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Title:
PROCESS OF ENHANCEMENT OF STIGMASTEROL AND HECOGENIN CONTENT IN IN VITRO ROOT CULTURES OF CHLOROPHYTUM BORIVILIANUM THROUGH POLYPLOIDY
Document Type and Number:
WIPO Patent Application WO/2015/102018
Kind Code:
A1
Abstract:
This invention relates to Chlorophytum borivilianum, popularly known as Safed Musli. Currently, the active nutrition enhancing ingredients such as Stigmasterol and Hecogenin are extracted from the tuber of the product which is available only during three months in a year. Furthermore, the quantum content of the active ingredients in the tuber does not facilitate stand alone extraction. Therefore, this invention provides for employing polyploidy in respect of the above plants whereby the active ingredient content in the plant material is substantially increased. Furthermore, this invention provides for a yearlong industrial process to extract the active ingredients in vitro root culture instead of the well developed tuber of the natural plants.

Inventors:
GAYATHRI BATHOJU (H.no. 3-4-114/A, Sai Chitra NagarRamanthapur, Hyderabad, Andhra Pradesh, IN)
Application Number:
IN2014/000755
Publication Date:
July 09, 2015
Filing Date:
December 08, 2014
Export Citation:
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Assignee:
GAYATHRI BATHOJU (H.no. 3-4-114/A, Sai Chitra NagarRamanthapur, Hyderabad, Andhra Pradesh, IN)
International Classes:
C07H1/08; A01H1/08; C07H15/256; C07J11/00
Domestic Patent References:
WO2007113646A22007-10-11
Foreign References:
US3510400A1970-05-05
US2774714A1956-12-18
Other References:
K AGEETHA ET AL: "New basic chromosome number in Chlorophytum borivilianum Santapau and Fernandes", JOURNAL OF SPICES AND AROMATIC CROPS, 1 January 2004 (2004-01-01), pages 121 - 123, XP055178953, Retrieved from the Internet [retrieved on 20150324]
KAUSHIK NUTAN: "Saponins of Chlorophytum species", PHYTOCHEMISTRY REVIEWS, KLUWER, NL, vol. 4, no. 2-3, 1 January 2005 (2005-01-01), pages 191 - 196, XP002473190, ISSN: 1568-7767, DOI: 10.1007/S11101-005-2607-5
NEELU JOSHI ET AL: "Cytological Characterization of a Rare Medicinal Herb 'Safed Musli' (Chlorophytum borivilianum Sant. et Fernad.)", CYTOLOGIA, vol. 71, no. 2, 1 March 2006 (2006-03-01), pages 153 - 159, XP055178957
None
Attorney, Agent or Firm:
MANOJ KUMAR D (KRISHNA AND SAURASTRI ASSOCIATES, No. 17 Sheshadri Road, Gandhi Nagar,,Bangalore 9, Karnataka, 56000, IN)
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Claims:
I Claim:

1. A process for extraction of steroidal saponins from saponin bearing plants of family Liliaceae, Amerillidacea and Dioscoreaceae, wherein the raw material for the process is obtained after: in vitro induction of polyploidy in shoot cultures;

allowing the regeneration of the shoot cultures;

iii. selecting and promoting the growth of the polyploid plants; and iv. Rooting of polyploid plants and growth of root cultures

wherein the steroidal saponins are extracted from in vitro root cultures.

2. The process according to claim 1 , wherein the saponin bearing plants of family Liliaceae, Amerillidacea and Dioscoreaceae among which Chlorophytum borivilianum is selected.

3. The process according to claim 1 , wherein the steroidal saponins selected is hecogenin and Stigmasterol.

4. The process according to claim 1 , wherein the process comprises: i. Treatment of in vitro grown shoot cultures to colchicine, wherein the concentration of colchicine ranges from 0.001 to 0.2%;

ii. Incubating the treated shoot cultures in micro propagation media (2 mg/l BAP) for varied time period ranging from 12 hrs - 3 days, in the absence of light;

iii. Transfer of the shoot cultures to multiple shoot formation media (2.5 mg/l BAP+ 0.5 mg/l NAA) and allowing for plant regeneration for three successive generations;

iv. Transfer of the shoot cultures onto rooting media (3 mg/l IBA) for development of roots;

v. Selection of genetically stable polyploid plants;

vi. Establishment of root cultures; and vii. Extraction and quantification of saponins from root cultures wherein the polyploid plant has enhanced properties evincing increased root biomass as well as increased saponin content.

