Indian Patents. 278934:"A PROCESS FOR EXTRACTION OF BIOACTIVE PSORALEN COMPOUND"

Title of Invention	"A PROCESS FOR EXTRACTION OF BIOACTIVE PSORALEN COMPOUND"
Abstract	The present invention relates to a process for the extraction of Psoralen from in vitro grown tissues of Psoralea. The Psoralen obtained by the process of the present invention is in the range of 1500-2600 µg/g fresh wt. in seeds; in the range of 200-500 µg/g fresh wt. in leaves, nodes and roots; and in the range of 150-160 µg/g fresh wt. in entire plant.

Full Text	A Process for the Extraction of Bioactive Psoralen Compound FIELD OF INVENTION The present invention relates to a process for the extraction of high quantities Psoralen compound from in vitro grown tissues of Psoralea. The present invention also relates to the extraction of Psoralen from the seeds, in vivo and in vitro explant parts of Psoralea corylifolia which is bioactive as a biopesticide, biofungicide and in the treatment of vitiligo. BACKGROUND OF INVENTION Nomenclature and taxonomic position: Psoralea corylifolia Linn, commonly known as Babchi is an endangered and medicinally important plant belonging to the family Fabaceae. Distribution: The genue Psoralea L. comprises 120 species distributed in the five continents. These plants are mainly found in South Africa [45 species], North America [30 species] and Australia [15 species]. However, Psoralea plants are also present in Asia, South America, North Africa, and the Mediterranean region. P. corylifolia is indigenous to India. Economic importance: This plant is well recognized in Chinese and Indian folkloric medicine. The seeds are used in indigenous medicine as laxative, aphrodisiac, anthelmintic, diuretic and diaphoretic in febrile conditions. They have been especially recommended in the treatment of leucoderma, leprosy, psoriasis, and inflammatory diseases of the skin and are prescribed both for oral administration and for local external applications in the form of a paste or ointment. Naturally occurring Psoralens, such as 8-Methoxypsoralen [8-MOP], 5-Methoxypsoralen [5-MOP], and substituted Psoralens have been identified as phytoalexins. They are part of complex defensive response of plant against fungal and insect challenges. Furthermore Psoralens are powerful phototoxic agents in animals and humans. Psoralen is increasingly used in photochemotherapy for management of such disorders as vitiligo, psoriasis and mycosis fungicides. Psoralens comprise the most important class of photochemical reagents for the investigation of nucleic acid structure and function [Figure. 1]. They have been use for determining the structure of both cDNA and RNA in viral, bacterial and mammalian systems and also for studying functional questions, such as the role of small nuclear RNAs in processing heteronuclear RNA. Both Psoralens and Isopsoralens intercalate into double-stranded nucleic acid and undergo covalent photo-cycloaddition at either the furan or ' coumarin ends. Psoralen also reacts with single stranded nucleic acid, but to a much smaller extent than with double stranded structure. Psoralens have some reactivity with proteins and lipid membranes. (Figure Removed) Figure: 1: Chemical structure of Psoralen Many workers have tried to extract Psoralen from different plant parts of Psoralea. Only a few of them have been successful in extracting Psoralen and that too only from seeds and dried roots of Psoralea in very small quantities. Thus Psoralen still remains a very expensive compound. Table 1 shows the quantities of Psoralen obtained by earlier workers. None of these workers have reported extraction of Psoralen using in vitro methods. Table 1: Phytochemical analysis for Psoralen from Psoralea spp. done by earlier workers. (Table Removed) The present invention discloses a process for the extraction of high quantities of Psoralen from different plant parts (in vitro and in vivo explants). The Psoralen obtained by the extraction process of the present invention is economical and ecofriendly compared to the Psoralen obtained by conventional extraction process adopted in prior arts. OBJECTS OF THE INVENTION Primary object of the present invention is to provide a novel process for the extraction of Psoralen compound from seeds and in vitro grown tissues of Psoralea. which is economical and safe. Another object of the present invention is to disclose a process for the extraction of Psoralen from seeds and in vivo and in vitro explant parts of P. Corylifolia. Yet another object of the present invention is to extract Psoralen in high quantities. Still another