Medicinal Plants And Plant Derived Pharmaceuticals

Published Date: 02 Nov 2017

Production of pharmaceuticals in plants for therapeutic purposes shows great promise, with some Plant made Pharmaceuticals in clinical trials and many others under investigation. Plant production systems are low cost and can easily be expanded as compared to the cell culture systems used now-a-days for the production of biological therapeutics (Thomas et al., 2002). Twelve traditional medicinal plants Agapanthus campanulatus, Antidesma venosum, Becium obovatum, Cucumis hirsutus, Cyperus textilis, Diospyros lycioides, Dissotis princeps, Gladiolus dalenii, Haworthia limifolia, Protea simplex, Vernonia natalensis, Watsonia tabularis that are commonly used for the treatment of gastro-intestinal ailments, were, when collected from South Africa and evaluated for the presence of phytochemical components such as flavonoids, gallotannins, condensed tannins, other phenolic compounds, alkaloids and saponins. All the plant species evaluated contained some phenolic compounds. Phenolic compounds are of important pharmacological value, some having anti-inflammatory properties (Bruneton, 1995).

In inflammatory responses, some molecular targets of pro-inflammatory mediators are inhibited by phenolic compounds such as condensed tannins, gallotannins, and flavonoids. (Sharma et al., 1994; Iwalewa et al., 2007). These aforementioned plant materials are antimcrobial and show genotoxicit which was reported earlier (Fawole et al., 2009). Many common cultivated plants are also the source of compounds used as building blocks in the semisynthesis of pharmaceuticals. There are several phytochemicals derived from soybean (Glycine max) like sterols, sitosterol, stigmasterol, and campesterol (Barbara et al., 2008). Medicinal herbs in clinical trials show dominance of antioxidant constituents in phenolic compounds displaying positive and linear correlation between antioxidant activity and total phenolic content. Major phenolic compounds were phenolic acids, quinones, stilbenes, flavonoids, coumarins, curcuminoids, lignans, and tannins. Future expectation from the relationship of anticancer/antioxidant activity and phenolic compounds is to get comprehension of the mechanism of action of Chinese medicines used traditionally (Caia et al., 2004).

Diosgenin, a steroid derived from Mexican yams (Dioscorea spp), is used in the semi synthesis of medical steroid. Morphinan alkaloids produced by opium poppy (Papaver soniferum) are thebaine, morphine, codeine, noscapine and papaverine. Semi-synthetic drugs derived from opium poppy are fentanyl, dihydrocodeine, and oxycodone which are frequently used as cough suppressants, sedatives and strong analgesics. (Barbara et al., 2008). Natural products are becoming popular in modern society where synthetic products have shown their injurious effects on environment and human health. According to the scientific research on the healing properties and bioactivity of natural compounds, especially of plant derived compounds, is getting to be intense, in the West as well as in developing countries of rich floral biodiversity (Louw et al., 2002).

Plants continue to serve as medicine in past and present. But the modern pharmacy has born from botanical medicine and most botanical therapeutics is obtained from pharmaceutically important plants, cultivated for production of greater amount of bioactive components (Schmidt et al., 2008). Traditional medicines are still being used as conventional treatments of different diseases (Alviano & Alviano, 2009). In Europe and USA, about 50% of the prescribed products are originating from natural products including plants or their derivatives (Cordell, 2002; Newman et al., 2003). Only 1-10 % out of the 250,000 – 500,000 plant species on Earth have been studied chemically and pharmacologically (Verpoote, 2000). In the Middle East, 700 species of the discovered plants are known for their medicinal values (Azaizeh et al., 2006).

Crocus sativus Linn. (Iridaceae) is used widely in tropical and subtropical countries for a variety of purposes in both household and for medicinal purposes (Bhargavak, 2011). Saffron in filaments is the dried, dark red stigmata of Crocus sativus flowers and it is used as a spice, food colorant, and a drug in medicine (Abdullaev, 2002). The stigmas of the plant are used for they contain a variety of chemical constituents like the crocetin, crocin and other flavanoids which make them suitable to possess diversified medicinal properties for treating various ailments (Bhargavak, 2011). It is a therapeutic and promising herb of pharmaceutical industry due to its varying medicinal properties ranging from mild fever to cancer and DNA repair (Wani et al., 2011). A growing body of research has demonstrated that saffron extract itself and its main constituents, the carotenoids, possess chemopreventive properties against cancer (Abdullaev, 2002).

Plants pharmaceuticals are going to be the next major commercial development in biotechnology and they will be of great value due to their large scale easy and safe production and long time storage capacity; they are also useful for the developing world in having low-cost drugs and vaccines (Julian et al., 2005). New drugs have been produced through medicinal plants which are becoming valuable and useful sources showing splendid results for the treatment of almost all of the diseases.

M. oleifera is very important for its medicinal value. Various parts of a single plant, such as the leaves, roots, seed, bark, fruit, flowers and immature pods of Moringa oleifera can act as cardiac and circulatory stimulants, possess antitumor, antipyretic, antiepileptic, anti-inflammatory, antiulcer, antispasmodic, diuretic, antihypertensive, cholesterol lowering, antioxidant, anti-diabetic, hepato-protective, antibacterial and antifungal activities, and is being applied as the treatment of different ailments in the indigenous system of medicine, particularly in South Asia (Anwar, et al., 2007). Echinacea is an herbal medicine and also known as the purple coneflower that has been used for centuries, traditionally as a treatment for the common cold, coughs, upper respiratory infections, bronchitis and some inflammatory conditions (Percival, 2000).