5. The process according to claim 4, wherein the enhanced properties comprise increased concentration of steroidal saponins as well as increase in the root biomass.

6. The process according to claim 5, wherein the steroidal saponins Hecogenin and Stigmasterol are extracted from the in vitro root cultures.

7. The process according to claim 6, wherein the hecogenin content at least 2.2 fold increases and the stigmasterol content is at least 14.5 fold increases in polyploid roots than the diploid roots of Chlorophytum bonvilianum root cultures.

Description:
Field of the invention

PROCESS OF ENHANCEMENT OF STIGMASTEROL AND HECOGENIN CONTENT IN IN VITRO ROOT CULTURES OF CHLOROPHYTUM BORIVILIANUM THROUGH POLYPLOIDY

The present invention mainly relates to Chlorophytum borivilianum and more particularly to the enhancement of Stigmasterol and Hecogenin content in vitro root cultures of Chlorophytum borivilianum through polyploidy.

5 Background of the invention

Plants are an excellent source for discovery of novel products with pharmaceutical importance in drug development. Plants form an integral part of human civilization, as most of them are used in the traditional system of medicine. India has a rich biodiversity with various agro climatic conditions.0 Every year India exports 32,600 tons of medicinal plants. Indian agricultural practices must evolve to meet the increasing demand and satisfy the growing and diversified needs of different stakeholders starting from production to the consumption chain.

Due to wide range of use, certain plant species are getting extinct, which5 in turn causes loss of genetic diversity and habitat destruction. The usage of plant based medicines is limited due to two major reasons, one of which is the lack of reproducibility as 40% of plant extract activity is not authenticated, when they are re-extracted and re-sampled.

Other reason includes, the secondary metabolites that are medicinally0 important are produced by the plants, which are otherwise difficult to synthesize via organic synthesis. Modern Pharmacopoeia constitutes between 25-50% of plant-derived medicines and has become a main base for the production of many alternative and modern medicines. The demand for medicinal plants is increasing in developing and developed countries as an assortment of organic5 compounds are produced by the plants useful in the production of pharmaceuticals.

Polyploidy is well known in the art which is a technique of enhancing plant production potential. Genomic manipulation increases the secondary metabolic production rate as well as improves the quality of the biochemical profile.0 Artificial polyploidy is used in a variety of plant species as an alternative technique to increase secondary metabolite production. Because of this increase in the complement of chromosomes, polyploidy plants are often bigger.

The vigor of the determinate plant parts are often enhanced by polyploidy, wherein the biomass or secondary metabolite of these plants constitutes the economic value. Polyploids also provide a wider germplasm base for breeding studies. Chromosome duplication using colchicine has long been used in plant breeding programs. In most plants, artificial polyploidy is often accompanied by increased cell size, leading to larger reproductive and vegetative organs.

Chlorophytum borivilianum is an important medicinal plant. Stigmasterol and Hecogenin are the two main constituents of this plant where in they are mostly used as an aphrodisiac, and also have the property of preventing different types of cancers.

Further the active ingredients of Chlorophytum borivilianum are also has aphrodisiac activity, erectile properties, immunomodulatory activity, antioxidant activity, anthelmintic activity, antiulcer activity, Anti-mutagenic anti-tumour and chemomodulatory activity, anti stress activity, anti diabetic activity, antimicrobial activity, larvicidal activity, anti viral activity, Anti-obesity, health-promotional benefits and health-restorative benefits and etc. So, the requirement of Chlorophytum borivilianum active ingredients found to be high due to its high pharmaceutical values. Furthermore, the roots are widely used as a natural "sex tonic" and are an integral part of more than 100 herbal drug formulations. Although Indian forests may rich in Chlorophytum borivilianum the demand is increasing rapidly in Indian and international drug markets. Moreover the field grown plants are failed to supply the required amount of stigmasterol and Hecogenin. Currently, the active nutrition enhancing ingredients are extracted from the tuber of the product which is available only during three months in a year. Furthermore, the quantum content of the active ingredients in the tuber does not facilitate stand alone extraction.