object of the present invention is to extract bioactive Psoralen. SUMMARY OF THE INVENTION The present invention discloses a process for the extraction of Psoralen from in vitro grown tissues of Psoralea comprising the steps of (a) soaking the in vitro grown tissues in alcohol; (b) homogenizing the soaked tissue to obtain a crude extract; (c) mixing the crude extract with alcohol; (d) centrifuging the mixture obtained from step (c) to obtain a pellet and supernatant; and (e) filtering the supernatant to obtain Psoralen. In another embodiment of the present invention the Psoralea is Psoralea corylifolia. In yet another embodiment of the present invention the in vitro grown tissues are seeds, leaves, nodes, roots, callus, embryonal axis derived plant or entire plant. In still another embodiment of the present invention the alcohol is selected from a group comprising methanol, ethanol, propanol or combinations thereof. In another embodiment of the present invention the soaking is for 24 h in dark and the homogenizing is performed using pestle and mortar. In yet another embodiment of the present invention the centrifuging is performed for 10-30 min at 12000 rpm at room temperature and the filtering is performed using a milipore filter. In still another embodiment of the present invention the Psoralen is in the range of 1500-2600 µg/g fresh wt. in seeds; in the range of 200-500 µg/g fresh wt. in leaves, nodes and roots; and in the range of 150-160 µg/g fresh wt. in entire plant. In another embodiment of the present invention the Psoralen is bioactive as a biopesticide, biofungicide and in the treatment of vitiligo. DESCRIPTION OF THE INVENTION The present invention relates to an economical process for the extraction of bioactive Psoralen compound in high quantities from the seeds, in vivo and in vitro explant parts of Psoralea corylifolia. Source ofexplants: The plant materials of Psoralea corylifolia L. are collected from the Homeopathic Pharmacopoeia Laboratory [HPL], Ghaziabad. The twigs [20 cm long] are cut and immediately stored in an antioxidant solution [5% citric acid or abscorbic acid or poly vinyl pyrollidone] kept in a polyethane bag and brought to the laboratory in University of Delhi. Twigs are cut into 1-2 cm long nodal segments, which are used, as a source of mature nodal explants. Juvenile explants are excised from in vitro grown shoots, which are initially established from mature nodal segments. Source of seeds: One year old seeds are procured from the seeds division National Bureau of Plant Genetic Resources (NBPGR) Pusa, New Delhi and fresh mature as well as green seeds are procured from HPL, Ghaziabad. Sterilization of twigs: Twigs are cut [1-2 cm long], excised from mature plants and washed thoroughly under running tap water for 20-30 min to remove the debris from the surface and leachates. After washing the twigs are defoliated and cut into single nodes. These nodal segments are immersed in 1% [w/v] solution of bavistin, a fungicide [BASF Pharma, Mumbai] and kept on a rotary shaker for 15 min at 150 rpm in order to facilitate better surface contact of the fungicide with the explants. Subsequently, they are thoroughly washed under running tap water 6 or 7 times to remove the traces of bavistin. These are surface sterilized with 0.5% [w/v] freshly prepared mercuric chloride for 10 min on rotary shaker at 200 rpm. Finally, they are washed 4 or 5 times with sterilized distilled water under laminar airflow cabinet and stored in the same for inoculation. Sterilization of seeds: Fresh green seeds excised from field grown plants are thoroughly washed under running tap water for 30 min and surface sterilized with freshly prepared 0.5% [w/v] mercuric chloride [HgCL2]. The seeds are washed 4 or 5 times with sterilized distilled water under laminar airflow cabinet. The seed coat and fruit wall are removed with the help of forceps and scalpel. Green cotyledons are inoculated in B5 medium supplemented with 3% sucrose and auxins. General culture media, glassware's and surgical instruments are sterilized by autoclaving at 121°C for 15 min at 105 Kg/cm2 [=15 lb/psi] pressure. Culture media: B5 medium is employed for raising cultures of explants. For preparation of medium analytical grade [AR] salts [Qualigens or Glaxo fine Chemicals, Mumbai] are used o prepare the stock solutions. Macro and Micronutrient solutions are prepared 20 times concentrated while those of organic and iron salts are 100 times concentrated. They are stored in refrigerator. Growth regulators and heavy metals used during this investigation are procured from