NIGELLA

TECHNICAL FIELD: The present invention relates to production of cell, tissue and organ cultures of plants belonging to the genus Nigella (Ranunculaceae) with the objective of production of the biopolymer, melanin. More specifically the present invention relates to a method for mass production of natural melanin pigments by cell, tissue or organ cultures of plants belonging to the genus Nigella.

BACKGROUND OF THE INVENTION The biopolymer melanin

The biopolymer known as melanin is a natural ubiquitous pigment. It is present in most genera including mammals, invertebrates, insects, fungi, bacteria and plants [1, 2, 3]. It may also be chemically synthesized [4, 5, 6]. Melanins from animal and synthetic sources have been extensively studied for their prospective uses in cosmetics, plastics, pharmacological and biomedical applications [7, 8, 9]. Sepio-melanin from the marine animal Sepia offocinalis, the squid, is the most studied example of this pigment [10, 11, 12]. Melanin plant-pigments naturally present in various plants (mostly in the seed coats) have been successfully extracted via chemical extraction methods [13, 14, 15]. Use of melanin in various applications and methods of its production via various ways have been the subject of numerous articles and former patents. However, naturally occurring melanin sources usually yield meager quantities in comparison to the start up volume of the raw material. The extraction methods may involve hazardous chemicals. The melanin source is sometimes objectionable because of fear of contamination by disease-causing agents (e.g. melanin from bacterial or animal sources) or the process is slow and rather expensive with a low yield as it is in the case of production of synthetic melanin. In most of the synthetic melanin production methods and of extraction of the naturally occurring melanins the end product pigment is only alkali soluble (at pH > 9.5). This requires development of melanin solublization methods for most of the applications [3]. Presently, commercially available melanins sell at prohibitively high prices. The industrial, health and cosmetics scopes of application of melanin are quite large but have, up to now been hindered by these limitations of non-cost-effectiveness and lack of a copious safe resource.

The genus Nigella L.

Nigella is a genus of annual plants of about 14 species belonging to the family anunculaceae (the Buttercup family), which is widely distributed over southern Europe, the middle East, north and eastern Africa and southwest Asia [16]. The species grow up to 20-90 cm tall, with white, yellow, pink, pale blue or pale purple flowers with 5-10 petals. The fruits are capsules composed of follicles, each containing numerous seeds, usually black or brown in colour, due to the presence of melanin in the seed coat. Melanin at concentrations of up to 15% has been shown to exist in the seed coats of Nigella sativa L. [13, 14]. The seed of Nigella sativa L is a common, culinary, non-toxic edible oil seed that has been in use as a food ingredient for millennia. The well known, cultivated, members of the Nigella genus are Nigella sativa L. (whose seed is a spice known as "Black cumin"), Nigella damascena and Nigella hispanica (both are decorative flowering plants collectively known as "Love in a mist" in the US). Other less known members are: Nigella integrifolia, Nigella arvensis, Nigella ciliaris, Nigella negellastrum, and Nigella orientali. All members of the genus have been shown by Skvarla and Nowike (1979) [17] to have Nigella integrifolia as a monotypic representative of the genus. Earlier Reinders (1943) [18] studied the species crossing in the genus Nigella and pointed out the overall genetic characteristics of the group. Schmauder and Doebel (1991)

[19] reported the in vitro culture, regeneration and formation of secondary metabolites by the Nigella genus while Banerjee and Gupta (1976) [20] reported on the embryogeneses and differentiation in Nigella sativa leaf callus in vitro; all of these reports did not mention melanin as a constituent of these plants at any stage of the culture process or at any later stage of the growth of the plant towards its final stage of production of seeds.

SUMMARY OF THE INVENTION

Callus and suspension cultures of cells, tissues and organs of the genus Nigella (Ranunculaceae) producing melanin, as an induced component and a process for extraction of melanin therefrom are provided. The callus and suspension cultures producing melanin in a high content can be induced and proliferated by culturing cells, tissues or organs of a plant belonging to the genus Nigella (Ranunculaceae) using germinated axenic seedlings in particular. Explants from leaves, stems or roots may successfully be used for this purpose. The cultures are advantageously carried out in the presence of a particular combination of plant growth regulators in a growth medium. A callus or suspension culture containing at least melanin as an effective component can be stably provided in this way, making it possible to effectively produce melanin from the callus or the suspension.

