COMPLEMENTARY ANALYTICAL TECHNIQUES PAPER, THIN-LAYER, HIDE-POWER, AND COMBINED METHODS FOR CHARACTERIZATIONOF TANNIN IN PLANTS

epicatechin and some unidentified phenolics were present. However, dihydrofisetin and robinetin, which were used as standards, were not detected. Astringency values shows that the Acacia mellifera (0.18), Acacia seyalvar.fistuala (0.18), Pithecellobium dulce (0.15), Acacia senegal (0.14), Acacia farnesiana (0.13), Calotropis procera (0.13)barks could be used in place of A. mearnsii (international commercial tannin materials) (0.16) because the degree of relative astringency or the ability of their tannin to combine with protein is close to that of A. mearnsii ; in other words these six species can give leather with characteristics comparable with that of A. mearnsii.


ISSN: 2320-5407
Int. J. Adv. Res. 9 (10), 777-785 778 the main material of a wide range of artefacts and adapted to very diverse functional needs such as footwear, bookbinding, saddles, harness, liquid vessels, cases and caskets coverings or seating furniture and carriages upholstery. Beyond its utilitarian function, it was also used as support material for artistic and decorative paintings, wall hangings and screen coverings.
Condensed tannins, or proanthocyanins, are natural polyphenolic oligomers made of flavan-3-ol units. They are recognized as suitable natural substitutes in the formulation of wood adhesives (Yazaki and Collins, 1994;Roffael et al. 2000;Pizzi, 2008), foamed resins (Lacoste et al., 2013) and heavy metal removal systems. Industrially used tannins are mostly extracted from the bark of black wattle (Acacia mearnsii [De Wild.]) and the heartwood of Quebracho (Schinopsislorentzii [Engl.]). The bark of softwood species has also been reported as a valuable source of condensed tannins (Krogell et al., 2012). In Switzerland, 425,000 m 3 of bark was produced in 2013, the majority of which was burned for energy production (Lacoste et al., 2013 This investigationpurposes to expand the knowledge aboutvegetable tanning materialsthathad significant concentrations of these compounds. This information is important to recognize tannin structure, knowledge, deprivation susceptibility or state in demand to bring out suitable techniques, and if required, to choosesuitable one for tannin vegetable materials.

Preparation of sample
Fresh plant parts (bark) (0.3-2.0 kg) from different species growing in Khartoum area, Blue Nile, and South Kordofan (Dalang), were used for this study ( Table 1). The conformation of the identity of the plant species is done by Soba Forestry Research Center Herbarium. The samples were air-dried and reduced to powder with a star mill. The fractions passing through 40-mesh and retained on 85-mesh sieve were collected, thoroughly mixed and kept in airtight containers.

Analysis of Tannins Extraction Using ALCA-Palsy Method
Cold water extracts (2 litres) were obtained with an ALCA (American Leather Chemist Association)-Palsy apparatus (Doat, 1978). The presence of tannins was detected by the gelatin salt test and their types were identified using the iron-alum and formaldehyde-HCl test (SLTC. 1965).
Tannic acid, catechin, gallic acid, epicatechin, fisetin, dihydrofisetin and robinetin were used as standard compounds (R f ×100) for the above chromatographic analyses. Samples were prepared by hydrolyzing 5 g raw materials with 2M HCl using reflux for 30 min. The effluent was then cooled and filtered; ethyl acetatethen used to extract the produced filtrate. The aqueous layer was heated to remove any trace of solvent and extracted with a small volume of amyl alcohol. The solvent extracts were concentrated to thick syrup under vacuum (Harborne,1998).