Sigma-AIdrich Chemical Co., USA and Qualigens, respectively. For preparing one-litre medium, 100 ml each of Macro and Micronutrient stock solutions and 10 ml each of organic and iron stock solution are used. The basal media are used either as such or supplemented with growth regulators such as N6- benzyladenine [BA], 2,4-Dichorophenoxy acetic acid [2,4-D]. As a source of carbon, 3% [w/v] sucrose [Daurala, DCM, meerut] is used in all experiment unless mentioned specially. The media are gelled with 0.8% agar [Qualigens, Bombay]. The pH of media is adjusted to 5.8 using IN NaOH or IN HCI before autoclaving. Approximately 20 ml media is dispensed in each 2.15 x 15 cm test tube [Borosil] plugged with non-absorbent cotton wrapped in muslin cloth. The test tubes containing media are autoclaved at 1.06 kg cm"2 at 121 °C for 15 min. Raising and incubation of cultures: Cultures are incubated in continuous light of 400-500 mW/cm2 by cool day light fluorescent incandescent tubes [40 W, Philips, Calcutta]. The cultures are maintained in a culture room at the temperature of 25±2°C and 5± 10% relative humidity. The explants are sub-cultured after every 30 to 32 d interval on the same but fresh medium. Psoralen Extraction Samples preparation: The tissues [seeds, leaf, node, root, entire plant and callus] are soaked in ethanol for 24 h, under dark and then homogenized using pestle and mortar. They are allowed as such in the pestle till the time ethanol gets evaporated. After evaporation of ethanol, the semisolid form of extract is mixed in methanol [HPLC grade]. This mixture is transferred to centrifuge tube and centrifuged for 10-30 min to 12000 rpm at room temperature. The pellet is discarded and the supernatant is filtered using 0.22 µM milipore filter and this is used as sample solution of which 20 µl is injected in to column [C18 Zorbex ODS] for HPLC analysis. Psoralen Standard: 1 mg of pure Psoralen powder [Sigma Aldrich Chemical Company, USA], is dissolved in few drops of ethanol and the final volume is made to 1 ml with methanol. This is used as standard solution during HPLC analysis. Isolation through HPLC: The HPLC unit of SHIMADZU-4A type, equipped with ultraviolet [UV] detector and printer plotter is operated under the following parameters: Column : C18 Column packing : Zorbex ODS [Octadicyl si lane] Solvent Methanol [HPLC grade] Injection volume : 20 µl Flow rate 0.5 ml/min Detection at : UV 244 nm Quantification: Estimation of Psoralen quantity is done manually. Different dilutions of Psoralen standard solution are analyzed using HPLC. The quantity of unkown sample is compared with known concentration of Psoralen. The present invention is illustrated and supported by the following experiments and examples. These are merely representative examples and optimization details and are not intended to restrict the scope of the present invention in any way. Example-1 Psoralen Content in Seeds and their parts: Field ripe seeds [old], fresh mature seeds, immature green seeds, green seed coat [without cotyledons] and green cotyledons [without seed coat] of P. corylifolia are analysed for their Psoralen content. A comparative study of Psoralen estimation of different parts mentioned above shows that green cotyledons [without seed coat] contain a maximum of 2594 ug/g of fresh wt. followed by fresh mature black seeds, fresh green seeds, seed coat [without cotyledons], and one year old seed contain 2512, 2135, 2080 and 1920 ug/g fresh wt. respectively [Table 2]. Table 2: HPLC analysis of Psoralen content in field ripe seeds, immature seeds, and different parts of seed of P. corylifolia. (Table Removed) Example- 2 Psoralen Content in in vivo plant parts: Quantitative analysis of Psoralen (HPLC analysis) of different plant parts like leaf, node, root and entire plant reveals that maximum quantity of Psoralen is present in roots [241.82 ng/g fresh wt.] followed by entire plant, leaf and nodal segment containing 165.0, 99.38 and 85.56 µg/g fresh wt. respectively [Table 3]. Table 3: HPLC analysis of Psoralen from different in vivo and in vitro plant parts of P. corylifolia. (Table Removed) Example- 3 Psoralen Content in in vitro plant parts: Similar to in vivo plant parts, maximum quantity of Psoralen is detected in roots [463.0 µg/g fresh wt.]