Therefore there is a need in the art with the method/process of enhancing the stigmasterol and Hecogenin content in vitro root cultures of Chlorophytum borivilianum to solve the above mentioned limitations. Objective of the invention

This invention relates to Chlorophytum borivilianum, popularly known as Safed Musli. Currently, the active nutrition enhancing ingredients such as Stigmasterol and Hecogenin are extracted from the tuber of the product which is available only during three months in a year. Furthermore, the quantum content of the active ingredients in the tuber does not facilitate stand alone extraction. Therefore, this invention provides for employing polyploidy in respect of the above plants whereby the active ingredient content in the plant material is substantially increased. Furthermore, this invention provides for a yearlong industrial process to extract the active ingredients in vitro root culture instead of the well developed tuber of the natural plants.

The main objective of the present invention is to develop a new, unique, colchiploid Chlorophytum borivilianum (GN-1) with increased amount of Stigmasterol and Hecogenin by optimization of conditions for the colchicine treatment and continuous production of the secondary metabolites throughout the year. The present invention uses the polyploidy technique which has been proved to increase the secondary metabolites in Chlorophytum borivilianum.

Nature of the invention

This invention teaches a yearlong industrial process for extraction of active ingredients of Stigmasterol and Hecogenin from the in vitro root culture of the plant Chlorophytum borivilianum after inducing polyploidy, and the root culture of the polyploidy induced plant (Tetraploid) has higher Stigmasterol and Hecogenin content in comparison with the normal (Diploid) plant.

Summary of the invention An aspect of the present invention is to address at least the above- mentioned problems and/or disadvantages and to provide at least the advantages described below.

Accordingly, in one aspect of the present invention relates to a process for extraction of steroidal saponins from saponin bearing plants of family Liliaceae, Amerillidacea and Dioscoreaceae, where the raw material for the process is obtained after: in vitro induction of polyploidy in shoot cultures, allowing the regeneration of the shoot cultures, selecting and promoting the growth of the polyploid plants, and Rooting of polyploid plants and growth of root cultures, where the steroidal saponins are extracted from in vitro root cultures.

Other aspects, advantages, and salient features of the invention will become apparent to those skilled in the art from the following detailed description, which, taken in conjunction with the annexed drawings, discloses exemplary embodiments of the invention. Brief description of the drawings

The above and other aspects, features, and advantages of certain exemplary embodiments of the present invention will be more apparent from the following description taken in conjunction with the accompanying drawings in which: Figure 1 shows the survival percentage of plants on different concentration of colchicine for different time duration according to one embodiment of the present invention.

Figure 2 shows the polyploid plant with 0.1 % colchicine for 72 hours treatment and (rooting) according to one embodiment of the present invention. Figure 3 shows the normal in vitro leaf according to one embodiment of the present invention.

Figure 4 shows the polyploidy leaf (short and increased width) according to one embodiment of the present invention.

Figure 5 shows the average height and width of control and polyploidy plant according to one embodiment of the present invention.

Figure 6 shows the diploid number of in vitro control plant using flow cytometry according to one embodiment of the present invention. Figure 7 shows the confirmation of doubling of chromosomal number in 0.1% colchicine treated plants by flow cytometry according to one embodiment of the present invention.

Figure 8 shows the study of stomata number and division's in vitro diploid plant and polyploid plants according to one embodiment of the present invention.

Figure 9 shows the measurement of number of stomata per box in vitro control (diploid) plant according to one embodiment of the present invention.

Figure 10 shows the number of stomata per box in polyploidy according to one embodiment of the present invention. Figure 11 shows the measurement of number of stomata per box in polyploidy (different and clear view) according to one embodiment of the present invention.

Figure 12 shows the measurement of stomatal size (45 divisions) of a diploid control leaf and (97 divisions) of a polyploid leaf according to one embodiment of the present invention.

Figure 13 shows the root cultures of polyploidy plant culture initiation and culture after 25 days according to one embodiment of the present invention.

Figure 14 shows the estimation of stigmasterol and Hecogenin in three successive generations in diploid and polyploidy according to one embodiment of the present invention.

Figure 15 shows the average amount of stigmasterol and Hecogenin present in polyploid plant in comparison to in vivo and in vitro plants.

