An Analysis of the Chemical Composition of Different Varieties of Betel Leaves, and Purification and Characterization of Invertase and Polyphenol Oxidase from Doga Variety

Abstract

Betel leaf (Piper betel) is a tropical creeper and most important herb in Bangladesh. The nutrient compositions such as protein, lipid, total sugar, reducing sugar, non­reducing sugar, starch, chlorophyll, crude fibre, ash, moisture, phenol, vitamin-C, vitamin-B1, vitamin-B2, P-carotcne and minerals wch as iron, phosphorus and calcium of four varieties of betel leaves were compared. The enzymes such as amylase, invertase and cellulase activities in the betel leaves were also investigated at differe����t maturity stages. Activities of protease, P-galactosidasc, polyphenol oxidase, catalase and peroxidase were also measured. In general premature betel leaf contained about 86.87-87.99 gm% moisture, 2.96-3.08 gm% ash, 1.848-1.93 gm% fibre, 0.492-0.672 gm% phenol, 143.24-186.16 mg% chlorophyll, 1.29-1.43 gm% total wgar, 1.226-1.359 gm% non-reducing sugar and 4.98-6.28 gm% starch 2.87-3.34 gm% protein and 0.175-0.37 gm% lipid while mature betel leaf contained about 85.33-85.94 gm% moisture, 3.22-3.26 gm% ash, 2.095-2.11 gm% fibre, 0.547-0.704 gm% phenol, 255.65-320.76 mg% chlorophyll, 3.74-4.92 gm% protein, 0.98-1.63 gm% lipid, 3.22-3.58 gm% total sugar, 0.497-0.576 gm% reducing sugar, 2.587-2.900 gm% non-reducing sugar, 5.60-7 .05 gm% starch, 297-341 mg0/ii vi tamin-C, 120-131 mg% P-carotcne, 32.24- 36.02 mg% vitamin-B1, 0.63-0.78 mg% vitamin-B'.!, 216-231 mg% calcium, 9.15- 9.78 mg% iron and 145-157 mg% phosphorus. The amount of total sugar, non­reducing sugar an_d strnch conl'cnts were increased moderately from premature to over mature stage. No detectable amount of reducing sugar was found in premature stage but its content was increased thereafter upto over mature stage. The highest amount of invertase was found in Doga variety (64.00-82.32 Units per gm) and the lowest in Kai Hangla variety (26.67-40.32 Units per gm) in all the maturity stages whereas the acr-ivity of invertase was increased with the changes of maturity. The activities of amylase was maximum in Kal Bangla variety (premature 60.23 Units, mature 75.78 Units and over mature 70.51 Units per gm) and minimum in Dudhswar variety (premature 32.49 Units, mature 58.70 Units an<l over mature 53.35 Units per gm). Shail variety contained the highest cellulase activity while Kal Bangla variety contained the lowest. The activities of amylase and cellulase increased upto mature stage and then decreased drastically in over inature stage. The activities of protease and P-galactosidase were lowest in Dudhswar variety and highest in Doga variety but the protease activity increased with the increase in maturity while P-galac!'osidase activity decreased from premature to over mature st·age. The activities of catalase, peroxidase and polyphenol oxidase were found to be varied between 49.50 to 57.08 Units, 28.24 to 37.45 Units and 20.9 to 22.4 Units per gm respectively in different varieties of betel leaves at mature stage. Two invertases (AIV 1 and ,\ IV 11) as well as polyphenol oxic.lase were purified from Doga variety betel leaves at mature stage by successive chromatogrnphies on DEJ\E-Cellulose followed by Cl\il-Ccllulose and Sephac.lcx G-75 column. The molecular weights (Mr) were found to be 94 kDa and 93.5 kDa for the enzyme /\IV I, 72 kDa and 71.5 kDa for J\TV IT and 48 kDa and 45.5 kDa for polyphenol oxidase (PPO) as measured by Cd filtration and SDS-Polyacrylamide gel electrophoresis respectively. In the presence of 2-mcrcaptoethanol, i\ IV I showed two identical subunits with f\fr of about 46 kDa indicating that the enzyme is a homodimer while the enzyme AIV I I is heterodimer with L\fr of 40.5 kDa and 31 kDa. On the other hand, the enzyme polyphenol oxidase is monomer in nature. The purified invertases, .A lV I and AIV II were glycoprotein with neutral sugar content of 14.6% and 19.4% respectively. The Km values of .AIV J, J\IV II and PPO were also determined. The cn7.ymes ATV I, i\lV II and PPO showed the following characteristics such as optimum pH 4.5, 5.5 and 6.2; optimum temperature 37°C, 3°C and 3211C respectively. The activities of all three enzymes were gradually decreased with the increase in concentration of various chemicals studied. 7.n2+ and i\g·' produced inhibitory effects while Ca2+ and M1121 produced no effects on betel leaf invertases. The activities of both .AIV I and ;\IV 11 were increased moderately in the presence of Mg2+, K+ and Cu2+ salts. The activities of PPO were increased in the presence of metallic salts of Ca2+ and Cu2+ but its activit-y was decreased in the presence of salts of Fe2+ and fvig2+. The activities of J\IV I, 1\JV II and PPO were decreased drastically in the presence of l-lgCl2 an<l the activities were almost completely inhibited by 5 mM HgCl2 suggesting the -SH group containing amino acids arc present at or near the active sites. Remarkably, the betel leaves extract showed antibacterial activities but contained no anti fungal activities. The betel leaf extracts are toxic in nature as detcct����d by brine shrimp lethality bioassay and the LD50 values for different varieties were found to be as follows: 7 .02 ����Lg/ ml for Dudhswar, 10.75 µg/ml for Shail, 17.25 ����Lg/ml for Doga and 10.52 ����Lg/ml for Kal Bangla varieties.