium (t4.3+, p < 0.05) in colored and decolorized decoctions though the soluble magnesium from Hibiscus sabdariffa and red Sorghum bicolor decoctions was significantly (t4.3 , p < 0.05) higher than from the decolorized.
4. Discussion The ash content ranged from 3.5 to 7.2% with the least percentage ash recorded for the red Sorghum bicolor stalk while the highest was recorded for both Sobo and Theobroma cacao (Table 1). The range of 6.5–19.4% had been reported for some Nigerian vegetables (Awoyinka et al., 1995). The pH value (Table 1) indicated that Sobo decoction was acidic (pH 3.2), which is a consequence of the fermentation of the Hibiscus sabdariffa calyx before sale in Nigeria. The solubility of minerals such as iron and calcium increases in acidic solution, thereby promoting their availability (Champagne and Rao, 1985; Adewusi et al., 1999). pH also plays an important role in the binding of minerals by both dietary fibre and
19
phytate (Brune et al., 1992; Vauquero, 1993). Even in the absence of dietary fibres, iron had been found to precipitate progressively as the pH increases (Reinhold et al., 1982). It is therefore not surprising that the low pH of the decoction of Sobo and the stalk of the red Sorghum bicolor would explain in part the increased solubility and availability of the minerals analyzed in this study. The ascorbic acid content (Table 1) was highest in Sobo (625 mg/kg) and may contribute to the low pH (3.2) of this decoction. The level of ascorbic acid in the stalk of red Sorghum bicolor (375 mg/kg) was higher than the white variety (250 mg/kg); this seemed to affect the pH of the decoctions accordingly. Ascorbic acid is heat labile and since these samples have been subjected to various degrees of heat treatment, the ascorbic acid of the decoction could have been reduced. It has been demonstrated that ascorbic acid is one of the ligands that promote absorption of iron from meals (Hallberg et al., 1986; Oladipo et al., 2004) and could therefore account in part for the availability of iron and other minerals induced by the decoctions. Phytate, which chelates minerals (Cheryan, 1980; Adewusi and Falade, 1996), was not detected in any of the herbs analyzed (Table 1). The tannin content in whole samples was lower than the range of 27–92 g/kg catechin equivalent reported for tea (green and black) and coffee powder (Brune et al., 1991) and lower still in all the decoctions and below detection level in the Sobo decoction. The absence of tannin in the decoction of Sobo and its presence below lethal level in other decoctions indicated that tannin from these herbs is not likely to play any significant role in mineral availability of composite meals. The range reported here for the total anthocyanin was lower than the range recorded in barley and wheat bran but
Table 4 Effects of colored compounds on mineral solubility and availability from the herbs Sample
Soluble mineral (mg/dm3 )
Available mineral (mg/dm3 )
COL.
DECOL.
DF (%)
COL.
Iron Hibiscus sabdariffa Sorghum bicolor (red) Sorghum bicolor (white)
39.0 ± 0.3a 2.3 ± 0.1 1.5 ± 0.3
30.6 ± 0.5b 2.2 ± 0.2 1.3 ± 0.1
22 4 13
4.8 ± 0.1 0.8 ± 0.1 0.3 ± 0.1
Zinc Hibiscus sabdariffa Sorghum bicolor (red) Sorghum bicolor (white)
10.0 ± 0.2 1.0 ± 0.1 2.0 ± 0.6
8.5 ± 1.7 1.0 ± 0.2 1.5 ± 0.3
15 – 25
Calcium Hibiscus sabdariffa Sorghum bicolour (red) Sorghum bicolor (white)
3527 ± 21 208 ± 41 226 ± 0
3505 ± 63 189 ± 0 224 ± 1
Magnesium Hibiscus sabdariffa Sorghum bicolor (red) Sorghum bicolor (white)
1341 ± 28a 1086 ± 48a 776 ± 13
1160 ± 11b 1005 ± 7b 777 ± 36
0.6 9 0.8 13 7 –
DECOL.
Available (%) DF (%)
COL.
DECOL.
4.3 ± 0.1 0.6 ± 0.0 0.2 ± 0.0
10.4 22.5 26.6
12 34 20
14 28 31
1.3 ± 0.1 0.3 ± 0.1 0.3 ± 0.1
1.3 ± 0.1 0.3 ± 0.1 0.2 ± 0.0
4 0 15
13 26 13
15 26 15
466 ± 11 3.2 ± 0.4 5.6 ± 0.1
495 ± 5.6 3.2 ± 0.1 4.2 ± 0.8
+6 1 25
13 1.5 2.4
14 1.6 2.8
323 ± 6 13 ± 4 40 ± 3
344 ± 4 12.4 ± 1 35 ± 6
+6 5 13
24 1.2 5.2
29 1.2 4.5
Mean ± standard deviation of triplicate analysis. Values with superscript letters (a and b) are significantly different at (t9.9 , p < 0.01), other values are not significantly different. DF (%) is expressed as percentage difference in available and soluble mineral in colored and decolorized decoction. Available mineral (%) is expressed as a fraction of soluble mineral in colored and decolorized decoctions. COL.: colored decoction. DECOL.: decolorized decoction.