Figure 16 shows the HPLC analysis of the control (diploid) roots according to one embodiment of the present invention. Figure 17 shows the HPLC analysis of authentic stigmasterol and

Hecogenin according to one embodiment of the present invention. Figure 18 shows the HPLC analysis of polyploid plant for stigmasterol and Hecogenin according to one embodiment of the present invention.

Persons skilled in the art will appreciate that elements in the figures are illustrated for simplicity and clarity and may have not been drawn to scale. For example, the dimensions of some of the elements in the figure may be exaggerated relative to other elements to help to improve understanding of various exemplary embodiments of the present disclosure.

Throughout the drawings, it should be noted that like reference numbers are used to depict the same or similar elements, features, and structures. Detail description of the invention

The following description with reference to the accompanying drawings is provided to assist in a comprehensive understanding of exemplary embodiments of the invention as defined by the claims and their equivalents. It includes various specific details to assist in that understanding but these are to be regarded as merely exemplary. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the invention. In addition, descriptions of well-known functions and constructions are omitted for clarity and conciseness. The terms and words used in the following description and claims are not limited to the bibliographical meanings, but are merely used by the inventor to enable a clear and consistent understanding of the invention. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present invention are provided for illustration purpose only and not for the purpose of limiting the invention as defined by the appended claims and their equivalents.

It is to be understood that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a component surface" includes reference to one or more of such surfaces. By the term "substantially" it is meant that the recited characteristic, parameter, or value need not be achieved exactly, but that deviations or variations, including for example, tolerances, measurement error, measurement accuracy limitations and other factors known to those of skill in the art, may occur in amounts that do not preclude the effect the characteristic was intended to provide.

Discussed below, and the various embodiments used to describe the principles of the present disclosure in this patent document are by way of illustration only and should not be construed in any way that would limit the scope of the disclosure. Those skilled in the art will understand that the principles of the present disclosure may be implemented in any suitably arranged communications system. The terms used to describe various embodiments are exemplary. It should be understood that these are provided to merely aid the understanding of the description, and that their use and definitions in no way limit the scope of the invention. Terms first, second, and the like are used to differentiate between objects having the same terminology and are in no way intended to represent a chronological order, unless where explicitly stated otherwise.

Plant secondary metabolites are the molecules, which belong to a diverse group involved in plant's adaptation to the environment. These groups of compounds are termed in different ways as phytochemicals, plant secondary metabolites, plant xenobiotics etc. Plants are devoid of an immune system and motility, but have elaborate alternative defense strategies, which involve huge varieties of secondary metabolites as tools to overcome stress, constraints environmental changes and survival. Micro propagation or an efficient in vitro protocol is considered as a prerequisite for the production of colchiploids, transgenic plants or in vitro root culture initiation.

Plant extracts (the active ingredient) obtained from the plant substances is estimated to be present in approximately one quarter of all the prescribed drugs. These are necessary for manufacturing modern drugs e.g. β-ionone, solasodine, diosgenin etc. Although synthetic drugs have their own market, herbal drugs have an enormous demand, owing to the public awareness that the phytopharmaceuticals are considered safer in disease prevention and treatment

Secondary metabolites: Some substances referred to as secondary metabolites may not be involved directly in the plant growth but play a key role in adaptation of plant to their environment. Secondary metabolites display large diversity of chemical types and interactions that underlay the impressive multiplicity of protective functions ranging from toxicity and light/ UV shielding to signal transduction. These secondary metabolites present in the medicinal plants have biological activities, which act as a base in preparation of modern drugs. Plant secondary metabolites can be grouped into four major categories viz. alkaloids, terpenoids, flavonoids and phenolic compounds. These plant secondary metabolites are synthesized and sequestered in different parts of the plant like seeds, bark, leaves, flowers, fruits/roots, rhizomes.

Chlorophvtum borivilianum: Chlorophytum borivilianum (Santapau and Fernandes) belonging to the family Liliaceae is commonly called as safed musli. Chlorophytum borivilianum is an endogenous medicinal plant to India and is distributed in eastern part of India. Due to its aphrodisiac properties safed musli is considered as an important medicinal plant. Chlorophytum borivilianum is a widely grown and commercially exploited species. Farmers in India cultivate safed musli on a commercial scale as it has high economic value. The tubers are rich in carbohydrates, alkaloids, vitamins, proteins, polysaccharides, steroids and saponins. The rhizomes are used as a base in making herbal tonics in ayurvedic therapy.