Nigella sativa is frequent natural remedy used widely for its aromatic seeds(Kalonji) as spice, as condiment and as carminative, Nigella seeds play different roles as antihypertensive liver tonic, diuretic, digestive, emmenagogue, anthehelmintic, appetite stimulant, analgesic, antidiarrheal, antibacterial and good remedy for skin disorders. As a result, Kalonji is widely studied for its biological propereties like it is anticancerous, renal protective, antioxidant, immunomodulator, antidiabetic, analgesic, anti-inflammatory, antihypertensive, antimicrobial, hepatoprotective, spasmolytic, and bronchodilator. (Gilani, 2004).

Ginseng (Panax ginseng) is having a long history in China as a traditional herbal medicine. (Wang et al., 2010; Su et al., 2010; Kaufman et al., 2002; Kim et al., 2002; Keum et al., 2000; Attele et al., 1999). In past, only root of ginseng was used but recent researches have claimed for it to be possessing similar medicinal properties in its aerial parts. Ginseng stem shows more susceptibility to diseases and environmental issues in its long period of growth but molecular mechanism of its vulnerability is still unclear. (Wang et al., 2009) But proteomic approaches like 2-DE give better understanding of the pathogenocity of ginseng during study of healthy and pathogen susceptible tissues consequently improving ginseng productivity (Sun, et al., 2011). A protein derived from Chinese herb Tian-hua-fen, from the root tubers of Trichosanthes kirilowii of family Cucurbitaceae is Trichosanthin, which is an abortifacient and also proved to be the treatment of placental cancer, trophoblastic tumours, including molar pregnancy and invasive mole. (Tsao et al., 1986).

Pharmaceutical proteins

Pharmaceuticals practiced as disease therapy are built on organic molecules obtained from microbes or organic chemistry. They include most antibiotics, hormones, analgesics, and others. More attention is being given to larger complex protein molecules as therapeutic agents. Proteins are larger molecules, made up of long chain of smaller subunits known as amino acids. (Suslow et al., 2002). Proteins subunits: amino acids are just like 26 letters of alphabet, linked together in various combinations of 20 or more amino acids, no matter what is the length of proteins whether 100 or 1,000 amino acids ("letters") long (Thomas et al., 2002).

The proteins and peptides have been extensively used as active therapeutic ingredients since the mid-1990s, aided by improvements in modern recombinant DNA technology and biotechnological manufacturing. All new drug approvals in the United States and the European Union contain one-third share of proteins, but these macro-molecules have several levels of structure, referred to as primary, secondary, tertiary, and quaternary structures, which are the basis for multiple interaction pathways that lead to protein instability and to avoid biological activity loss and possible immunogenic effects of the structurally or chemically altered protein molecules, some stable processes have to be designed (Schiffter, 2011). There are 200 pharmaceutical proteins analyzed clinically to date. More than half of these are undergoing research and development, and about 100 are still being tested clinically, but about a dozen are already available for sale in market. Their target remedies are tumors, heart diseases, epidemic and autoimmune diseases (Blohm et al., 1988). Proteins are the heart of cell biology, used as remedies for several diseases. Insulin, a small peptide was the first protein discovered as a revolution for diabetes treatment. Similarly, antigens for vaccinations are also often proteins (Thomas et al., 2002).

Interest in pharmaceutical proteins, is expanding from basic research to drug marketing and these developments are starting with genetic engineering and biotechnology, which contribute to the discovery of new proteins on a larger scale. Genetically engineered proteins, which are chemically and biologically exactly defined, can be used as active substances for new drug products and in research to get new knowledge of therapeutic use, based on the concept of therapy with endogenous proteins (Blohm et al., 1988).

Animal Proteins:

In every human culture, animals have been used for the treatment and cure of several illnesses and diseases (Eraldo & Costa-Neto, 2005). Recombinant proteins can be obtained from several transgenic animals but only two systems are working now-a-days. One is milk having a protein, human antithrombin III, obtained from transgenic animals and has been studied for 20 years, to get the agreement of EMEA (European Agency for the Evaluation of Medicinal Products) in 2006. Second system is chicken egg white which is used in producing transgenic birds, after improvement in methods (Roberts, 1996). These systems and a few more systems have generated a variety of recombinant proteins experimentally. Recombinant proteins include monoclonal antibodies, blood factors, vaccines, hormones, cytokines, growth factors, enzymes, collagen, milk proteins, fibrinogen and others (Houdebine, 2009).

Revolutionary new opportunities for the production of novel proteins in milk have been created by the development of methods for gene transfer. Exploitation of these opportunities depends upon selection and cloning of milk protein genes and identification of the sequences that govern tissue specific hormonally induced expression in the mammary gland. Studies with three genes, ovine β-lactoglobulin, rat β-casein and whey acidic protein of rat and mouse, suggest that they may all meet this requirement. Fragments of the ovine β-lactoglobulin, murine whey acidic protein and rabbit β-casein genes have directed production of novel proteins in the milk of transgenic mice, sheep, rabbits and pigs. The proteins were biologically active and usually co-migrated with authentic proteins (Wilmut et al., 1991).

Livestock animals are of great use throughout humankind's history. Farm animals bovine and porcine are of great value for production of insulin (for treatment of diabetes), gelatin (for pharmaceutical and other purposes), as well as horse and sheep antibody against natural venoms, toxins, drugs and microbial peptides. Recently, they are being harnessing as bioreactor for production of biopharmaceutical related products through gene farming with efficiency far greater than any conventional microbial or cell-culture production systems. Only 16 transgenic cows would be covering the worldwide needs from human growth hormone. The transgenic, especially animal, technology would be solving a several biopharmaceutical products disadvantages, such as cost, biosafety, immunogenicity and the availability dimensions (Redwan, 2009).