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higher than 22.5 mg/kg reported for wheat flour (Abdel-Aal and Huel, 1999). Anthocyanins have been reported to form complexes with metal ions, especially iron, magnesium, zinc and calcium, and this could affect their availability, but the present study has revealed that the presence of these water soluble pigments did not seem to affect mineral availability from the herbs. The content of iron in the herbs (Table 3) indicated that Sobo had the highest total iron while the least was recorded for the red sorghum stalk. The values recorded for the herbs were higher than 28 mg/kg total iron reported for cowpea leaves (Potter, 1983) and the range reported for green beans (Martinez et al., 1998). Iron was found to be more soluble and available from the decoction of Sobo probably with a low pH value, which is an indication of a high level of organic acids resulting from the fermentation of the calyx (Adewusi et al., 1999). Theobroma cacao root recorded the least soluble and available iron despite its high total iron content probably due to the high level of fibre, most especially cellulose, which has been reported to bind minerals (Reinhold et al., 1982). The high pH of its decoction (pH 6.5) could also contribute to the low solubility of its iron as indicated earlier (Clydesdale and Camire, 1983). The result also showed that iron was more soluble and available from the stalk of red Sorghum bicolor compared to the white cultivar possibly reflecting the difference in their pH (6.7 and 5.5 for white and red cultivars, respectively) and tannin (0.20 and 0.03 mg/dm3 for white and red cultivars, respectively). Decolorization did not seem to have a significant role in mineral availability (Table 4) since there was no significant difference (p < 0.05) between available iron from the decolorized and colored decoctions. The total zinc content recorded for Sobo compared favourably with 24 mg/kg total zinc reported for amaranthus (Adewusi et al., 1999). The highest soluble and available zinc was also recorded for Sobo while the least was recorded for Theobroma cacao. Theobroma cacao sample contained the highest tannin content (Table 1), which has been reported to inhibit the speciation of zinc and may thus be responsible for low zinc availability from the decoction. It is interesting to note that the decoction of Theobroma cacao root which recorded the least solubility and availability for zinc showed the highest percentage zinc availability (92%). The implication is that the little amount of zinc that was soluble was readily available. This decoction needs to be further investigated. The observed total calcium in this study was higher than the 7792–9135 mg/kg values reported earlier for amaranthus (Adewusi et al., 1999). The highest calcium solubility and availability was recorded for Sobo as reported earlier for iron and possibly for the same reasons (Adewusi et al., 1999). Other decoctions recorded less than 5% available calcium, which may be a consequence of their tannin and fibre content and more importantly the high pH of the decoctions in which calcium is expected to precipitate (Clydesdale and Camire, 1983). As observed in iron, colored compounds (Table 4) did
Fig. 1. Membrane stabilizing activity of the decoctions of Hibiscus sabdariffa calyx, white Sorghum bicolor and red Sorghum bicolor stalk.
not seem to have any significant effect on the availability of calcium. The highest solubility and availability of magnesium was recorded in the Sobo decoction. Magnesium forms water soluble complexes which could be responsible for its high solubility as well as availability of this mineral in Sobo. Colored compounds do not seem to have any significant role in magnesium availability from these decoctions as observed in iron and calcium. Theobroma cacao recorded the highest total magnesium but its solubility as well as availability was the lowest. This is expected since the roots’ main constituent, cellulose, is well known to bind minerals. The result of the bovine erythrocyte membrane stabilizing activity of the decoctions indicated that Hibiscus sabdariffa and red and white Sorghum bicolor stalks exhibited moderate membrane stabilizing activity (Fig. 1) which could be biphasic especially at high concentration of the extracts. The extract of the Theobroma cacao root exhibited a very high membrane stabilizing activity comparable to the membrane stabilization activity of the Olax subscorpioides (87.5%), Aspilia Africana (85%) and Fagara zanthoxyloides (83.8%) (Oyedapo and Famurewa, 1995; Oyedapo et al., 1997) extracts against heat
Fig. 2. Membrane stabilizing activity of the decoctions of Theobroma cacao root and the standard drugs—Ibuprofen and Indomethacin.
O.S. Falade et al. / Journal of Ethnopharmacology 102 (2005) 15–22
and hypotonic induced stress and even marginally exceeded the activity of the standard drug Ibuprofen (Fig. 2). The haematopoietic activity of the decoction of Theobroma cacao root in vivo would therefore seem to be through a combination of two systems via the enhancement of mineral availability from foods as well as its membrane stabilizing activity
5. Conclusion The data reported here showed that decoctions of the herbs are capable of providing an alternative source of minerals as well as enhancing mineral availability from other food sources through the low pH of the decoctions. This could be especially true of the decoction from the fermented Hibiscus sabdariffa calyx which is now a popular drink called “Sobo” in Nigeria. The presence of ascorbic acid in the decoctions may be another mechanism whereby the herbs enhance mineral availability. There is a high positive correlation factor between the ascorbic acid content (Table 1) and the percent soluble minerals presented in Table 3: iron (r = 0.88), zinc (r = 0.93), calcium (r = 0.98) and magnesium (r = 0.92). This seems to implicate ascorbic acid in the speciation of the minerals in the herbs and hence their availability. This could prevent mineral deficiency diseases such as anemia. These findings give credence to the claim that the herbs could be effective in ameliorating iron deficiency anemia. Theobroma cacao root extract possesses the additional property of a high level of membrane stabilizing activity which could stabilize red blood cells from stress injury. This result will serve as a base data for nutritionists, traditional and orthodox medical practitioners who may be looking for an alternative source of iron for the treatment of anemia and some other mineral deficiency diseases. During the course of this study, a new herbal preparation “JUBI” incorporating Sorghum bicolor stalk has been introduced into the market in Nigeria as a support for the “management of moderate and severe anemia”. It was also reported that Hibiscus sabdariffa is a well known medicinal plant in Thailand (Chewonarin, 1999).
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