Phvtochemistrv of Chlorophvtum borivilianum: Chlorophytum borivilianum is well known for its saponin content. These saponins are present in roots, rhizomes as well as in the leaves. Major biochemical constituents are carbohydrates 42%, protein 5-10%, fiber 3-4% and saponins 17-20%, with flavonols 0.07% and the total polyphenolic content 0.25%. Saponins are considered as the major bioactives of Chlorophytum borivilianum with over all high content of 17-20%. Different saponins like Chlorophytoside I and borivilianosides of the F, G and H series were identified. Neotigogenin, Neo Hecogenin and Tokorogenin and immunostimulatory polysaccharides and other saponins are also present. Stigmasterol and phytosterol constitute the total weight of 0.9%. Fatty acids (linoleic acid, 11 and 14-Eicosadienoic acid, hexadecane 7) of Chlorophytum borivilianum is a rich source of 25 alkaloids. Vitamin C is present at 0.67% of dry weight.

Mechanism of polyploidy (uses of polyploidy): Polyploids arise when an unusual meiotic or mitotic catastrophe like non-disjunction causes formation of gametes having duplicate chromosomes as a complete set resulting in the formation of diploid gametes. It is evident from the recent investigations based on several workers reports that the production of polyploids is one of the breeding methods and an approach to enhance production of secondary metabolites in medicinal plants. Actinidia chinensis commonly known as kiwifruit have fruits with commercial significance that are too small selected this plant for the induction of polyploidy. Polyploids displayed increased size and altered shape of the fruit, the fruit load was found to be much higher in case of type A plants compared to diploid fruits. It was also noted that the induced autotetraploids were 50-60% larger then diploid progenitors.

Olea europaea is used as a model species for the in situ stomatal counting. In polyploids the guard cells which is a common characteristic of all plants was given alkali-induced blue fluorescence treatment that is the quick and handy method for the measurement of in situ stomatal complex number, size and distribution. Stomatal cell length was studied in four closely related taxa of Phylum in genus Phleum (P. nodosum and P. pratense) and two cytotypes of P. commutatum. It was found that the stomatal cells of the polyploid taxa P. pratense (42) and P. commutatum (28) are longer than their close diploid P. nodosum (14) and P. commutatum (14). This study led to the development of rapid and useful technique in identification of the cytotypes not only in the live but also from the herbarium material. The reported chromosome number of diploid Chlorophytum borivilianum is 2n=16. Based on the status of the prior art the effect of polyploidy to enhance the secondary metabolite content the same was used to enhance the Stigmasterol and Hecogenin in Chlorophytum borivilianum.

5 The present invention is improved by applying the procedure of polyploidy to Chlorophytum borivilianum and conditions optimized as to get polyploids of Chlorophytum borivilianum with increased amount of stigmasterol and Hecogenin in vitro grown root cultures which gives an opportunity for the commercial production of these two pharmaceutically important compounds 0 throughout the year. The present invention describes Chlorophytum borivilianum colchiploid GN-1 as a polyploidy with high morphological characteristics.

Figure 1 shows the survival percentage of plants on different concentration of colchicine for different time duration according to one embodiment of the present invention. The plants were selected from different 5 parts of India among which high yielding accessions were selected. In vitro cultures were established, and these micro propagated plants were used for the treatment with different amounts of colchicine and at different time intervals. Table 1 shows the study of the survival percentage of explants towards different concentrations of colchicine incubated at different time periods. 0

