Date Seeds Biochemical Treasures .I. Biologically Active Proteins

Introduction
Date seeds are the main by-product in the processing of pitted dates, dates paste, dates syrup and all fermentation products. They comprise between 12- 20 % of the date fruit total weight. However, they are used mainly for animal feed in spite of their content of protein (~7%), oil (10%), tannins, cellulose, hemicellulose and lignin. Since dates pitting process (for pitted dates and dates paste production) do not involve heat treatment, the seeds retain full biological activity of embryo, proteins and enzymes.

What are the biologically active proteins?
They represent a large and highly diversified group of proteins with various specific functions which are essential for the survival of all living organisms. All enzymes (the biological catalysts), receptors as well as many hormones, inhibitors, lectins (phytohemagglutinins in the old terminology), most stimulators and bio-carriers are proteins. Plant seeds contain a wide variety of enzyme inhibitors and lectins as an essential part of their biochemical defense mechanisms. Most α-amylase, trypsin, chymotrypsin and elastase inhibitors are small, thermally stable proteins with potent inhibitory activity against exogenous enzymes (i.e. insect, animal and microbial enzymes) with no activity towards the endogenous enzymes. In addition to the importance of protease inhibitors in affinity chromatography, they received great attention in the past 10 years due to their possible inhibitory activity against HIV protease-1 (essential enzyme for virus growth) which may contribute to the fight against AIDS.

Lectins are carbohydrate binding proteins or glycoproteins of non-immunological origin capable of cells agglutination and/or glycoconjugates precipitation. They are widely spread in nature among animals, plants and microorganisms. Agglutination of human and animal erythrocytes, leucocytes and platelets are very well studied activities. Many lectins are known as `potent mitogens where they act as division stimulators of lymphocytes and converting them from resting cells to growth and development. Furthermore, some lectins has the specificity of agglutination of cancer cells but not normal cells. They have been used in

-affinity chromatography of membrane receptor proteins
- probes in the study of cell membranes
- rapid identification of closely relate bacterial strains
- diagnostic blood samples analysis in serological and forensic laboratories
- cytogenetic studies

The present article is an attempt to shed some light on the occurrence of human salivary α-amylase, bovine trypsin inhibitor and lectins in the seeds of four date cultivars (Iraqi cultivars: Zahdi, Khastawi, Brame and Barhee).

Major Biologically Active Proteins in Date Seeds

Enzyme Inhibitors
The crude extracts of all seed samples contained trypsin and α-amylase inhibitory activities (Table 1). While Barhee seeds have the highest trypsin inhibitor

Table 1. Specific activity levels of salivary α-amylase and trypsin inhibitors in the seed crude extracts of four date cultivars

activity, Zahdi seeds contained the lowest activity. α-Amylase inhibitor activity was at the highest level in Zahdi seeds and the lowest in Brame seeds. However, the relatively low α-amylase inhibitor activity could indicate that the inhibitor may have lower specificity towards the human salivary α-amylase. Further detailed study is required on the purification and characterization of these inhibitors.

Lectins
The crude extracts of the four date cultivars showed variable hemagglutination activity on the four major human blood groups (Table2). Zahdi cultivar seeds extract have the highest specific activity towards (O+) group while Barhee seeds extract showed the highest specific activity towards (B+). The highest specific activity of Khastawi and Brame cultivar extracts was towards (AB+) group.

Table 2. Hemagglutination Activity of Date Seed Crude Extracts on the Four Major Human Blood Groups.

However, there are no similar studies on other date cultivars.

Main Characteristics of Zahdi Cultivar Seeds Lectin
The lectin was purified to homogeneity by a very efficient two-steps procedure based on affinity chromatography using Sepharose-4B column. The binding of the lectin to the Sepharose-4B column indicates that it is a galactose specific lectin (i.e. it binds D-galactose residues). The purified lectin has optimal hemagglutination activity at pH 7.0 and it is most stable at pH 7.5. The hemagglutination activity of the purified lectin on various blood types is shown in Table 3. While it has comparable activity on human (O+ group) and rabbit, it has extremely low but comparable activity on calf and sheep blood.

Table (3). Hemagglutination Activity of the Purified Lectins from Various Origins.

The purified lectin has a very strong agglutination activity on several pathogenic bacteria (Staphylococcus aureus, Enterobacter aeroginosa and Salmonella typhimurium). This property requires deeper investigation to explore this lectin activity on the binding to major viruses (e.g. HIV and hepatitis viruses). The Con A lectin is known to bind the HIV and inactivate it (i.e. cell protection from the infection).

Summary
Date palm seeds are not utilized efficiently as major dates processing by-product in spite of their content of unstudied constituents. This article concentrates on the main types of biologically active proteins in the seeds of four major date cultivars. Trypsin inhibitor, α-amylase inhibitor and lectin activities are present in the seed extracts of four cultivars. However, the extracts has variable specificity towards the human main blood groups. The purified lectin from Zahdi cultivar seeds has agglutination activity on certain pathogenic bacteria.

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