In a first aspect, the invention relates to a process for producing melanin by culturing cells originating from cells, tissues and/or organs of plants belonging to the genus Nigella of the Ranunculaceae family, which process comprises the following steps:

- preparing cell, tissue and/or organs of genus Nigella L. to produce proliferating living cultures;

- cultivating said cultures in a nutrient medium for inducing proliferating living calli - optionally producing proliferating suspension cultures by culturing said calli in a nutrient medium;

- inducing melanin production in said calli or suspension cultures; and

- recovering melanin from the cultures.

Other aspects of the invention are a callus or suspension culture obtained in a process according to the first aspect which contains at least melanin that is readily water soluble and melanin obtainable by a method according to the first aspect.

Preferred embodiments are set out in the dependent claims.

According to the present invention cultures of cells, tissues and organs of Nigella plants, containing melanin pigment, can effectively be induced and proliferated using various growth media. Furthermore, melanin can be isolated from these cultures as a water soluble or alkali soluble powder. Accordingly, the present invention is available for use in the field of manufacturing cosmetics, plastics, pharmacological and general biomedical applications. The following is a non-exhaustive list of US patents concerned with possible applications of melanins: 5047447, 5057325, 5256403, 5286979, 5310539, 5384116, 5451254, 5538752, 5641508, 5643554, 5817631 , 5827330, 5906610, 6103777, 6242415, 6440691, 6525019, 6576268, 6761452, 7029758.

DETAILED DESCRIPTION OF THE INVENTION The present invention has been made keeping in mind the drawbacks occurring in the prior art, and an object of the present invention is to provide a method capable of mass-producing high quality melanin. The present inventors have made intensive research into callus initiation from cells, tissues and organs of plants belonging to the genus Nigella and into proliferation of the callus and suspension cultures. As a result it has been found that cultured media of various species of the genus, produce melanin content at given densities depending on the species. It was specifically found that Nigella sativa undergoes continuous and copious production of melanin on culture, as described in detail in the example below. It was thus determined that melanin can be efficiently obtained by culturing specific cells, tissues and organs originating from a specific species belonging to the genus Nigella, whereby the present invention was achieved. Another objective achieved by the present invention is that the melanin produced in the cultured media is water soluble. Depending on the method of extraction of melanin from the cultures either water soluble or alkali-soluble melanins may be obtained.

Namely, according to the present invention, a process is provided for producing melanin by culturing cells, organs and tissues originating from plants belonging to the genus Nigella, which comprises: a) A step of preparing a living cell, tissue and organ cultures; b) A step of culturing the cell, tissue and organ cultures obtained in step a) in a nutrient medium suitable for inducing a callus, to thereby induce a living callus; c) A step of culturing the callus cells obtained in step b) in a nutrient medium suitable for proliferating calli and suspension cultures and their somatic cells and roots; d) A step for induction of production of melanin by the callus and suspension cultures produced in step c), whereby the calli and suspension cultures produce a dark pigment (melanin) after undergoing repeated sequential (6-8) subcultures; e) A step for induction of production of melanin by the roots induced in callus subcultures obtained in step c), whereby roots growing in unaltered same media produce dark pigments (melanin) within 2-3 weeks. f) A step for induction of production of melanin by somatic embryos produced by the calli in step c), whereby the somatic embryogenesis cells produce dark pigments (melanin) after 6-8 weeks. g) A step of recovering the water soluble melanin produced from the cultured products obtained in step d), step e) and step f).

The main features of the new method are as follows:

1. Presents a method for production of melanin using cell, tissue, and organ cultures of plant species belonging to the genus Nigella for the first time.

2. The melanin produced in the culture media is soluble in water in contrast to synthesized or melanins extracted from various other sources 3. Using different methods of extraction either water soluble melanin or alkali soluble melanin may be obtained.

4. The method facilitates large scale commercial production of both water-soluble and alkali soluble melanin from a natural plant source.

The phrase "plants belonging to the genus Nigella", as used in the present invention, meaning plants belonging to the genus Nigella of the family Ranunculaceae (e. g. as defined by the US Dept. of Agriculture - Plant Classification Data Base [ 1 ] ) and suitable for the object of the present invention (i.e. production of melanin). Typical examples thereof include, but are not restricted to, Nigella Sativa, Nigella damascena and Nigella hispanica.

According to the present invention, living cells, tissues and organs are prepared from specific parts of these plants in a first step. Consequently, plant materials put into culture using explants of leaves, stems, roots, flowers, fruits or seeds can be used as is, or the plant materials may include those prepared from them in a form suitable for the culture; for example, in the case of seeds, clean germinating tissues isolated from surface sterilized seeds, which are the preferable mode for the present invention, may be used.

The above-described preparation is part of the concept of including treatments in which the plant materials for culture are isolated as they are and alive, and are surface sterilized. Typical examples of such preparations involve, but are not limited to, steps in which, first the materials collected from plants of the genus Nigella are sterilized, the raw materials are then aseptically divided into pieces, and thereafter, are transferred onto media solidified by agar or gellan gum suitable for inducing a callus as described herein below.

The living tissues obtained in the above described stage are then cultured on nutrient media suitable for callus induction, and the callus thus derived is transferred to a nutrient medium suitable for the proliferation of callus cells. The primary components to be used in the nutrient media include water; mineral nutrients, e.g. nitrogen (ammonium salts and nitrates), phosphorus, potassium, calcium, magnesium, sulfur, etc.; sugars, e.g., sucrose, glucose, fructose, maltose, etc.; organic substances such as vitamins and amino acids; naturally originating substances such as coconut milk; and optionally, gelling agents, e.g., agar, gellan gum, alginic acid, and agarose.

The basic formulations of mineral nutrients known as basic media include Murashige & Skoog medium [21] (hereinafter referred to as "MSO medium" or "MS medium"), Schenk and Hildebrandt medium [22], Gamborg's B5 medium[23], White's medium [24], Nagata & Takebe medium [25], Nitsch & Nitsch [26] medium and woody plant medium. To these basic media are further optionally added auxins such as 1 -naphthaleneacetic acid (hereinafter referred to as "NAA"), indole-3 -acetic acid, indole-3-butyric acid, and 2,4- dichlorophenoxyacetic acid (Hereinafter referred to as "2,4-D"); cytokinins such as benzylaminopurine, zeatin, and 6-furfurylaminopurine (kinetin); and gibberellins as plant growth regulators. As the nutrient media suitable for inducing the callus according to the present invention, media in which auxins, cytokinins and/or gibberellins are added to the above-described basic media are preferable.

As the nutrient media suitable for proliferating suspension culture cells, the above-described basic media with cytokinins and auxins added thereto are used, with the media using the MS medium as the basic medium and having kinetin as the cytokinin and NAA or 2,4-D as the auxin added thereto being particularly preferable. The pH levels of such media are adjusted to values from 5 to7, preferably 5.5 to 6.0, with an appropriate acid or alkali.

Each of the above-described cultures using these media can be carried out at a temperature in the range of 15-25°C with or without light-irradiation. The general method for induction of production of melanin from the genus Nigella calli, cell cultures, cell suspensions, tissues and organs are as detailed in the steps d-g in section 1.3. In particular, the cultures for the proliferation of the callus cells are preferably carried out in a liquid medium, in which the medium containers are placed on rotary shakers.

The isolation of melanin thus obtained from the cultured products can be carried out by following a number of different methods requiring knowledge of melanins solubility in the different aqueous pH phases or in different solvents. The term "cultured products" used herein is intended to include calli, cultured cells and clumps of cultured cells, cultured tissue, cultured organs and media used for the culture. Although not restricted thereto, the melanin can be isolated from such cultivated products by separating cali or cultured cells from the media, drying and pulverizing same, extracting melanin from the resulting powder by altering pH conditions and precipitation; extracting melanin from the resulting powder with an appropriate organic solvent, washing the organic phase with water, drying the organic solvent over anhydrous sodium sulfate, magnesium sulfate or calcium chloride, and then evaporating the solvent. Different mechanical separation methods may also be employed such as filtration, homogenizing, ultrasonication and centrifugation. If necessary, the melanin thus isolated can be purified by various chromatographic purifications or techniques akin to re-crystallization methods. Dry melanin powders can be obtained by various dehydration techniques including treatments at elevated temperatures (up to 70°C), freeze drying and spray drying.