. Thus other in vitro plant parts viz. node, leaf, entire plant and embryonal axis derived plant contain 315, 227.84, 157.22 and 4.94 µg/g fresh wt. Psoralen, respectively [Table 3]. Interestingly the quantity of Psoralen is significantly higher in all the in vitro plant parts. Except entire plant, the quantity of Psoralen enhanced (in in vitro) to almost double in roots and 3 to 4 times in leaf or node, respectively (Table 3) when compared to Psoralen content in in vivo plant parts. Example- 4 Psoralen Content in callus derived from different explants: HPLC analysis of the callus of different parts cultured on B5 medium containing 10µM BA shows a maximum of Psoralen in callus derived from cotyledons. The quantity of Psoralen is maximum in entire cotyledons which is markedly reduced in callus derived from it (Table 2 and Table 4). However, colyledonary callus if reared on l0µM BA and 250 mg/1 proline shows marked elevation in Psoralen quantity over control. At 5% sucrose 750 ng/g fresh we. Of Psoralen is achieved in cotyledonary callus which is three times more over control (Table 4). Table 4: HPLC analysis of Psoralen content in callus from different explants of P. corylifolia. (Table Removed) Example- 5 Bioactive Seed extract of P. corylifolia: 10 g seeds of P. corylifolia are soaked in 50 ml ethanol for 24 h in dark and homogenized using pestle and mortar. The crude extract is again dissolved in 10 ml ethanol for dilution. This mixture is filtered through a common filter paper. The filtrate is used as a biopesticide, biofungicides and in the treatment of vitiligo [200 mg/ seed/ml/ of ethanol]. Example- 6 Nodal explants cultured under metal stresses: In vivo nodal explants are cultured on B5 medium containing 5µM BA and heavy metals [ZnSO4, Pb(NO3)2, CuSO4 and AgNO3]. Maximum quantity of Psoralen is found with 100 mg/1 ZnSO4. However, in case of Pb(NO3)2 lower concentration enhances the quantity of Psoralen and higher quantities decreases the quantity of Psoralen. Addition of CuSO4 to the regenerative medium increases the quantity of Psoralen. Except for AgNO3 the quantity of Psoralen increases in the nodal explants reared on metal containing media (Table-5). Table 5: Estimation of Psoralen in nodal segments of P. corylifolia cultured at different concentrations of heavy metals in B5 + 5µM BA medium. (Table Removed) Thus, the present invention lays down a process, for the extraction of bioactive, high quantities of Psoralen using biotechnological approach. The main advantages of the present invention are: 1. The present process extracts Psoralen from all plant parts of P. corylifolia in high quantities. 2. The process of the present invention is environmentally compatible and safe. 3. The Psoralen obtained by the extraction process of the present invention is effective as a biopesticide, biofungicide and in the treatment of vitiligo. 4. The process of the present invention extracts Psoralen in high quantities from nodal explants cultured under metal stress. We Claim: 1. A process for the extraction of Psoralen from in vitro grown tissues of Psoralea comprising: a) soaking said in vitro grown tissues in alcohol; b) homogenizing the soaked tissue to obtain a crude extract; c) mixing said crude extract with alcohol; d) centrifuging the mixture obtained from step (c) to obtain a pellet and supernatant; and e) filtering said supernatant to obtain Psoralen. 2. The process as claimed in claim 1 wherein said Psoralea is Psoralea corylifolia. 3. The process as claimed in claim 1 wherein said in vitro grown tissues are cotyledons, leaves, nodes, roots, callus, embryonal axis derived plant or entire plant. 4. The process as claimed in claim 1 wherein said alcohol is selected from a group comprising methanol, ethanol, propanol or combinations thereof. 5. The process as claimed in claim 1 wherein said soaking is for 24 h in dark and said homogenizing is performed using pestle and mortar. 6. The process as claimed in claim 1 wherein said centrifuging is performed for 10-30 min at 12000 rpm at room temperature and said filtering is performed using a milipore filter. 7. The Psoralen obtained by the process as claimed in any preceding claim wherein said Psoralen is in the range of 1500-2600 µg/g fresh wt. in seeds; in the range of 200-500 µg/g fresh wt. in leaves, nodes and roots; in the range of 150-160 µg/g fresh wt. in entire plant and upto 1200 µg/g fresh wt. in callus. 8. The Psoralen obtained by the process as claimed in any preceding claim wherein said Psoralen is bioactive as a biopesticide, biofungicide and in the treatment of vitiligo. 9. A process for extraction of Psoralen from in vitro grown tissues of Psoralea substantially as hereinbefore described and with reference to the foregoing examples. 10. Psoralen obtained from in vitro grown tissues of Psoralea substantially as hereinbefore described and with reference to the foregoing examples.