Traditional Chinese Medicine (TCM) has been increasingly practised in many countries of the world. Various forms of TCM have been advocated to treat numerous diseases and ailments in humans and animals. It is estimated that at least a quarter of the world’s human population use medical practices based on TCM (Jones & Vincent, 1998). It is claimed that about 13% of the medicines used by TCM are derived from animals (Coker, 1995). At present, about 40% of all prescription drugs are substances originally extracted from plants, animals, fungi and microorganisms (Wilson, 1995).

Plant proteins:

Plants have many advantages over established production technologies for the large-scale expression of recombinant proteins, but several challenges remain to be addressed in terms of improving yields and product quality (Khanzada et al., 2008). Twelve traditional medicinal plants Agapanthus campanulatus, Antidesma venosum, Becium obovatum, Cucumis hirsutus, Cyperus textilis, Diospyros lycioides, Dissotis princeps, Gladiolus dalenii, Haworthia limifolia, Protea simplex, Vernonia natalensis, Watsonia tabularis that are commonly used for the treatment of gastro-intestinal ailments, were, when collected from South Africa and evaluated for the presence of phytochemical components such as flavonoids, gallotannins, condensed tannins, other phenolic compounds, alkaloids and saponins. All the plant species evaluated contained some phenolic compounds. Phenolic compounds are of important pharmacological value, some having anti-inflammatory properties (Bruneton, 1995).

Like animals, many plant species contain carbohydrate-binding proteins, which are commonly referred to as either lectins or agglutinins. Lectins are proteins that bind to specific carbohydrate structures and can thus recognize particular glycoconjugates among the vast array expressed in animal tissues. Most animal lectins can be classified into four distinct families (1): C-type lectins (including the selectins); P-type lectins; pentraxins; and galectins, formerly known as S-type or S-Lac lectins (Douglas et al., 1994). Animal lectins undoubtedly fulfill a variety of functions, many could be considered in general terms to be recognition molecules within the immune system. More specifically, lectins have been implicated in direct first-line defense against pathogens, cell trafficking, immune regulation and prevention of autoimmunity (Anon, 2002). Generally speaking, lectins are proteins that bind reversibly to specific mono- or oligosaccharides. Lectins from different plant species often differ with respect to their molecular structure and specificity. It is important, therefore, to realize that all plant lectins are artificially classified together solely on the basis of their ability to recognize and bind carbohydrates. Molecular, biochemical, cellular, physiological and evolutionary arguments indicate that lectins have a role in plant defense. A circumstantial argument in favor of a defense role of plant lectins is their marked stability under unfavorable conditions. Most lectins are stable over a wide pH range, are able to withstand heat, and are resistant to animal and insect proteases (Peumans & EIS, 1995). Plant proteins can be classified as plant antifungal proteins which are further divided into different groups comprising chitinases and chitinase-like proteins, chitin-binding proteins, cyclophilin-like proteins, defensins and defensin-like proteins, deoxyribonucleases, embryo-abundant protein-like proteins, glucanases, lipid transfer proteins, peroxidases, protease inhibitors, ribonucleases, ribosome-inactivating proteins, storage 2S albumins, lectins, and thaumatin-like proteins (Wong et al., 2010).

A novel mannose-binding lectin (designated OJL) was purified from rhizomes of Ophiopogon japonicus. It was inhibitory to herpes simplex virus type II (HSV-II) and displayed an antifungal activity against Gibberella saubinetii and Rhizoctonia solani (Tian et al., 2008). Cajanus cajan which is both a food crop and a cover/forage crop having high levels of proteins and important amino acids like methionine, lysine and tryptophan (Pal. et al., 2011). Guduchi (Tinospora cordifolia), a widely used plant in folk and Ayurvedic systems of medicine is well known for its immunomodulatory activity; however, the presence of an immuno-modulatory protein (ImP) in guduchi has not been investigated. Ayurvedic medicines are prepared using guduchi because it contains an immune-modulatory protein in guduchi stem showing lympho-proliferative and macrophage e-activating perties (Aranha et al., 2012).

The extracts of some medicinal plants such as Hygrophila auriculata, Croton tiglium, Abrus precatorius, Moringa oleifera, Withania somnifera, Solanum nigrum and Psoralea corylifolia were studied against pathogenic fungal strains of Aspergillus tamarii, Rhizopus solani, Mucor mucedo and Aspergillus niger. They were extracted, purified and treated with trypsin and found loss of antifungal activity in many of them, which confirmed the presence of proteins and peptides in them (Aranha et al., 2012). Some of the wild nuts and seeds used as food in several parts of the world have considerable promise as protein source (Amubode & Fetuga, 1983). Tamarindus indica contain crude protein of high levels (31.08) than the levels reported earlier (Ishola et al., 1990; Bhattacharya et al., 1994; Siddhuraju et al., 1995). Tamarindus indica also contains a high level protein with many essential amino acids which is very helpful in building strong and efficient muscles (Prakash, 1988).

Family Iridaceae: pharmaceutically important compounds:

Family Iridaceae is abundant in its morphological and anatomical characters, as well as in its secondary metabolites. Several varied phytochemicals have been described in the family containing almost all common classes of Flavonoids (Harborne & Williams). Genus Iris comprises of over 300 species and is famous for its ornamental and medicinal value (Mirheidar & Moaref, 1996). Plant species of genus Iris (Iridaceae) were detected to have rich sources of secondary metabolites in the past, especially of flavonoids and have a long history of medicinal use in several places of the world (Hui et al., 2010).