EXAMPLES

The present invention will now be described in greater detail by description of a working example. The example shall not be construed as limiting the invention, which is that of the appended claims.

Materials and Methods:

Seeds of three cultivars (Ethiopian, Indian and Arabian) of Nigella Sativa L. were purchased from a local market, surface sterilized and germinated on Heller- support filter paper dipped in liquid medium. Leaf, stem and root explants were cut from the germinated axenic seedlings and cultured on agar solidified nutrient medium in petri dishes. The medium contained the inorganic salts of Murashige and Skoog and B5 vitamins as basal medium [21, 27], supplemented with sucrose (3%), Kinetin, and/or 2,4-D, NAA and 0.8% bactoagar. The cultures were incubated under controlled dark conditions.

Callus Induction:

Calli were induced from the three types of explants on 2,4-D or NAA containing media after 4 to 6 weeks regardless of the cultivar. The calli were friable, loose and yellow. They were further maintained by sub culturing onto a similar medium every 4 weeks. Suspension cultures were initiated by transferring 1.0-2.0 g of callus to liquid medium of similar composition (100 ml/500 ml flask) and shaking at 100 rpm on a rotary shaker.

Root Formation: Extensive root formation occurred after the second subculture in the callus that was initiated on NAA containing medium only, and irrespective of the explant type. However, this process ceased after the sixth subculture. When the formed roots were transferred to a liquid medium devoid of growth regulators, they multiplied and elongated. Regular transfers to new media maintained the growth of the roots. When the roots were not transferred to new medium, they released dark brownish-black pigments (melanin) into the medium within 2-3 weeks.

Induction of Somatic Embryogenesis:

Somatic embryos were induced upon sub culturing the callus that was initiated on 2,4-D containing medium onto medium containing NAA. The embryos were produced in clusters at high frequency. Embryo proliferation continued through the 20th subculture and was interrupted by asynchronous development into shoots. When the embryogenic callus was transferred to a new liquid medium it proliferated into new clusters of embryos, but ceased to develop beyond the globular and heart-shaped stages. The embryogenic cultures also produced dark pigments (melanin) after 8 weeks on the same medium.

Production of Dark Pigments:

The cultures of N. sativa produce dark pigments (melanin) after 6-8 weeks of culture under dark or light conditions. The production of so called pigments does not depend on the morphogenetic status of the culture i.e. in differentiated as well as undifferentiated cultures, nor on the physical state of the medium (solid vs. liquid), or the type of cultivar. In liquid medium the cultures release most of the pigments into the medium while retaining little in the tissues. In solid medium the cultures retain most of the pigments and release little into the agar medium. The color of the medium darkens with time and varies from brown to black. The pH of the medium becomes more alkaline towards the end of culture period (8 weeks) and may reach 9.5 in a highly dark medium. The release of pigments is probably a result of the chemical stress inflicted on the growing cultures by the depletion of a nutrient factor(s) from the medium, thus affecting nitrogen metabolism and pH.

Separation of Dark Pigments: a- From callus cultures grown on agar media:

The darkened callus tissues were collected after 8 weeks from the agar-solidified medium and the fresh weight determined. A solution of 0.5-1. ON NaOH was added just enough to cover the tissues and the mixture was left for 2 days at room temp. The dissolved pigments in the aqueous medium were separated from the cells by filtration. The pH was brought down to 7.0 by adding HCI acid. The solution was then divided into 100 ml batches, transferred to 250ml round bottom flasks and freeze-dried. Pigments diffused into the agar medium were treated similarly.

b- From suspension cultures:

Cell suspension cultures, embryogenic cultures and root cultures were harvested after 8 weeks when growth has completely ceased. The dissolved pigments were separated by vacuum filtering. The filtered tissues retained a dark black color while the liquid appeared brownish black. The density of the liquid was recorded and was close to 1 g/ml. The pH varied from 6.8 to 9.5 depending on cultivar. The separated dark brown solution was either directly freeze- dried or treated with cone. HCl to obtain a precipitate.

c- From dark pigments:

It is assumed that the dark pigments that formed in the callus tissues or released in the liquid medium are complex phenolic substances that constitute melanin [2] . Melanin, a complex phenolic molecule, does not dissolve in organic solvents. It does dissolve in alkaline pH and precipitates in acidic pH [1]. Therefore either of the following methods is used in this case: Extraction from tissues (callus, filtered cells, embryos, or roots) using NaOH or NH4OH, followed by acid precipitation (HCl), pH~2, or neutralizing the pH to 7.0 and washing with double distilled water (ddH20) before freeze-drying.

• Acid precipitation from liquid medium: 0.5-1.0 N HCL is added to an equal volume of liquid medium. When the pH reaches 3.5 to 3.0 the melanin precipitates, and leaving it overnight will ensure maximum precipitation. The precipitated melanin can be separated with the help of a separation funnel, by simply decanting off the light colored upper layer or centrifuging. Washing with ddH20 will help reduce acidity before freeze drying.

• Direct freeze drying of the medium separated from the suspension cultures. This will bypass the acid precipitation step but may require longer freezing time and may result in the formation of a mixture of culture medium substances together with melanin.

Tables 1-a) and Table 1 -b) provide descriptions of two production processes (Process 1 and Process 2 for calli and liquid suspension cultures, respectively) involved in the production and separation of melanin from the various cultures of Nigella saliva L. The yields of the calli are as shown in the tables. Table 2 shows the yields of melanin from liquid suspension cultures of various cultivars used in this example.

Characterization of the Melanin pigment:

The following set of tests, following known methods in the literature [1, 2, 3], confirmed the presence of melanin in the cultures. They have all successfully indicated the presence of melanin in samples obtained as described above.

1. Precipitation test: Melanin dissolves in alkaline pH (> 9.5) and precipitates in acidic pH (3.0 - 3.7).

2. Potassium permanganate (K ₂ Mn0 ₄ ) test: Melanin is an electron donor and would reduce many oxidizing agents. A test was developed for melanin pigments where addition of few drops of light violet colored solutions of K ₂ Mn0 ₄ to a light brown melanin solution at alkaline pH (~ 12.5) produces a dark green color; indicative of reduction of K ₂ Mn0 ₄ in basic media.

3. UV-Visible Light absorption: Melanin absorbs light continuously in an increasing manner in the range of visible to UV light producing a characteristic smooth spectral line reported by many authors [1, 2]. Melanin has a distinctive IR spectrum. A small percentage of melanin powder in KBr pellets produces the spectrum using an IR-FT spectrometer.

Electron Paramagnetic Resonance (EPR): Melanin has the unusual property of harboring persistent organic free radicals as part of its basic composition. These radicals easily lead to paramagnetic resonance absorption at room temperature. Their numbers in melanin increase upon UV irradiation or on thermal treatment. The EPR technique is a sensitive technique and is capable of detecting minute quantities of free radicals in a medium. An x-band EPR spectrometer indicated the presence of free radicals in all samples of the melanin extracts made from the above described cultures. A fairly strong single line signal of around 1018 spins/g at g-value ~ 2.00 and of width 4-7 G at room temperature was obtained. Under UV radiation of the samples in situ additional large number of free radicals were shown to be generated as shown by an increase in the intensity of the signals by about 25%.

6.

Table 1. Methods for production and separation of melanin from cultures Table 1-a) Process 1 : Pigment Separation from Suspension Cultures

Table 1-b) Process 2. Pigment Separation from callus and agar-solidified media

Table 2. Freeze-dry masses of Melanin obtained from Suspension Cultures of three

Cultivars oiNigella sativa L.

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1 17-130. Universita degli Studi di Parma, Parma, Italy (1992).

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