Full Text

A Process for the Extraction of Bioactive Psoralen Compound
FIELD OF INVENTION
The present invention relates to a process for the extraction of high quantities Psoralen compound from in vitro grown tissues of Psoralea. The present invention also relates to the extraction of Psoralen from the seeds, in vivo and in vitro explant parts of Psoralea corylifolia which is bioactive as a biopesticide, biofungicide and in the treatment of vitiligo. BACKGROUND OF INVENTION
Nomenclature and taxonomic position: Psoralea corylifolia Linn, commonly known as Babchi is an endangered and medicinally important plant belonging to the family Fabaceae.
Distribution: The genue Psoralea L. comprises 120 species distributed in the five continents. These plants are mainly found in South Africa [45 species], North America [30 species] and Australia [15 species]. However, Psoralea plants are also present in Asia, South America, North Africa, and the Mediterranean region. P. corylifolia is indigenous to India.
Economic importance: This plant is well recognized in Chinese and Indian folkloric medicine. The seeds are used in indigenous medicine as laxative, aphrodisiac, anthelmintic, diuretic and diaphoretic in febrile conditions. They have been especially recommended in the treatment of leucoderma, leprosy, psoriasis, and inflammatory diseases of the skin and are prescribed both for oral administration and for local external applications in the form of a paste or ointment.
Naturally occurring Psoralens, such as 8-Methoxypsoralen [8-MOP], 5-Methoxypsoralen [5-MOP], and substituted Psoralens have been identified as phytoalexins. They are part of complex defensive response of plant against fungal and insect challenges. Furthermore Psoralens are powerful phototoxic agents in animals and humans. Psoralen is increasingly used in photochemotherapy for management of such disorders as vitiligo, psoriasis and mycosis fungicides.
Psoralens comprise the most important class of photochemical reagents for the investigation of nucleic acid structure and function [Figure. 1]. They have been use for determining the structure of both cDNA and RNA in viral, bacterial and mammalian systems and also for studying functional questions, such as the role of small nuclear RNAs in processing heteronuclear RNA. Both Psoralens and Isopsoralens intercalate into double-stranded nucleic acid and undergo covalent photo-cycloaddition at either the furan or
' coumarin ends. Psoralen also reacts with single stranded nucleic acid, but to a much smaller extent than with double stranded structure. Psoralens have some reactivity with proteins and lipid membranes.
(Figure Removed)
Figure: 1: Chemical structure of Psoralen
Many workers have tried to extract Psoralen from different plant parts of Psoralea. Only a few of them have been successful in extracting Psoralen and that too only from seeds and dried roots of Psoralea in very small quantities. Thus Psoralen still remains a very expensive compound. Table 1 shows the quantities of Psoralen obtained by earlier workers. None of these workers have reported extraction of Psoralen using in vitro methods.
Table 1: Phytochemical analysis for Psoralen from Psoralea spp. done by earlier workers.
(Table Removed)
The present invention discloses a process for the extraction of high quantities of Psoralen from different plant parts (in vitro and in vivo explants). The Psoralen obtained by the extraction process of the present invention is economical and ecofriendly compared to the Psoralen obtained by conventional extraction process adopted in prior arts. OBJECTS OF THE INVENTION
Primary object of the present invention is to provide a novel process for the extraction of Psoralen compound from seeds and in vitro grown tissues of Psoralea. which is economical and safe.
Another object of the present invention is to disclose a process for the extraction of Psoralen from seeds and in vivo and in vitro explant parts of P. Corylifolia.
Yet another object of the present invention is to extract Psoralen in high quantities. Still another object of the present invention is to extract bioactive Psoralen. SUMMARY OF THE INVENTION
The present invention discloses a process for the extraction of Psoralen from in vitro grown tissues of Psoralea comprising the steps of (a) soaking the in vitro grown tissues in alcohol; (b) homogenizing the soaked tissue to obtain a crude extract; (c) mixing the crude extract with alcohol; (d) centrifuging the mixture obtained from step (c) to obtain a pellet and supernatant; and (e) filtering the supernatant to obtain Psoralen.
In another embodiment of the present invention the Psoralea is Psoralea corylifolia. In yet another embodiment of the present invention the in vitro grown tissues are seeds, leaves, nodes, roots, callus, embryonal axis derived plant or entire plant.
In still another embodiment of the present invention the alcohol is selected from a group comprising methanol, ethanol, propanol or combinations thereof.
In another embodiment of the present invention the soaking is for 24 h in dark and the homogenizing is performed using pestle and mortar.
In yet another embodiment of the present invention the centrifuging is performed for 10-30 min at 12000 rpm at room temperature and the filtering is performed using a milipore filter.
In still another embodiment of the present invention the Psoralen is in the range of 1500-2600 µg/g fresh wt. in seeds; in the range of 200-500 µg/g fresh wt. in leaves, nodes and roots; and in the range of 150-160 µg/g fresh wt. in entire plant. In another embodiment of the present invention the Psoralen is bioactive as a biopesticide, biofungicide and in the treatment of vitiligo.
DESCRIPTION OF THE INVENTION
The present invention relates to an economical process for the extraction of bioactive Psoralen compound in high quantities from the seeds, in vivo and in vitro explant parts of Psoralea corylifolia.
Source ofexplants: The plant materials of Psoralea corylifolia L. are collected from the Homeopathic Pharmacopoeia Laboratory [HPL], Ghaziabad. The twigs [20 cm long] are cut and immediately stored in an antioxidant solution [5% citric acid or abscorbic acid or poly vinyl pyrollidone] kept in a polyethane bag and brought to the laboratory in University of Delhi. Twigs are cut into 1-2 cm long nodal segments, which are used, as a source of mature nodal explants.