Quantitative Analysis
The extracts were quantitatively analyzed for total and soluble solids, non-tannins and tannins by the official hidepowder method (Jamet,2000) (hide-powder batch C28). A modification of the hide-powder method, i.e., the combined method (Swain and Goldstein,1964) was also used. Total phenolic materials in the extract were measured using the Folin-Denis'smethod (Folinand Denis, 1915). Freshly hydrated chromated hide-powder equivalent to 3.0 g oven-dried was prepared. Tannin was then allowed to absorb onto the hide powder, after which the remaining phenolic materials were determined. The catechin number (Stiasny number) was determined according to the method by Yazaki and Hillis (Folin and Denis, 1915). For this 100 ml extract were filtered through a glass fritted funnel (G4) and poured into a conical flask. Stiasny reagent (5 ml of HCl + 10 ml of 37% formaldehyde) was added into the flask and then the mixture was allowed to stand for 24 hours at room temperature (30-35 °C). Then the precipitate was filtered on a tared crucible (G4) before being dried to constant weight at about 100 ± 5 °C to obtain the weight of catechin (Folin and Denis, 1915).

Results and Discussions:-
Tannins are phenolic compounds of relatively high molecular weight. They are classified as condensed and hydrolysable tannins. The hydrolysable tannins are readily hydrolyzed by acids, alkalis or enzymes (tannases) into a sugar or a related polyhydricalcohol (polyol) and a phenolic carboxylic acid (Pizzi, 2008). Depending on the nature of the phenolic carboxylic acid, hydrolysable tannins are subdivided into gallotannins and ellagitannins. Hydrolysis of gallotannins yields gallic acid while hydrolysis ofellagitannins yields hexahydroxy diphenic acid which is isolated asellagicacid (Pizzi, 2008). Hydrolysable tannins are considered as one of the most potentantioxidants from plant sources. They are ready to form complexes with reactive metals, avoiding free radical generation whichresults in oxidative damage of cellular membranes and DNA (Lacoste et al., 2013). Hydrolysable tannins, in addition, clean free radicals within the body by neutralizing them before cellular damage occurs (Hagerman, 1998; Gülcinet al., 2010).

Formaldehyde-HCl and Iron Alum Test
From the formaldehyde-HCl and iron alum test, the whole twelve species screened were of the condensed type except Acacia seyalvar. fistuala, and Acaciaseyalvar. seyal, Casuarina equistifolia, and Pithecellobium dulce were of mixed hydrolysable-condensed (gallo-catechol) type. The gallic acid and catechin number test results supported these assignments ( Table 2). The quantitative data indicated that fiveparts (bark) of twelve species, when extracted, contained more than 10% (oven-dry basis) of tannins, the level of commercial interest. Of these 12 species, 6species had an acceptable extraction ratio (tannin to non-tannin) of 1.0-4.5. The tannin purity or the ratio of tannin/soluble solids was good, >0.5, for 7species of the twelve species studied (Table 2). However, the type of tannin present and the part extracted are also important.

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Different parts of species bark, leaves, and fruits had the same type of tannin but in different proportions. Usually, the tannin content was higher in the barks (Acacia mearnsii, Acacia seyalvar. fistuala, Acacia seyalvarseyal, Acacia Senegal, Pithecellobium dulce, andCasuarinaequistifolia) ( Table 2). The catchin numbers indicated that all the studied species contained condensed tannin in varying amounts (0.6-45.7), while the presence of both gallic acid and catechin means that the tannin is of mixed type (hydrolysable-condensed) (gallo-catechol) ( Table 2).

Thin-layer and paper Chromatography
Thin-layer and paper chromatography with different solvent systems confirmed the presence of catechin and gallic acid, and showed that tannic acid, fisetin, epicatechin and some unidentified phenolics were present. However, dihydrofisetinandrobinetin, which were used as standards, were not detected ( Table 3).

