Journal of Plant Biology Research 2013, 2(1): 25-37
eISSN: 2233-0275 pISSN: 2233-1980 http://www.inast.org/jpbr.html
REGULAR ARTICLE
Extraction and Preparation of Psoralen from different Plant Part of Psoralea Corylifolia and Psoralen Increasing with some Elicitors *
Ebrahim Alinia Ahandani1 , Mohamed Ragab Abdel Gawwad2, Alimohammad Yavari3 1
2
Department of Plant Biology, Faculty of Science, Urmia University, Iran. Natural Sciences Dept., Faculty of Engineering and Natural sciences, International University of Sarajevo, Bosnia and Herzegovina. 3 Department of Horticulture,Faculty of Agriculture,Urmia University, Iran.
ABSTRACT
Psoralen as a medicinal material was assessed from different (in vivo and in vitro) plant parts of Psoralea corylifolia by means of HPLC set. In comparison to in vivo plant parts the quantity of psoralen in in vitro plant parts were less than in vivo plant parts. The highest amount of psoralen (3047µg/g fresh wt.) in in vivo condition was shown in brown seed. Quantitative analysis of psoralen was done from the callus derived from different plant parts showed that a maximum of 2501.7µg/g fresh weight of psoralen was recorded in callus derived from cotyledons. Estimation of psoralen was carried out from shoots derived from different callus which indicated the maximum amount of psoralen (562.298) was detected in plant drivated from node callus. This is the first report for identification of psoralen in the callus of Psoralea corylifolia. Tryings were done to enhance of psoralen by some elicitor (proline ,yeast extract, myzo- inositol and sucrose) in different mediums. For the first time a comparative study of mature nodal, juvenile nodal explants and cotyledonary callus cultures which elicitated with different organic elicitor was done which revealed that maximum quantity of psoralen was present in cotyledonary callus cultures. A certain and distinguished variation in the psoralen content in juventile, mature nodal and cotyledonary callus cultures has been earned when they were elicited medium supplemented with different elicitors in various range of concentration .Higher amount of psoralen content (2761.8µg/g fresh wt.) even among all of the organic elicitors tried was found at 300mg/l yeast extract in cotyledonary callus cultures as shown past and we had respection to found it. Keywords: Psoralen, Psoralea corylifolia, HPLC set and Elicitors.
INTRODUCTION Psoralea corylifolia is one of the family of Fabaceae. The genus Psoralea includes 130 species, distributed in the tropic and sub tropic of both hemispheres (Willis, 1966). Psoralea corylifolia usually distinguished by Babchi is an endangered and potentiated important plant. It is one of the highly important medicinal plants, which has been included in the series of threatened plants (Bhattacharjee, 1998; Jain, 1994). Psoralea corylifolia is high source of bioactive compounds, which endows the plant with immense value for its application in *
Corresponding author: Ebrahim Alinia Ahandani Corresponding author e-mail
[email protected]
pharmaceuticals, health and body care products. It is applicable for various biomedical applications. The seed is antibacterial, aphrodisiac, astringent, cardiac, cytotoxic, anthelminthic, deobstruent, diaphoretic, diuretic, stimulant, stomachic and tonic (Anonymous, 1989; Joshi, 2000). It is also used externally to cure different skin ailments including leprosy, leucoderma , hair loss and treating vitiligo and psoriasis (Anonymous, 1989; Joshi, 2000, Wamer et al., 1982.Triplex-formation oligonucleotides attached with a photoreactive psoralen molecule can be applied to induce site
J. Plant Bio. Res. 2013, 2(1): 25-37
specific DNA damage and control gene expression (Ping et al., 2005). Seed extract of P. corylifolia inhibit tumour and cancer growth (Latha et al., 2000). Psoralen has been assessed to inhibit the in vitro growth of three human tumor cell lines representing different tumor types, MCF-7 (breast cancer), NCI-H460 (nonsmall cell lung cancer) and SF-268 (CNS cancer). The result showed the efficiency of the psoralen in inhibition of cancer (Oliveira et al., 2006).Biologically active compounds using was first initiated by Rakhmankulov and Korotkova (1975). They reported that the seeds and roots were the richest sources of furanocoumarins (psoralen and angelicin). Cappelletti et al. (1984) reported the presence of psoralen and angelicin mainly in the pericarp of fruits of P. corylifolia. There was a considerable variation in content and ratio of furanocoumrains. P. plumose had the highest amount of psoralen 274.4mg (0.27%) and 302.2 mg angelicin (0.30%) per 100g dry fruits with a ratio of 0.82:1 and is considered to be a useful potential source of furocoumarins.A reversedphase high performance liquid chromatographic method or HPLC was developed by Dong et al. (2003) to determine the contents of psoralen and angelicin from some medicinal herbs. The seeds of Psoralea corylifolia showed the highest content of psoralen (7.8mg/g) and angelicin (2.3mg/g) between the tested herbs. Yang and Qin (2006) studied the chemical constituents of the fruits of Psoralea corylifolia. L. The constituents were extracted and purified by column chromatography. Six compounds were extracted and identified as psoralen, isopsoralen, psoralidin, bavachalcone, daidzein and bavachin. Rajput et al. (2008) isolated psoralen by column chromatography from the methanol extract from Psoralea corylifolia seeds. Ruan et al. (2007) isolated a psoralen with other compounds. Qiao et al. (2006) found two new benzofuran glycosides, nemed psoralenoside and isopsoralenoside, Liu et al. (2004) isolated psoralen and isopsoralen from Psoralea corylifolia by high-speed counter-current chromatography (HSCCC). Due to the complex bioactivity of psoralen, its biosynthesis pathway of psoralen may enable us to influence its formation in direct way, example by metabolic pathway engineering. The biosynthetic pathways to the linear furanocoumarin (psoralen) involved precursors and enzymes cofactors.