Juvenile explants are excised from in vitro grown shoots, which are initially established from mature nodal segments.
Source of seeds: One year old seeds are procured from the seeds division National Bureau of Plant Genetic Resources (NBPGR) Pusa, New Delhi and fresh mature as well as green seeds are procured from HPL, Ghaziabad.
Sterilization of twigs: Twigs are cut [1-2 cm long], excised from mature plants and washed thoroughly under running tap water for 20-30 min to remove the debris from the surface and leachates. After washing the twigs are defoliated and cut into single nodes. These nodal segments are immersed in 1% [w/v] solution of bavistin, a fungicide [BASF Pharma, Mumbai] and kept on a rotary shaker for 15 min at 150 rpm in order to facilitate better surface contact of the fungicide with the explants. Subsequently, they are thoroughly washed under running tap water 6 or 7 times to remove the traces of bavistin. These are surface sterilized with 0.5% [w/v] freshly prepared mercuric chloride for 10 min on rotary shaker at 200 rpm. Finally, they are washed 4 or 5 times with sterilized distilled water under laminar airflow cabinet and stored in the same for inoculation.
Sterilization of seeds: Fresh green seeds excised from field grown plants are thoroughly washed under running tap water for 30 min and surface sterilized with freshly prepared 0.5% [w/v] mercuric chloride [HgCL2]. The seeds are washed 4 or 5 times with sterilized distilled water under laminar airflow cabinet. The seed coat and fruit wall are removed with the help of forceps and scalpel. Green cotyledons are inoculated in B5 medium supplemented with 3% sucrose and auxins. General culture media, glassware's and surgical instruments are sterilized by autoclaving at 121°C for 15 min at 105 Kg/cm2 [=15 lb/psi] pressure.
Culture media: B5 medium is employed for raising cultures of explants. For preparation of medium analytical grade [AR] salts [Qualigens or Glaxo fine Chemicals, Mumbai] are used o prepare the stock solutions. Macro and Micronutrient solutions are prepared 20 times concentrated while those of organic and iron salts are 100 times concentrated. They are stored in refrigerator. Growth regulators and heavy metals used during this investigation are procured from Sigma-AIdrich Chemical Co., USA and Qualigens, respectively.
For preparing one-litre medium, 100 ml each of Macro and Micronutrient stock solutions and 10 ml each of organic and iron stock solution are used. The basal media are used either as such or supplemented with growth regulators such as N6- benzyladenine [BA], 2,4-Dichorophenoxy acetic acid [2,4-D].
As a source of carbon, 3% [w/v] sucrose [Daurala, DCM, meerut] is used in all experiment unless mentioned specially. The media are gelled with 0.8% agar [Qualigens, Bombay]. The pH of media is adjusted to 5.8 using IN NaOH or IN HCI before autoclaving. Approximately 20 ml media is dispensed in each 2.15 x 15 cm test tube [Borosil] plugged with non-absorbent cotton wrapped in muslin cloth. The test tubes containing media are autoclaved at 1.06 kg cm"2 at 121 °C for 15 min.
Raising and incubation of cultures: Cultures are incubated in continuous light of 400-500 mW/cm2 by cool day light fluorescent incandescent tubes [40 W, Philips, Calcutta]. The cultures are maintained in a culture room at the temperature of 25±2°C and 5± 10% relative humidity. The explants are sub-cultured after every 30 to 32 d interval on the same but fresh medium.
Psoralen Extraction
Samples preparation: The tissues [seeds, leaf, node, root, entire plant and callus] are soaked in ethanol for 24 h, under dark and then homogenized using pestle and mortar. They are allowed as such in the pestle till the time ethanol gets evaporated. After evaporation of ethanol, the semisolid form of extract is mixed in methanol [HPLC grade]. This mixture is transferred to centrifuge tube and centrifuged for 10-30 min to 12000 rpm at room temperature. The pellet is discarded and the supernatant is filtered using 0.22 µM milipore filter and this is used as sample solution of which 20 µl is injected in to column [C18 Zorbex ODS] for HPLC analysis.
Psoralen Standard: 1 mg of pure Psoralen powder [Sigma Aldrich Chemical Company, USA], is dissolved in few drops of ethanol and the final volume is made to 1 ml with methanol. This is used as standard solution during HPLC analysis.
Isolation through HPLC: The HPLC unit of SHIMADZU-4A type, equipped with
ultraviolet [UV] detector and printer plotter is operated under the following parameters:
Column : C18
Column packing : Zorbex ODS [Octadicyl si lane]
Solvent Methanol [HPLC grade]
Injection volume : 20 µl
Flow rate 0.5 ml/min
Detection at : UV 244 nm
Quantification: Estimation of Psoralen quantity is done manually. Different dilutions of Psoralen standard solution are analyzed using HPLC. The quantity of unkown sample is compared with known concentration of Psoralen.
The present invention is illustrated and supported by the following experiments and examples. These are merely representative examples and optimization details and are not intended to restrict the scope of the present invention in any way.
Example-1
Psoralen Content in Seeds and their parts:
Field ripe seeds [old], fresh mature seeds, immature green seeds, green seed coat [without cotyledons] and green cotyledons [without seed coat] of P. corylifolia are analysed for their Psoralen content. A comparative study of Psoralen estimation of different parts mentioned above shows that green cotyledons [without seed coat] contain a maximum of 2594 ug/g of fresh wt. followed by fresh mature black seeds, fresh green seeds, seed coat [without cotyledons], and one year old seed contain 2512, 2135, 2080 and 1920 ug/g fresh wt. respectively [Table 2].
Table 2: HPLC analysis of Psoralen content in field ripe seeds, immature seeds, and different parts of seed of P. corylifolia.
(Table Removed)
Example- 2
Psoralen Content in in vivo plant parts:
Quantitative analysis of Psoralen (HPLC analysis) of different plant parts like leaf, node, root and entire plant reveals that maximum quantity of Psoralen is present in roots [241.82 ng/g fresh wt.] followed by entire plant, leaf and nodal segment containing 165.0, 99.38 and 85.56 µg/g fresh wt. respectively [Table 3].
Table 3: HPLC analysis of Psoralen from different in vivo and in vitro plant parts of P. corylifolia.
(Table Removed)