Methods of Determination of Tannins
The tannin content determined by the hide-powder method was highest (39.8) forAcacia mearnsii followed by (28.8%) for Pithecellobium dulce bark, and for Acacia seyalvar. seyalandAcacia seyalvar. fistuala, Casuarina equistifoliabark (24.8,23.7,10.2% respectively) ( Table 2). These data were compared with those obtained from the spectroscopic method of Swain and Goldstein (Hagerman,1998) and also with two methods for total phenolic (Yazaki and Hillis,1998; Hagerman and Butler 1978) ( Table 4). In the first comparison, the correlation between total phenolics and tannin content was high (r 2 = 98.7%, n = 24, p < 0.01). In the second case, the phenolic content by the Hagerman and Butler method (Judd, et al., 2007;Talhouk et al., 2007) was approximately half that of Folin-Denis's assay, but the correlation between the two assays was still high (r 2 = 70.9%, n = 24, p < 0.01). The combined method also gave slightly lower values of tannin content and extraction rates (Table 4). Care should be taken when comparing tannin content determined bydifferent methods as the isolation procedures may affect the proportion and types of phenolic present (this due to different method have different ways of determination and isolation). The relative astringency values for most of these tannins were quite close to that of A. mearnsii tannin, but much higher values were obtained for Acacia mellifera and Acacia seyalvar. Fistualabark. However, theAcacia melliferabark has low tannin contents (17.9%) ( Table 4).

Stringency Factor
Astringency values shows that the Acacia mellifera(0.18), Acacia seyalvar. Fistuala(0.18), Pithecellobium dulce (0.15), Acacia senegal(0.14), Acacia farnesiana(0.13), Calotropis procera(0.13)barks could be used in place of A. mearnsii(international commercial tannin materials) (0.16) because the degree of relative astringency or the ability of their tannin to combine with protein is close to that of A. mearnsii; in other wards thesesix species can give leather with characteristics comparable with that of A. mearnsii.

Precipitation of Protein
The protein precipitation curve for the tannins from A. mearnsii bark (international commercial tannin materials) andAcacia senegal, Acacia seyal var. fistuala, Cassiasiamea, Albizzia amara, bark reflected their different nature and relative astringency ( Figure 1). The fairly gradual solubilization of A. mearnsiitannins (wattle) and Cassia siamea, Albizzia amara, Acacia senegal, and Acacia seyal var. fistualabarktannins indicated greater reactivity. It seemed probable that the highly astringent and strongly binding tannin would react with animal hide protein so firmly and rapidly that the penetration of the materials would have to be controlled by selection of pH and concentration. Thus, the resulting leather might be hard and coarse. In contrast the less astringent tannin (mixed type) obtained from the Acacia seyal var. fistualabark and Cassia siameabark mixed with Calotropis procerabarkshould penetrate the hide more extensively and the reaction should not be weaker in terms of poorer tanning or greater vulnerability to microbiological damage.  --783 * Adsorbent: Polyamide precoated plate (10x10 cm); solvent system: acetone-propanol-water (5/4/1); detection: UV/254nm; FeCl 3 . **Adsorbent: Whatman paper no.2; solvent system: acetic acid-conc. HCl-water (10/3/30); detection: UV/254nm; strong ammonia vapor.

Conclusions:-
The Complementary analytical technique was shown to be very efficient in the characterization of tannins from plants species. The twelve indigenous and exotic species studied only five contained more than the 10% tannin needed for commercial exploitation. The highest tannin content exotic species, but of limited distribution in Sudan, was Acacia mearnsii bark (black wattle) (39.8%) followed by the four indigenous species of Pithecellobium dulce bark (28.7%), Acacia seyalvar.seyalbark (24.8%), Acaciaseyal var. fistualabark (23.7%), and Casuarina equistifoliabark (10.2%) ( Table 2). All the tannins species studied contained catechin, but four species were of the mixedhydrolysable-condensed(gallo-catechol) type (Acacia seyal var. fistuala, Acaciaseyal var. seyal, Casuarina equistifolia, and Pithecellobium dulce). The benefit of the Complementary analytical technique, related to the conventional extraction systems for polyphenols, had similar yield of polyphenols attained with a lesser solvent feeding and a shorter removal time.