The present study showed the evaluation of the psoralen content in (i) in vivo, in vitro plant parts and callus derived from different plant parts of P. corylifolia. (ii) in vitro elicitation of psoralen employing precursors of the psoralen biosynthetic pathway.
MATERIALS AND METHODS Explant's Source Mature nodal explants were collected from a field grown mature plants of P. corylifolia in Guilan province located in north of Iran. Juvenile nodal explants were coped with in vitro grown shoots of P. corylifolia from Guilan Agricultural & Natural resources Research Center that was located in Rasht county of Guilan province. Culture media: Mature nodal and Juvenile nodal explants were cultured on B5 + 5µM BA medium. Cotyledons coped with green seeds of P. corylifolia were inoculated on MS + 10µM BA + 5µM IBA medium. Cultures were incubated in continuous light of 400-500µw/cm2 by cool day light fluorescent incandescent tubes (40W, Philips, Kolkata). The cultures were maintained in a culture room at the temperature of 25±2oC and 55±10% relative humidity. Callus developed on this medium was used for identification, evaluation, characterization of psoralen and precursors treatment. Sample preparation and assessment of psoralen in Psoralea corylifolia Method for extraction of psoralen was shifted by Singh (2003). The fresh samples (1g, each) of plant tissue were crushed with liquid nitrogen carefully and were soaked in ethanol for 24h under dark and then homogenized using pestle and mortar. They were followed as such in the pestle till the time than that ethanol gets evaporated. After evaporation of ethanol, the semisolid form of extract was mixed in methanol (HPLC grade). This mixture was transferred to centrifuge tube and centrifuged for 15min at 12000rpm at room temperature there. The supernatant was filtered using 0.22m Millipore filter and Pellet was discarded and the. The HPLC unit, equipped with UV detector and printer plotters was operated and done under the following parameters: Column packing: Zorbex ODS (Octadecyl silane); Column: C18; Solvent: Methanol (HPLC grade); Flow rate: 0.5ml/min; Injection volume: 20µl; Detection: UV 244nm for psoralen content.
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J. Plant Bio. Res. 2013, 2(1): 25-37
RESULTS AND DISCUSSION HPLC determination of psoralen from various plant parts of P. corylifolia HPLC chromatogram of psoralen standard showed retention time at 5.43 (fig.1 A). HPLC determination of psoralen in in vivo condition in different plant parts such as root, leaf, node, buds, bracts ,flower and brown seeds have determined that maximum amount of psoralen was present in brown seed (3047µg/g fresh wt.) followed by flower, buds, bracts, node, leaf and root segment (Table 1and Fig 1.B). The maximum quantity of psoralen was detected in in vitro node explants (746.13µg/g fresh wt.) followed by leaf and root (Table 1). In comparison to in vivo plant parts the amount of psoralen in in vitro plant parts were less than in vivo plant parts. Quantitative assessment of psoralen was done by the callus derived from different plant parts showed that a maximum of 2601.8µg/g fresh wt. of psoralen was recorded in callus derived from cotyledons (Table 1and Fig 1.C). The amount of psoralen in node, leaf and root derived callus was 1852.2, 1447 and 1058µg/g fresh wt., respectively (Table 1). HPLC analysis of psoralen was done from shoots derived from different callus of the respective psoralen quantities detected were 562.298 and 537.19ug/g fresh wt. in node callus and leaf callus, respectively (Table 1). Thus, our results revealed that the quantity of psoralen in in vivo, in vitro plant parts, callus derived from cotyledons and plant derived callus were various. It could be possible that cotyledons of seed include a specific gene responsible for synthesis of psoralen material. With loosing of cotyledons, this particular gene is not fully expressed or less expressed in plant derived callus. It can be due to development of secondary metabolism mechanism in in vivo and callus derived from cotyledons, too. Against of Rakhmankulov and Korotkova (1975) and Innocenti et al. (1997) the quantity of psoralen in root was the fewer amounts in comparission to other parts of P. corylifolia plant. Some Elicitors The existence of comparatively higher content of secondary metabolites in medicinal plants has endowed them as an important source of phytomedicines. It is therefore, imperative to develop ways for increasing the bioactive