Example- 3
Psoralen Content in in vitro plant parts:
Similar to in vivo plant parts, maximum quantity of Psoralen is detected in roots [463.0 µg/g fresh wt.]. Thus other in vitro plant parts viz. node, leaf, entire plant and embryonal axis derived plant contain 315, 227.84, 157.22 and 4.94 µg/g fresh wt. Psoralen, respectively [Table 3]. Interestingly the quantity of Psoralen is significantly higher in all the in vitro plant parts. Except entire plant, the quantity of Psoralen enhanced (in in vitro) to almost double in roots and 3 to 4 times in leaf or node, respectively (Table 3) when compared to Psoralen content in in vivo plant parts.
Example- 4
Psoralen Content in callus derived from different explants:
HPLC analysis of the callus of different parts cultured on B5 medium containing 10µM BA shows a maximum of Psoralen in callus derived from cotyledons. The quantity of Psoralen is maximum in entire cotyledons which is markedly reduced in callus derived from it (Table 2 and Table 4). However, colyledonary callus if reared on l0µM BA and 250 mg/1 proline
shows marked elevation in Psoralen quantity over control. At 5% sucrose 750 ng/g fresh we. Of Psoralen is achieved in cotyledonary callus which is three times more over control (Table 4).
Table 4: HPLC analysis of Psoralen content in callus from different explants of P. corylifolia.
(Table Removed)
Example- 5
Bioactive Seed extract of P. corylifolia:
10 g seeds of P. corylifolia are soaked in 50 ml ethanol for 24 h in dark and homogenized using pestle and mortar. The crude extract is again dissolved in 10 ml ethanol for dilution. This mixture is filtered through a common filter paper. The filtrate is used as a biopesticide, biofungicides and in the treatment of vitiligo [200 mg/ seed/ml/ of ethanol].
Example- 6
Nodal explants cultured under metal stresses:
In vivo nodal explants are cultured on B5 medium containing 5µM BA and heavy metals
[ZnSO4, Pb(NO3)2, CuSO4 and AgNO3]. Maximum quantity of Psoralen is found with 100
mg/1 ZnSO4. However, in case of Pb(NO3)2 lower concentration enhances the quantity of
Psoralen and higher quantities decreases the quantity of Psoralen. Addition of CuSO4 to the
regenerative medium increases the quantity of Psoralen. Except for AgNO3 the quantity of
Psoralen increases in the nodal explants reared on metal containing media (Table-5).
Table 5: Estimation of Psoralen in nodal segments of P. corylifolia cultured at different concentrations of heavy metals in B5 + 5µM BA medium.
(Table Removed)
Thus, the present invention lays down a process, for the extraction of bioactive, high quantities of Psoralen using biotechnological approach.
The main advantages of the present invention are:
1. The present process extracts Psoralen from all plant parts of P. corylifolia in high quantities.
2. The process of the present invention is environmentally compatible and safe.
3. The Psoralen obtained by the extraction process of the present invention is effective as a biopesticide, biofungicide and in the treatment of vitiligo.
4. The process of the present invention extracts Psoralen in high quantities from nodal explants cultured under metal stress.