2.5 0.5 0.1 48 48

PMC1 2.5 0.5 72 46

PMC2

PMC3 2.5 0.5 24 75

2.5 0.5 0.01 48 50

PMD1 2.5 0.5 72 45

PMD2

PMD3 2.5 0.5 24 95

2.5 0.5 0.001 48 85

2.5 0.5 72 85

The figure 2 shows the polyploid plant with 0.1 % colchicine for 72 hours treatment and (rooting) according to one embodiment of the present invention. The present invention uses the colchicine for the induction of polyploidy in the 5 present study. In vitro grown Chlorophytum borivilianum shoot cultures were excised and used for induction of polyploidy. Different concentrations of colchicine ranging from 0.001% - 0.2% were used for the development of colchiploids / Polyploids. Liquid media with colchicine was used for the experimentation. 0 Different time period (optimization): After inoculating the shoots with 0.001, 0.01 , 0.1 and 0.2 % of colchicine, the explants were incubated for different time intervals of 24 hours, 48 hours and 72 hours in the dark. These concentrations were dissolved in the media, which is normally used for the micro propagation, but media is devoid of agar. After the required time period these plants were5 later on transferred on to the normal MS media with 2.5 mg/l BA and 0.5 mg/l NAA which is the normal media for the in vitro plant regeneration of Chlorophytum borivilianum, these were allowed to grow on this media for few generations and are later on transferred onto the rooting media with IBA.

Study of growth pattern: After sub culturing on to the semi solid media the0 physical appearance (Morphological) of the plants was observed. The leaf diameter, height, width and color of the colchiploid were studied for the regenerants.

The figure 4 shows the polyploidy leaf (short and increased width) according to one embodiment of the present invention. The figure 5 shows the average height and width of control and polyploidy plant according to one embodiment of the present invention. The present invention shows the polyploid plant exhibited short stature in comparison to the control plants height of control plant is 21.53±1.08 cm, whereas polyploid plant displayed height 7.14±0.15 cm. The maximum width of the leaf was found to be (18.72±0.82 mm) in case of polyploid plant when compared to that of the control leaf 7.14±0.1 mm.

Table 2 shows the study of the average height an average width leaf in control as well as the colchiploid plant

The figure 6 shows the diploid number of in vitro control plant using flow cytometry according to one embodiment of the present invention.

Figure 7 shows the confirmation of doubling of chromosomal number in 0.1% colchicine treated plants by flow cytometry according to one embodiment of the present invention. These polyploids were studied for three successive generations to confirm their genetic stability, using flow cytometry and stomatal studies. The flow cytometry confirmed doubling in the number of the chromosome number when compared to that of the control plant (2n). This chromosomal doubling was stable through successive generations proving that the polyploid plants were genetically stable. Figure 8 shows the study of stomata number and division's in vitro diploid plant and polyploid plants according to one embodiment of the present invention.

Comparison of measured stomatal apertures and number of stomata of colchiploid and normal plant: Leaves of control and colchiploid plants were placed on the slide and fixed with the cover slip and analyzed for the stomatal cell studies. This is a convenient and rapid method for in situ measurements of the size, number and distribution of stomatal complexes. The number of stomata in the box was measured and the divisions on the stomata were counted which depicts the size of the stomata. The surface of the sample covered by each image was 0.308 mm "1 .

Comparison of measured stomatal apertures and number of stomata in polyploid and control plant: The stomata in the polyploid plant have shown doubling in their number compared to that of diploid control and the stomatal size was also seen to be double to that of the control plant. This was stable through further generations. Table 3 shows the study of stomatal number and divisions in vitro control plant.

Method of evaluation between different samples: Images from different leaf samples were processed to study the accuracy and reproducibility of the method. The number of stomata was expressed per surface unit (objects mm "2 ).

Stability of ploidy level: The stability of ploidy for three successive generations was studied. Ploidy levels were confirmed for regenerants and the amount of phytochemical (Hecogenin and stigmasterol) produced each time was estimated.

Confirmation of ploidy: Flow-cytometry: - The leaves of diploid plant and polyploid were taken and each sample was prepared for analysis using Partec Cystain UV precise P- 05-5002 kit. [Approximately 0.5 cm 2 of leaf tissue or plant material was placed in a disposable petri dish, 400 μΙ of extraction buffer was added to it. The material is chopped for 30-60 seconds with the help of sharp blade and incubated for 30 sec - 1 minute in the same extraction buffer. After incubation sample was filtered through 50 μιτι Cell tries disposable filter into the vial.1.6 ml of staining solution was added to the test tube and incubated for 30-60 seconds. The sample was analyzed by flow cytometer in blue fluorescence channel with Fluorescence excitation: UV (excitation below lamda=420nm) and Fluorescence emission: Blue for DAPI (wavelength between =435 and =500nm. (Partec CyStain UV precise P -05-5002)].

Stomatal study: An epidermal impression was made by coating the leaf surface with nail varnish. The dried layer of nail varnish was peeled off by using cello tape and stick onto a slide to observe under a microscope.

Microscopy, Measurement and Data analysis:

Figure 9 shows the measurement of number of stomata per box in vitro control (diploid) plant according to one embodiment of the present invention.

Figure 10 shows the number of stomata per box in polyploidy according to one embodiment of the present invention.

Figure 11 shows the measurement of number of stomata per box in polyploidy (different and clear view) according to one embodiment of the present invention. The number of stomatal cells observed in each box and number of divisions on the stomata were measured and noted under 40X and 100X. The differences in the stomatal cells of normal plants and colchiploid plants were studied.

Bright field microscope was used to examine the stomatal cells on the leaf peel. Shoot tips from in vitro raised plants were used as the explants to carry out the polyploidy experiments. As colchicine is known to induce polyploidy, it was used in the present experiment for induction of polyploids on the similar lines. Shoot base explants of Chlorophytum borivilianum were used for induction of polyploids of the different concentrations of colchicine tested 0.001% and 0.01% showed better survival rate in comparison to higher concentration, i.e. 0.1% and 0.2%. As exhibited in table 1 higher concentrations of colchicine 0.2% proved to be fatal. The plants (0.2%) resulted with such treatment could not sustain themselves after few days. Plants treated with 0.1%, 0.01% and 0.001% concentrations of colchicine could survive and produced rooting after one subculture generation.

Confirmation of ploidv: These polyploids were studied for three successive generations to confirm their genetic stability, using flow cytometry and stomatal studies. The flow cytometry confirmed doubling in the number of the chromosome number when compared to that of the control plant (2n). This chromosomal doubling was stable through successive generations proving that the polyploid plants were genetically stable. Figure 12 shows the measurement of stomatal size (45 divisions) of a diploid control leaf and (97 divisions) of a polyploid leaf according to one embodiment of the present invention. The stomatal length in polyploids (97 divisions) was found to increase up to 2 times when compared to that of diploids (45 divisions). The frequency of the stomatal cells per unit area of the leaf deceased with increase in the ploidy level. The confirmation of polyploidy was done using flow cytometry.

Study of stability of ploidv for three successive generations: Study of the ploidy levels of control and induced polyploids for three successive generations confirmed the tetraploid status of induced polyploids. Table 5 shows the ploidy confirmation for three successive generations was done by flowcytometry and stomatal cell studies

Figure 14 shows the estimation of stigmasterol and Hecogenin in three successive generations in diploid and polyploidy according to one embodiment of the present invention.

The figure 15 shows the average amount of stigmasterol and Hecogenin present in polyploid plant in comparison to in vivo and in vitro plants.

The figure 16 shows the HPLC analysis of the control (diploid) roots according to one embodiment of the present invention.

Figure 17 shows the HPLC analysis of authentic stigmasterol and Hecogenin according to one embodiment of the present invention. Figure 18 shows the HPLC analysis of polyploid plant for stigmasterol and

Hecogenin according to one embodiment of the present invention.

Table 6 shows the percentage amount of stigmasterol in control, in vitro and polyploidy plants in each generation.

Phvtochemicals analysis of the polyploid plant:

The figure shows the phyto chemical analysis of Polyploids exhibited 14.5 fold increase in stigmasterol content and 2.2 fold increase of Hecogenin in comparison to the diploid control (Table 6, 7, 8; Fig. 14-18). Table 7 shows the percentage amount of Hecogenin in vivo diploid, in vitro diploid and polyploid plants each year Sample Amount of Amount of Amount of Average Hecogenin in I st Hecogenin in II nd Hecogenin in Hecogenin generation generation III rd generation (mg/g) (mg/g) (mg/g) (mg/g)

Control (in 10.94±0.2 10.43±0.33 11.59±0.48 10.98±0.58 vivo)

Control (in 33.88±0.34 35.93±0.71 34.77±0.39 34.86±1.02 vitro)

Polyploid 75.74±0.39 76.99±0.37 77.48±0.34 76.73±0.89 plant

Table 8 shows the comparison of production of stigmasterol and Hecogenin in field grown control, in vitro control and polyploid plants