These species were introduced as diuretic and expectorant at low doses while high doses were strong purgative and emetic besides being useful for pulmonary asthma, cancer, inflammation, liver, and uterus diseases, as well as haemorrhoid and gripe ((Mirheidar & Moaref, 1996; Purev et al., 2002). Over 90 flavonoid constituents have been discovered and characterized during the last decade (1999-2008) including 38 new compounds from 15 species of Iris (Hui et al., 2010). Phytochemical investigation of Iris species has resulted in the isolation of a variety of compounds including quinones, triterpenoids, flavonoids, isoflavonoids, and stilbene glycosides (Rahman et al., 2002). At generic level, anthocyanins in flowers are found generally. Quinonoid and xanthone pigments have also made distribution patterns systematically interesting. In Iris rhizome oils and in Crocus styles, occur some distinctive chemicals which are useful economically (Harborne & Williams, 2000).

Mangiferin is a unique C-glucosylxanthone having widespread distribution in nature unlike other xanthones. Iridaceae contain this biologically active chemical, having anti-inflammatory, anti-hepatotoxic and antiviral properties, which can be easily identified on paper or TLC and due to its characteristic ultraviolet spectrum and specific color reactions (Harborne & Williams 2000). The Iridaceae has not been much investigated pharmaceutically as other families, and plants may have several potentially important compounds (Ascough et al., 2009).

The genus Gladiolus (Family: Iridaceae) has 260 species of a perennial herb. In West Africa the corms of Gladiolus species are used in food and Traditional Medicine, often in combination with other plant materials. In South West Nigeria the corms called "baka" are used in treating gonorrhea, dysentery and other infectious conditions (Ameh, 2011). The methanol extract of Gladiolus psittascinus bulbs found to be very effective in decreasing blood glucose level in alloxan-induced diabetic rats (Adediwura & Kio, 2008). If this compound is identified, it could provide an important substitute for the management of non-insulin-dependent diabetes (Ascough et al., 2009).

Iris species are of great medicinal value as they are used for the treatment of cancer, inflammation, viral and bacterial infections (Hanawa et al., 1991). Saffron and its constituents are suggested as alternative anticancer agents, which alone and in combination with other synthetic substances may have the potential for the prevention and the treatment of certain forms of cancer (Chermahini, 2010). As a medicinal plant, saffron has traditionally been considered an anodyne, antidepressant, a respiratory decongestant, antispasmodic, aphrodisiac, diaphoretic, emmenagogue, expectorant, and sedative. It was used in folk remedy against scarlet fever, smallpox, colds, asthma, eye and heart diseases, tumor, and cancer. Saffron can also be used topically to help clear up conquer sores and to reduce the discomfort of teething infants (Abdullaev & Mejia, 1997).

Plant tissues having secondary metabolites and lectins are sources of naturally bioactive molecules that control the pathogens causing diseases in plants and humans with antibacterial and antifungal activities and thus the ability of lectin selectively to bind microrganisms makes them potential tools to study pathogen species (Paiva, et al., 2010). Saffron possesses lectins (Oda &Tatsumi 1983; Escribano et al., 2000) and is suggested to have antitumor activity caused by lectins (Abdullaev & Mejia, 1997).

Family Iridaceae Protein Profile:

The Iridaceae is an ornamental plant family of about 1500 species and 85 genera, which has an almost worldwide distribution. For its size, it is particularly rich in its phytochemistry, having a wealth of chemical structures, especially of the phenolic type (Hegnauer 1963; 1986). In family Iridaceae, 116 species were investigated and found to be containing free amino acids and γ-glutamyl peptides like subfamily Iridoideae, which contain 3-(3-Carboxyphenyl)-alanine, 3′-carboxyphenylglycine and γ-glutamyl peptides frequently but these are not found in subfamilies, Ixioideae and Sisyrinchoideae. Different amounts of aromatic amino acids also occur in species within the Irideae and Tigrideae and γ-glutamyl peptides are also found distinctly in Irideae (Larsen et al., 1981). A ribosomal inactivating protein (RIP) was isolated from Iris bulbs which were having piscicidal, antineoplastic, antioxidant, anti-tumor, antiplasmodial and antituberclosis potential (Hideyuki et al., 1995, Miyake et al., 1997; Bonfils et al., 2001).

Sparaxis:

Sparaxis is a genus of Iridaceae and subfamily Ixioideae described by Ker Gawler in 1802, comprises just 15 species (Goldblatt 1992; Goldblatt & Manning, 1999). Species extend from the Agulhas Peninsula in the south of the subcontinent to the Bokkeveld Plateau and western Karoo in the north, a range that falls entirely within the southern African winter-rainfall zone (Bond & Goldblatt, 1984). Several species of the genus Sparaxis of Iridaceae family produce flowering bulbs which give flowers out of corms in their active season of spring and have a rest period in the summer (Scagel, 2004). The genus Sparaxis in the Iridaceae family contains several species of flowering bulbs used in landscape and cut flower production. Sparaxis species grow from corms with their active period of growth and flowering in the spring and a rest period in the summer. Natural vegetative multiplication rate of Sparaxis species is low and limited to annual corm replacement (daughter corm) and production of cormels at leaf bases. (Scagel, 2004). Species of Sparaxis are seasonal, corm-bearing geophytes of small to moderate size, with spicate inflorescences typically 10-30 cm high, but up to 60cm in S. auriculata (Goldblatt et al., 2000).