We Claim:
1. A process for the extraction of Psoralen from in vitro grown tissues of Psoralea
comprising:
a) soaking said in vitro grown tissues in alcohol;
b) homogenizing the soaked tissue to obtain a crude extract;
c) mixing said crude extract with alcohol;
d) centrifuging the mixture obtained from step (c) to obtain a pellet and
supernatant; and
e) filtering said supernatant to obtain Psoralen.
2. The process as claimed in claim 1 wherein said Psoralea is Psoralea corylifolia.
3. The process as claimed in claim 1 wherein said in vitro grown tissues are cotyledons,
leaves, nodes, roots, callus, embryonal axis derived plant or entire plant.
4. The process as claimed in claim 1 wherein said alcohol is selected from a group
comprising methanol, ethanol, propanol or combinations thereof.
5. The process as claimed in claim 1 wherein said soaking is for 24 h in dark and said
homogenizing is performed using pestle and mortar.
6. The process as claimed in claim 1 wherein said centrifuging is performed for 10-30
min at 12000 rpm at room temperature and said filtering is performed using a milipore
filter.
7. The Psoralen obtained by the process as claimed in any preceding claim wherein said Psoralen is in the range of 1500-2600 µg/g fresh wt. in seeds; in the range of 200-500 µg/g fresh wt. in leaves, nodes and roots; in the range of 150-160 µg/g fresh wt. in entire plant and upto 1200 µg/g fresh wt. in callus.
8. The Psoralen obtained by the process as claimed in any preceding claim wherein said Psoralen is bioactive as a biopesticide, biofungicide and in the treatment of vitiligo.
9. A process for extraction of Psoralen from in vitro grown tissues of Psoralea
substantially as hereinbefore described and with reference to the foregoing examples.
10. Psoralen obtained from in vitro grown tissues of Psoralea substantially as
hereinbefore described and with reference to the foregoing examples.

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Patent Number

278934

Indian Patent Application Number

998/DEL/2006

PG Journal Number

01/2017

Publication Date

06-Jan-2017

Grant Date

04-Jan-2017

Date of Filing

17-Apr-2006

Name of Patentee

UNIVERSITY OF DELHI

Applicant Address

DELHI-110007,INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	VEENA AGRAWAL	DEPARTMENT OF BOTANY, UNIVERSITY OF DELHI, DELHI-110007,INDIA

PCT International Classification Number

A01N

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA