MATERIALS AND METHODS

Different stages of Parkia roxburghii pods were collected from a plant grown at Iroisemba near the Central Agricultural University campus and classified them into 3 groups based on the mean thickness of the pod measured at the site of seeds as tender (6 mm and below), immature (6.1- 10 mm) and mature (more than 10 mm). All the pods were scrapped to remove the outer green peel. The margin of the pods were neatly removed with the help of a knife and cut 10 cm (approximately) in length. They were divided into three groups of approximately equal weights and subjected to different methods of cooking following the method of Bishnoi and Khetarpaul (1994) with slight modifications. In Ordinary Cooking (OC), the pods were placed in a round open beaker and cooked on a heater with 4 times double distilled water (w:v) until they became soft. In case of Pressure Cooking (PC), the pods were pressure cooked (3 lit pressure cooker) at the maximum pressure for 2 min. In this case, the sample to water ratio was 1:3 (w:v).The remaining third group was treated as control. All the processed/cooked pods were air dried and kept in the oven at 60±5°C for 48 h. The samples were then ground using a Remi grinder and subsequently sieved (1 mm). The powder samples were collected and kept for various analyses.

Estimation of flavonoids: Flavonoid was analyzed following the procedures of Chang et al. (2002) with slight modifications. A 50 mg of the powdered sample was transferred into a 50 mL centrifuge tube. Then 10 mL methanol was added and stirred for 4 h in a magnetic stirrer. The solvent was evaporated to about 5 mL at room temperature and centrifuged at 10,000 rpm for 15 min. The supernatant was collected and volume made up to 5 mL with methanol. 0.1 mL of the extract supernatant was transferred into a test tube, 0.1 mL of aluminum chloride and 0.1 mL of 1 M potassium acetate were added and volume made up to 3 mL with double distilled water. This was allowed to stand for 30 min in room temperature and its absorbance was measured at 415 nm in a double beam UV-Visual spectrophotometer. The flavonoid was quantified against a calibration curve prepared using Quercetin (10-100 μg mL^-1) in methanol.

Estimation of tannins, phytate phosphorus and cyanogenic substances: Tannins and Phytate Phosphorus (PP) were determined following the procedures of Schanderi (1970) and Wheeler and Ferrel (1971). Cyanogenics determination was carried out as per the method of AOAC (1975).

Estimation of alkaloids and saponins: Alkaloid and Saponin were determined following the procedures of Obadoni and Ochuko (2002) with slight modifications. A 5 g of plant sample was dispensed in 100 mL of 20% ethanol. The suspension was heated over a hot water bath maintained at about 50°C and stirred with a magnetic stirrer for 4 h. The mixture was centrifuged and the residue re-extracted with another 100 mL of 20% ethanol. The combined extracts were reduced to 40 mL over water bath at about 90°C. The concentrate was transferred into a 250 mL separator funnel and 20 mL of diethylether was added and shaken vigorously. The aqueous layer was recovered while the ether layer discarded. The purification process was repeated. A 60 mL of n-butanol was added. The combined n-butanol extracts were washed twice with 10 mL of 5% aqueous sodium chloride. The remaining solution was evaporated to almost dryness on a water bath. The last traces of moisture were removed by drying in an oven (85°C) to almost constant weight. The difference in weight represents the saponin content.

Estimation of trypsin inhibitor and amylase inhibitor: Trypsin inhibitor activity was determined as per the method of Kakade et al. (1974) as modified by Sotelo et al. (1999). Amylase inhibitor activity was determined following the method of Deshpande et al. (1982) and Α-amylase Inhibitory Activity (AIU) was expressed as enzyme inhibitor units in mg g^-1 sample.

Statistical analysis: Standard errors of Mean differences (S. Ed±) and Critical Differences (CD) were calculated as per standard statistical procedures.

RESULTS

Table 1 indicated changes in secondary metabolites in different stages of the pods. Flavonoid content in the tender stage (13.2 mg g^-1) was higher than the other stages which decreased to 0.9 and 0.2 mg g^-1 in OC and PC while in other stages it decreased to 0.6 and 0.2 mg g^-1 in immature and to 1.7 and 1.1 mg g^-1 in mature stages of the pod. The percentage removal of flavonoids in different stages of the pod due to OC and PC were 93.18 and 98.48% in TP, 94.39 and 98.13% in IP and 82.11 and 88.42% in MP (Fig. 2a).

Tannin content in tender (98.30 mg g^-1), immature (86.47 mg g^-1) and mature raw pods (56.27 mg g^-1) reduced to 12.81, 14.31 and 6.64 mg g^-1 by OC and to 16.69, 18.84 and 8.92 (mg g^-1) by PC (Table 1). The percentage removed due to OC and PC was 86.97 and 83.02% in TP, 83.45 and 78.21% in IP and 88.25 and 84.15% in MP (Fig. 2b).