Freesia:

Freesia is beautiful ornamental plant which is grown as pot plant and for its decorative cut flowers. Freesia requires optimum conditions for plant growth and development i.e. proper temperature and moisture both for cut and pot flowers (Startek et al., 2000, 2002; Startek, 2002). Freesia contains protease (FP)-A in its regular Freesia corms (Kaneda et al., 1997). When new corms were reproduced from original ones which were kept at 4°C for several months, were found to be having two proteases (FP-B and FP-C). FPs purified from the extracts of new corms was having, Mr of 24 kDa (A), 25 kDa (B), and 24.5 kDa (C) by SDS-PAGE, respectively.

Freesia has been studied for analyzing the soluble proteins during somatic embryogenesis through SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Six polypeptides were found, which might have some roles in the process of somatic embryo development. Three polypeptides (45, 53 and 55 kDa) were identified during development of embryogenic callus, globular embryoid, and embryoid with coleoptiles, except the embryoid with leaf (Abdullaev & Aguirre 2004). The N-terminal sequences of FPs were identical to those of papain with respect to the conservative residues of cysteine protease (Chermahini, et al., 2010).

Cysteine proteases is a large family of proteins responsible for the function of programmed cell death, therefore cysteine proteinase inhibitor may take part a role in regulation of programmed cell death during embryonic patterning (Sharifi et al., 2012). Cysteine proteinases take part in each step of plant physiology and development. They have their role in development, senescence, programmed cell death, storage and mobilization of germinal proteins and also play role in showing response to different types of environmental stresses. Plant cysteine proteinases have a similar role of wound healing, immunomodulation, digestive conditions, and neoplastic alterations in mammals as mammalian serine proteinases have (Salas et al., 2008).

Caspases is a group of proteases which resemble structurally with the cysteine proteases in having common cleaving peptide bonds and Asp residues. These are commonly found in the cytosol of all animals and take part in proteolytic pathways required for executing programmed cell death, or apoptosis. In mammals the caspases are also involved in a function of activating pro-inflammatory cytokines (Stennicke & Salvesen 1998). The sequence of FP-A was identical with those of FP-B within 20 residues of its N-terminal. It may be possible that FP-B was produced by some post-translational modifications from FP-A during the chilling. On the other hand, N-terminal sequence of FP-C was different from those of FP-A and FP-B. It was explained that FP-C was a new protease of Freesia corm (Chermahini et al., 2010). A protease, Freesia protease (FP)-A, was purified to electrophoretic homogeneity from regular Freesia (Freesia reflacta) corms in harvest time. At the harvest time, Freesia corms were having Freesia protease (FP)-A, having Mr of FP-A estimated to be 24 k by SDS-PAGE. The optimum pH of the enzyme was 8.0 using a casein substrate.

Aims of Study:

Therefore the present study was intended to analyze:

The effect of two temperature values (4 °C and 25 °C) and four different pH values (3.0, 5.0, 7.5 and 10.0) on protein extractions of the two plants i.e. Freesia and Sparaxis.

The total protein content of the two ornamental plants while little work have been done till now.

To prove that family Iridaceae is not only an ornamental family but also contains pharmaceutically important proteins as already discovered from its genera like Crocus, Gladiolus and Iris containing important proteins of great pharmaceutical value.

MATERIALS AND METHODS

Seed sample collection:

The research work was conducted in the Physiological Lab, Institute of Pure and Applied Biology, Bahauddin Zakariya University Multan. The corms of the two plants were collected from the local market of Lahore.

Sr. No.

Scientific Name

Organ used

Collected from

1

Freesia

Corms

Local market

2

Sparaxis

Corms

Local market

Table 1: Sample Collection

Freesia was named in honor of Friedrich Heinrich Theodor Freese (1795-1876), a German physician from Kiel. Even though Freesia looks different than Irises, they do share a common sparsely branched stem with few leaves and a loose spike-like raceme of fragrant flowers. The most common cultivar is Freesia refracta. Freesia grows on loamy or sandy soils and growing from 15 to 40 cm with conical or globose corms with medium to coarse textured fibers as shown in Figure 1: A.

Figure 1: A: Freesia corms, B: Sparaxis corms

The corms of Sparaxis need just two inches of soil over them and can often be planted by just poking them in with fingers. Sparaxis is a genus in the Iridaceae family that is endemic to South Africa and found mostly in the southwestern Cape and the western Karoo. They are easy to grow in Mediterranean climates. They have been in cultivation for a number of years and there are many colorful hybrids grown as well. Sparaxis flowers grow from fall planted corms, blooming in spring. The corms are smaller than most spring bulbs, often no bigger than ½ to 1 inch across as shown in Figure 1: B. The flowers bloom in various colors with most blooms having two or more colors on each petal.

Preparation of crude extracts:

The protein extractions for the two plants were performed at two different temperatures (4 °C and room temperature i.e., 25 °C) and then each extraction was further processed at four different pH values (3.0, 5.0, 7.5 and 10.0). For extraction at 4 °C, the samples were put either in ice boxes or in cold refrigerator to avoid heat shock.

Preparation of pH Solutions:

After collection of seed samples, the following buffer solutions were prepared as shown in Table 2.

pH

Chemicals Used

pH

pH Calibration

pH 3.0

Solution A: 0.1N Citric acid

Solution B: 0.1N Sodium citrate

82 ml of Solution A + 18 ml of Solution B were mixed together to get the pH 3.0.

The pH value was adjusted to 3.0 and the final volume was set to 500 ml with DW.

pH 5.0

Solution A: 0.1N Acetic acid

Solution B: 0.1N Sodium acetate

70 ml of Solution A + 30 ml of Solution B were mixed together to get the pH 5.0.

The pH value was adjusted to 5.0 and the final volume was set to 500 ml with DW.

pH 7.5

Solution A: 0.1N Na2HPO4

Solution B: 0.1N NaH2PO4

40.5 ml of Solution A + 9.5 ml of Solution B were mixed together to get the pH 7.5.

The pH value was adjusted to 7.5 and the final volume was set to 500 ml with DW.

pH 10.0

A: 0.1M Sodium carbonate

B: 0.1M Sodium bicarbonate

60 ml of Solution A + 40 ml of Solution B were mixed together to get the pH 10.0.

The pH value was adjusted to 10.0 and the final volume was set to 500 ml with DW.

Table 2: Buffer Solutions

Grinding:

The corms of each plant for investigation were grinded in order to break up the cell structure. The grinded material from each plant was put in four falcon tubes of 50 ml. Volume in the falcon tubes was raised to 40ml by adding four buffers. Each Falcon tube was labeled with the plant name and the respective pH value as 3.0, 5.0, 7.5 and 10.0.

Stirring:

A magnetic bar was put in each flask and was kept on stirring for two hours by covering the flasks with aluminum foil.

Figure 2: Flow chart showing the different steps involved in protein processing

Centrifugation:

After stirring, the corm extracts were poured in centrifuge tubes by weighing them equally on digital balance. The centrifuge machine was covered with lid and was centrifuged for 20 minutes at 10,000 rpm. After 20 minutes of centrifugation, samples were taken out and filtered with Whatman filter paper in order to get the dust free crude extract.

Protein Quantification by Bradford assay

The assay reagent was prepared by dissolving 100 mg (0.1 g) of Coomassie blue G-250 in 50 ml of 95 % ethanol. The solution was then mixed with 100 ml of 85 % of ortho-phosphoric acid and made up to 1 liter with distilled water and was stirred for four hours. The reagent was filtered through Whatman No.1 filter paper before storage in an amber bottle at 4 °C. It was stable for several weeks, but slow precipitation of dye occurred on prolonged storage. So the filtration of the stored reagent was necessary before use.

Standard Curve for Protein Estimation

Bovine serum albumin (BSA) at a concentration of 5 mg/ml in distilled water was utilized as a stock solution. It was stored frozen. The standard solutions were prepared through appropriate dilutions of the BSA stock and the final protein concentrations were as follows:

Sr. No.

BSA standard solution concentrations (µg/ml)

Total protein quantity (mg)

1

0

0.00

2

100

0.10

3

250

0.25

4

500

0.50

5

750

0.75

6

1000

1.00

7

1250

1.25

8

1500

1.50

Table 3: Standard Concentrations for BSA Curve

100 µl of these standard solutions were added in test tubes containing 5 ml of Bradford reagent to take the absorbance reading for standard curve. Blank reading was obtained by mixing 100 µl of distilled water to 5 ml of Bradford reagent to calibrate the spectrophotometer, for calibration first place black body and press 0% which will show maximum absorbance (A= 3.0) and zero transmittance (T=0), then place blank solution in spectrophotometer and press 100% which will show maximum T (100%) and minimum or no A. Similarly, 100 µl of all the twelve samples from two plant Ranunculus and Anemone, at their respective pH i.e., 3.0, 5.0 7.5 and 10.0 were mixed with 5 ml of Bradford reagent and the change in color was measured Spectrophotometrically (BMS Spectrophotometer, SN 204573) at 595 nm (Bradford, 1976).

Figure 3: Standard Curve of BSA

Sodium Dodecyle Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE):

Sodium dodecyle sulfate polyacrylamide gel electrophoresis is used to separate proteins from different samples collected from different places. Polyacrylamide gels are prepared for running the samples. The basic components required for gel preparation are given in the table 4. The standard procedure (Laemmli, 1970) was adopted for the preparation of the gels and to visualize the protein samples. The samples were run under both reduced and non-reduced conditions to analyze the presence of inter-chain disulfide linkages.

Sr. No.

Stock Solutions

Composition

1

30% Bisacrylamide and acrylamide solution.

30.0 g of Acrylamide + 0.8 g of Methylenebisacrylamide +

Bring the final volume to 100 ml with DW.

2

10% SDS

10.0 g of SDS + 100 ml DW.

3

4x Resolving Gel Buffer (1.5M Tris-HCl, pH 8.8,with 0.4% SDS)

To 110 ml of distilled water add 36.4 g of tris base + Add 8 ml of 10 % SDS solution + Adjust pH to 8.8 with 1 N HCl + Bring the final volume to 200 ml with DW.

4

4x Stacking Buffer (0.5 M Tris-HCl, pH 6.8, with 0.4% SDS)

To 110 ml of distilled water add 12.12 g of tris base + Add 8 ml of 10% SDS solution +

Adjust pH to 6.8 with 1 N HCl +

Bring to a final volume of 200 ml with DW.

5

4x Tris-Glycine Running Buffer without SDS:

36.0 g of tris base + 172.8 g of glycine +

Bring volume to 3 L with DW.

6

1x Tris-Glycine Running Buffer with SDS:

750 ml of 4x Tris-Glycine running buffer without SDS + 30 ml of 10 % SDS solution +

Bring volume to 3.0 L with DW.