In the present study plants treated with 0.1% concentration of colchicine exhibited doubling of the chromosomal numbers without being fatal to the plants. The time period of 72 h proved to be optimal for induction of tetraploids. Increase in the secondary metabolite content, stigmasterol (57.0±0.05 mg/g) and Hecogenin (76.73±0.89 mg/g) have been observed when compared to the in vitro control which have stigmasterol (3.93±1.33 mg/g) and Hecogenin (34.86±1.02 mg/g). Whereas in the case of in vivo control the stigmasterol content was found to be 3.59±0.01 mg/g and Hecogenin at 10.98±0.58 mg/g. There was 14.5 folds increase in stigmasterol content of polyploid plant when compared to the in vitro control and 15.87 fold increases when compared to in vivo control. Similarly in the case of Hecogenin2.20 fold increase when compared to in vitro control and 6.9 fold increase when compared to the in vivo control.

The stomatal length in polyploids (97 divisions) was found to increase up to 2 times when compared to that of diploids (45 divisions). The frequency of the stomatal cells per unit area of the leaf deceased with increase in the ploidy level. The confirmation of polyploidy was done using flow cytometry. Polyploidy could be induced with 0.1 % colchicine. Colchiploids with 4n number showed 14.5 fold increase in stigmasterol and 2.2 fold increase in Hecogenin. The above study paves the path for the further industrial development of the valuable root cultures of Chlorophytum borivilianum and new antimicrobial products can be developed.

Process of Extraction of Saponins

Lyophilized C. borivilianum control and polyploidy roots were extracted in 1 ml HPLC grade methanol. The extract was filtered through 0.22 m syringe driven before subjecting to HPLC (Schimadzu-LC-10AT VP) equipped with Supelco column (250x4.6 mm, C18, ODC with particle size of 5 μητι). Injection volume of 20 I was injected.

Standard stigmasterol and hecogenin were prepared at a concentration of 1mg/ml in HPLC grade methanol.

Stigmasterol (Retension time 4.5 min) was detected at 254nm with mobile phase methanol: water: acetic acid (70:30:1) at flow rate 1 ml/minute.

Hecogenin (Retension time 4.4 min) was detected at 270 nm with mobile phase comprising of methanol: water (70:30) at a flow rate of 1 ml/minute.

Novel Features of the invention:

• Induction of polyploidy in Chlorophytum borivilianum using colchicine. · Optimization of conditions for growth of colchiploids.

• Increase in stigmasterol content of 14.5 fold increase compared to in vitro control plants and 15.87 fold increase compared to in vivo control.

• Increase in Hecogenin content of 2.20 fold increase compared to in vitro control and 6.9 fold compared to in vivo control.

In vitro extraction of saponins throughout the year by propagation through liquid cultures. • Doubling the number of chromosome and confirmation of stability for further generations to check the genetic viability.

• Study of the stomatal number per box in control as well as colchiploid.

• Study of the stomatal size of control as well as colchiploid.

• Polyploid roots of Chlorophytum borivilianum requires normal room temperature to grow compared to control which needs BOD.

• Roots grow faster and are thicker compared to control roots.

• Protocol for industrial development of the product can be achieved in liquid cultures and bioreactors.

Although the invention has been described in terms of preferred embodiment, it is not limited thereto. Those skilled in this technology can make various alterations and modifications without departing from the scope and spirit of the invention. Therefore, the scope of the invention shall be defined and protected by the following claims and their equivalents.

FIGS. 1-18 are merely representational and are not drawn to scale. Certain portions thereof may be exaggerated, while others may be minimized. FIGS. 1-18 illustrate various embodiments of the invention that can be understood and appropriately carried out by those of ordinary skill in the art.

In the foregoing detailed description of embodiments of the invention, various features are grouped together in a single embodiment for the purpose of streamlining the disclosure. This method of disclosure is not to be interpreted as reflecting an intention that the claimed embodiments of the invention require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed embodiment. Thus, the following claims are hereby incorporated into the detailed description of embodiments of the invention, with each claim standing on its own as a separate embodiment. It is understood that the above description is intended to be illustrative, and not restrictive. It is intended to cover all alternatives, modifications and equivalents as may be included within the spirit and scope of the invention as defined in the appended claims. Many other embodiments will be apparent to those of skill in the art upon reviewing the above description. The scope of the invention should, therefore, be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. In the appended claims, the terms "including" and "in which" are used as the plain-English equivalents of the respective terms "comprising" and "wherein," respectively.