Fig. 2(a-f):	Percentage removal of secondary metabolites and enzyme inhibitors due to different processing methods in different stages of Parkia roxburghii pods, (a) Flavonoids (b) Tannins (c) Phytate phosphorus (d) Saponins (e)Trypsin inhibitors and (f) Amylase inhibitors

Table 1:	Effect of processing methods on some secondary metabolite substances in different developmental stages of Parkia roxburghii pods (mg g-1)
ND: Not detected. Different letters in a column indicate significance at 5% level and different letters with different colours indicate significance at 1% level

Phytate Phosphorus (PP) in different stages of the raw pod ranged from 30.9 to 40.5 mg 100 g^-1 which reduced to 25.2 mg 100 g^-1 in TP, 24.8 mg 100 g^-1 in IP and 34.2 mg 100 g^-1 in MP by OC and to 20.7, 17.1 and 25.2 mg 100 g^-1 by PC while percentage removed out of the original content by OC and PC was 21 and 35.11% in tender stage, 32.24 and 53.28% in immature stage and 15.56 and 37.78% in mature stages of the pod (Fig. 2c). Saponin content in tender (25.20 mg g^-1), immature (26.20 mg g^-1) and mature (28.95 mg g^-1) raw pods decreased to 16.20, 16.70 and 17.0 (mg g^-1) due to OC and to 15.20, 15.80 and 16.50 (mg g^-1) by PC (Table 1) while degree of removal of saponin due to different cooking methods ranged from 35.71 to 39.68% in TP, 36.26% to 39.69% in IP and 41.28 to 43.01% in MP, respectively (Fig. 2d). Alkaloids and cyanogenetic factors were not detected in the Parkia roxburghii pods.

Trypsin inhibitor content in terms of Trypsin Inhibitor Units (TIU) mg^-1 in tender (13.37), immature (7.77) and mature (6.94) raw pods decreased to 6.02, 5.81 and 4.05 TIU mg^-1 in OC and to 4.41, 3.88 and 3.02 TIU mg^-1 in PC (Table 2). Similarly, Amylase Inhibitor Units (AIU) in mg g^-1 in tender (4.70), immature (7.90) and mature raw pods (8.40) reduced to 3.40, 6.40 and 6.80 in OC and to 3.90, 5.90 and 6.90 in PC. Percent destruction of TI in tender, immature and mature stages of the pod recorded was 67.02, 45.24 and 32.3% of the original content (Fig. 2e) while destruction of AI due to different cooking methods was comparatively lesser and recorded 27.76% in tender, 25.32% in immature and 17.86% in mature stages, respectively (Fig. 2f).

Table 2:	Effect of different processing methods on trypsin inhibitor and amylase inhibitor content in different stages of the Parkia roxburghii pods

DISCUSSION

Though legumes are an excellent source of proteins and essential dietary minerals its utility to human is limited by the presence of certain secondary metabolites and enzyme inhibitors (Savage and Deo, 1989). Flavonoid content in different stages of the pod decreased due to different cooking methods, the maximum loss being recorded in pressure cooking (Table 1 and Fig. 2a). Processing methods reported to affect phenolic, flavonoid and antioxidant contents (DPPH, FRAP) of dry beans (Boateng et al., 2008). Xu and Chang (2009) also reported significant decreases in Total Flavonoid Content (TFC), DPPH free-radical scavenging activity and ferric-reducing antioxidant power by different processing methods. Tannins were removed greater than the other secondary metaabolites and recorded loss of 91.56% in tender, 90.98% in immature and 87.76% in mature stage of the Parkia pods (Fig. 2b). Processing methods are reported to be effective in reducing tannin content of winged bean, soya bean and common beans (Tan et al., 1984; Bressani et al., 1982; Elias et al., 1979). Higher loss of tannins may be due to its solubility in water as tannins are known to be water soluble. Tannin reduction in Parkia seeds may improve its nutritional value by increasing protein digestibility (Savelkoul et al., 1992). Though presence of high amounts of tannin interferes with the digestive system, presence of tannin in food sometimes gives body and fullness of flavour to the food (Valier, 1951).

Loss of Phytate Phosphorus (PP) is lesser compared to tannins and accordingly its removal out of the total content is also lesser recording 33.01 to 53.28% in different stages of the pod (Table 1 and Fig. 2c). Cooking methods significantly (p<0.05) reduced PP in P. roxburghii. This observation agrees with the results of Ologhobo and Fetuga (1984) and Sutardi and Buckle (1985). Loss of phytate is due to its solubility in hot processing water during cooking operations and also partly due to the activities of endogenous phytases (Lolas and Markakis, 1975). Reduction of Phytate may increase the bioavailability of protein and minerals of Parkia pods. Appreciable amounts of saponins are detected in P. roxburghii pods. Saponin content in the pods increases as the pod matures (Table 1). However, loss of saponins due to different processing methods increases with the age of the pods, maximum loss (43.01%) being recorded in MP (Fig. 2d). Saponins occur in a wide variety of food plants, bengal gram, soybean, navy beans, haricot beans but most of them are removed by processing methods (Fenwick and Oakenful, 1983; Oakenfull, 1981; Varsney, 1969).

Trypsin Inhibitor Units in (TIU mg^-1) and Amylase Inhibitor Units (AIU mg g^-1) decreased due to OC and PC (Table 2). No significant differences were observed in TIU and saponin content in different stages of the raw pods, however, AIU increased with the age of the pods. Percent destruction of TIU due to processing methods recorded up to 67.02% in the tender stage of the pod (Fig. 2e) while destruction of AI was comparatively lesser, the maximum loss being 27.76% in tender stage of the pod (Fig. 2f). Heat inactivation of enzyme inhibitors in legumes has been reported by many workers (Ravindran and Ravindran, 1988; Ogun et al., 1989).

CONCLUSION

Parkia roxburghii pods recorded lesser values of phytate phosphorus and enzyme inhibitors to compare with the conventional legumes. The mean values of all the anti-nutrients decreased due to processing and cooking methods as compared to raw pods. Pressure cooking brought higher reduction in anti-nutritionals than ordinary cooking. Tannin reduction in parkia seeds may improve its nutritional value by increasing protein digestibility. Reduction of phytate may increase the bioavailability of protein and minerals of Parkia pods. Alkaloids and cyanogenetic factors are not detected in Parkia roxburghii pods.