7

10 % Ammonium Persulfate (APS) Solution

0.1 g of Ammonium Persulfate +

Bring volume to 1 ml with DW.

8

N,N,N,N-Tetramethylethylenediamine (TEMED)

Catalog Number T9281: Ready to use as neat liquid.

9

4x Sample Buffer:

4 ml of glycerol + 2ml of 2-mercaptoethanol +

1.2 g of SDS + 5 ml of 4x Stacking Buffer + 0.03 g of bromophenol blue +Aliquot into 1.5 ml microcentrifuge tubes. Store at –20 °C.

10

CBB R- 250 Stainer

0.25% of CBB is added in methanol : acetic acid : water solution (50 : 10 : 40) to prepare the CBB stain. It is after that filtered through the Whatman No. 1 filter.

11

Destainer

Add methanol : acetic acid : water solution (50 ml: 10 ml : 40 ml) to prepare destainer.

Table 4: Basic Components of the SDS-PAGE

Electrophoresis:

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein.

Figure 4: Electrophoresis Module (Aurogene Company)

Assembling of the glass plates:

The glass plates were assembled vertically.

Plates were embedded into the casting frame and clamped properly.

The bottom ends of the glass plates were properly aligned.

Then they were place on the casting stand.

Resolving Gels Solution:

A point was marked on the glass plates, 1 cm below the comb into the glass plates at the comb teeth end, before actually being inserted into plates. Addition of APS and TEMED was carried out just before pouring and they were mixed well by swirling gently. The resolving gel was poured till the mark. 15 µl TEMED was added to the resolving gel solution and mixed up well while avoiding the production of air bubbles. Filing was performed rapidly earlier than the TEMED causing the solution to turn into sticky. Resolving gel was left to polymerize. It took 15-20 minutes but this differed with the freshness of the reagents used. More ammonium persulfate solution (APS) and TEMED was added, as polymerization took long time than this. Sometimes new reagents were prepared again.

Stock Solutions

12 % Gel

30 % Acrylamide Gel Solution

6.0 ml

10 % Ammonium Persµlfate Solution

150 µl

4x Resolving Gel Buffer

3.75 ml

Distilled Water

5.25 ml

Table 5: Resolving Gel

Stacking Gel Solution:

Stacking gel solution was prepared using the above recipe. Air bubbles were avoided. TEMED (67µl) was added to the stacking gel solution and mixed up well by means of a Pasteur pipette to fill up the glass plates up to the apex with stacking gel solution. Extra solution was removed on the top of the resolving gel with distilled water. A comb was placed in the stacking gel sandwich (making sure no air bubbles get trapped below the ends of the comb teeth, as these slow up sample progression) and allowed it to polymerize for 30 minutes.

Ingredients

Quantity

Distilled Water

4.2 ml

10 % Ammonium persµlfate solution

67 µl

30 % Acrylamide gel solution

0.65 ml

4x Stacking Buffer

1.6 ml

Table 6: Stacking gel solution

Gel Pouring:

The gels pouring was done carefully before the polymerization of the resolving gel upto a specific mark. The space above the resolving gel was left for stacking gel. The stacking gel ususally takes more time as compared to the resolving gel.

Samples Preparation:

To prepare reduced sample, mercaptoethanol (1 drop in 1.5 ml) was added into the samples and heated up in a water bath or heating block for 2 minutes at 95 °C to denature the samples. The samples were centrifuged in a microcentrifuge for 20 seconds at 12,000 rpm. The non-reduced samples were prepared without heating and addition of mercaptoethanol. The protein samples were ready to load. Protein markers are loaded in one of the ending lanes mostly.

Tris-glycine, Gel Running Buffer:

To run the SDS-polyacrylamide gels, tris-glycine buffer was selected. It is useful for separating proteins of a wide range (10-200 kDa). Minimum volumes cooling potential is at a minimum, which may affect resolution. Outer tank was filled to just flood the bottom of the glass plates to 500 ml. Inner tank was filled to above the wells upto 250 ml.

Loading the Samples:

The electrophoresis apparatus, containing cast gels was transferred into the main tank in the correct direction as indicated: positive electrode (+ve, red) on the module associated with positive electrode (+ve, red) on the tank; negative electrode (-ve, black) on the module align with negative electrode (-ve, black) on the tank. The external tank is filled up with 1x running buffer. It was easy to remove the comb at that time, since it was lubricated but carefully without breaking the well. Now the gel was ready to load the samples. The samples were loaded into the wells by using a pipette tip, taking care not to smash up the wells or bring in any air bubble. The unused wells were loded with 1x sample buffer prepared by 4-fold strength of 4x sample buffer with water. The loading tip was washed a few times with distilled water making sure that all the water was poured out before loading the samples. The loading tip was inserted into the well a few mm above from bottom and delivered the samples into the well. The syringe was rinsed with distilled water after loading for a few times.

Running the Gels:

The gel, gel apparatus and samples were got ready. The power supply was attached by putting the lid. The voltage was set up to 90-225V or 40-90A for 1 mm or 12 % thick gels and run for 1 hour. The dye front was not allowed to go out of the gel. The bands were suppressed downward in the form of single line dye front by putting on the power supply. The power supply was turned off when the loading dye reached the underneath of the gel. The electrophoresis module was taken away and buffer was emptied into the major tank. Buffer can be re-used but this may influence run value if sustained. The glass plates were taken off and spaced out the glass plates. The gel was frequently attached to one of the plates and was detached by first soaking in buffer and then softly exciting with a spatula. The gel was ready for staining process.

Staining the gel:

After running, the power supply was switched off and the gel plates were taken out to remove the gel. The gel was placed in the staining solution; Coomassie Brilliant Blue R-250 for 2 hours to overnight. The acetic acid was used to fix the CBB stain to the proteins in the gel. This avoids the proteins since being washed away though staining process.

De-staining:

The gel was destained by the same acetic acid-methanol solution without the dye until the bands were properly seen. It was done by calm shake up for about 30-40 minutes of de-staining as the stainer was freshly prepared. The de-staining solution was re-used freshly during de-staining process to make protein bands visible clearly. De-staining solution can also be re-used. In order to make bands on the gel more clearly, the de-staining solution was mixed with small amount of the powder of activated charcoal. The activated charcoal was allowable to settle down. The obvious de-staining solution filtered through the Whatmann No.1 filter and could be additionally used.

Visualizing the gel:

The approximate molecular weight of the visualised protein bands was determined by comparing them with the reference bands on molecular weight ladders (marker).

Figure 5: Protein Ladder (Cat. No. 10747-012)

Ammonium Sulfate Precipitation:

This table below indicates the amount of ammonium sulfate crystals, precipitation at 25 °C added to 1 liter of solution to produce a desired change in the presence of ammonium sulfate in the given seed sample. First choose the % age i.e., 0.0, 20.0, 40.0, 60.0 and 80.0 % and add the corresponding concentration of ammonium sulfate and centrifuge after each addition of salt, if the ppts formed, separate the ppts of Freesia and follow up the next concentration. In this way, samples of ammonium sulfate ppts of Freesia were prepared at different % age of ammonium sulfate. The ppts were found at 40.0, 60.0 and 80.0 % ammonium sulfate precipitation. Load the corms samples into the wells and look up for the protein to be purified.

%

10 

15 

20

25

30

33

35

40

45

50

55

60

65

70

75

80

85

90

95

100

0

56

84

114

144

176

196

209

243

277

313

351

390

430

472

516

561

610

662

713

767

10

 

28

57

86

118

137

190

183

216

251

288

326

365

406

449

494

540

592

640

694

15

 

28

57

88

107

120

153

185

220

256

294

333

373

415

459

506

556

605

657

20

 

29

59

78

91

123

155

189

225

262

300

340

382

424

471

520

569

619

25

 

30

49

61

93

125

158

193

230

267

307

348

390

436

485

533

583

30

 

19

30

62

94

127

162

198

235

273

314

356

401

449

496

546

33

 

12

43

74

107

142

177

214

252

292

333

378

426

472

522

35

 

31

63

94

129

164

200

238

278

319

364

411

457

506

40

 

31

63

97

132

168

205

245

285

328

375

420

469

45

 

32

65

99

134

171

210

250

293

339

383

431

50

 

33

66

101

137

176

214

256

302

345

392

55

 

33

67

103

141

179

220

264

307

353

60

 

34

69

105

143

183

227

269

314

65

 

34

70

107

147

190

232

275

70

 

35

72

110

153

194

237

75

 

36

74

115

155

198

80

 

38

77

117

157

85

 

39

77

118

90

 

38

77

95

 

39

Table 7: Ammonium Sulfate Saturation constant table

Ammonium Sulfate Precipitation of Freesia:

Ammonium sulfate concentrations are calculated from the table given above for 10 ml sample of Freesia for 20.0, 40.0, 60.0, 80.0 and 100.0 %.

Sr. No.

Percentage

Ammonium Sulfate (g)

Freesia Sample used (ml)

1

25.0 %

1.44

10.0

2

40.0 %

1.23

10.0

3

60.0 %

1.32

10.0

4

80.0 %

1.43

10.0

5

100.0 %

1.57

10.0

Table 7: Ammonium Sulfate percentage concentrations

For 20.0 % Ammonium sulfate ppt. 1.14 g of ammonium sulfate was added in 10.0 ml of Freesia sample in 20.0 ml falcon tube. Centrifugation of sample for one minute at 3000 rpm was carried out. It revealed that there was no ppt. formation. For 40.0 % ammonium sulfate ppt. Addition of 1.23 g of ammonium sulfate in same 10 ml Freesia sample was made, extracted above the ppts of 20.0 % ammonium sulfate ppt. Examined the falcon tube carefully without shaking. This time we got the ppts at the bottom, pour above sample into another falcon tube separate ppt. in 1.5 ml eppendorf tubes. In this case of 25.0 % ammonium sulfate ppt. of Freesia no ppt. was formed. Samples of ammonium sulfate ppt. at 40.0 % sample, washed with the corresponding buffer solution (3.0pH) to remove the material other than proteins such as carbohydrates, lipids and sugars etc. Then add 1.32 g ammonium sulfate in the same 10.0 ml Freesia sample solution. At this (60.0 %) concentration of ammonium sulfate, ppts were found, these ppts. were separated carefully and aliquot into 1.5 ml eppendorfs. Then 1.43 g of ammonium sulfate was added in the same 10.0 ml Freesia sample which now not exactly 10.0 ml because of ppt. formation. At 80.0 % ammonium sulfate precipitation, ppts did not form, Similar procedure was opted for 100.0 %. Loading samples were prepared by adding 7µl of sample buffer into 21µl of 60.0 % ammonium sulfate ppt. of Freesia making total of 28 µl of loading sample of ammonium sulfate precipitation. Same procedure for 80.0 % Ammonium sulfate ppt. of Freesia is opted; load the samples into the gel.