compounds and separate the phytochemical constituents and develop methods for its large scale in vitro products (Pandey, 2009). The main roles of plant secondary metabolites are to protect plants from attack by insects, pathogens and herbivores or to survive other biotic and abiotic stresses. Some strategies in culture for the production of the metabolites based on this principle have been developed to improve the yield of such plant secondary metabolites. These include treatment with different elicitors, signal compounds and abiotic stresses (Zhao et al., 2005). Many such treatments indeed effectively promote the production of a wide range of plant secondary metabolites, both in vivo and in vitro. The general cellular process and regulating principle operation of plant secondary metabolite biosynthesis is that, intra cellular signal or an extra cellular is perceived by a receptor on the surface of the endomembrane or plasma membrane. The elicitor signal perception initiates a signal transduction network that leads to activation or de novo biosynthesis of transcription factors which regulate the expression of biosynthetic genes involved in plant secondary metabolism. The resulting enzymes catalyze the biosynthesis of target secondary metabolites. According to the scientific information about the increasing of secondary metabolites through the elicitors we choice some organic elicitors such as yeast extract, proline, myo- inositol and sucrose. A marked variation in the psoralen content in mature and juvenile nodal and cotyledonary callus cultures have been assessed when they were elicited on medium supplemented with 1, 5, 25, 50, 100, 200 and 300mg/l yeast extract, proline and myoinositol. The mature, juvenile nodal cultures developed on B5 +5µM BA with different concentration of yeast extract. Psoralen content varied from 1412µg/g fresh wt. at 25mg/l, being minimum, to a maximum of 2271.37µg/g fresh wt. of psoralen at 200mg/l of yeast extract in mature nodal cultures (Table 2 Figs 3). 476.6µg/g fresh wt. (lowest amount) and 639.58µg/g fresh wt. (highest amount) was detected at 100mg/l and 300 mg/l of yeast extract, respectively in juvenile nodal cultures. On other levels, almost constant amount of psoralen, but higher than that of control, has been detected (Table 2 Figs 4). In case of cotyledonary callus cultures lower concentrations of yeast extract (1, 5 and 25 mg/l) failed to elevate the psoralen content but beyond this level a gradual increase in
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Table 1: Assessment of psoralen from in vivo, in vitro plant parts , callus derived from different plant parts and plant derived from different callus of P. corylifolia. plant parts
In vivo plant parts
In vitro plant parts
Area
Psoralen Quantity (µg/g fresh wt.
Area
Psoralen Quantity (µg/g fresh wt.)
Brown seed
163374
3047
Flower
121387
2272.2
Bud
120145
2251
Bracket
77888
1449
Node
59169
1103.3
39747.3
746.13
Leaf
56134
1042.7
22794
422
Root
52877
985
17837
319
Callus derived from different plant parts Area Psoralen Quantity (µg/g fresh wt.)
Cotyledon callus
139008
2501.7
Node callus
100262
1852.2
Leaf callus
78429
1447
Root callus
56729
1058
plant derived from different callus Area Psoralen Quantity (µg/g fresh wt.)
Leaf Callus
28534
537.19
Node Callus
30578.6
562.298
Figure 1 A-D: HPLC determination of psoralen from different sources of P. corylifolia. Chromatograms showing psoralen peaks* at retention time 5.4 min: A=Standard, B= Brown seed, C= Cotyledonary callus and D = Node explants.
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J. Plant Bio. Res. 2013, 2(1): 25-37
Table 2: Assessment of psoralen in mature nodal explants, Juvenile nodal explants and cotyledonary callus cultures of P. corylifolia elicited on medium supplemented with different concentrations of yeast extract after 30d of inoculation. Experiment was repeated twice. Yeast extract
Mature nodal explants Area
Psoralen Quantity
Juvenile nodal explants Area
(µg/g fresh wt.)
(mg/l)
Psoralen Quantity
Cotyledonary callus Area
Psoralen Quantity
(µg/g fresh wt.)
(µg/g fresh wt.) 0
83292
1546.1
83183
470.18
83181
1928.8
1
92108
1719.5
29669
549.53
95719.7
1815
5
78057
1456.9
29823
549.3
104920
1870.49
25
76562
1412
28489
533.4
99955
1961.78
50
101210
1893.18
28393
529.4
109147
2033
100
103210
2081.19
25669
476.6
114614
2139.31
200
121318
2271.37
29668
555.3
118819
2221.03
300
10877
2033.34
34269
639.58
153969
2761.8
Table 3: Assessment of psoralen in Mature nodal explants, Juvenile nodal explants and cotyledonary callus cultures of P. corylifolia elicited on medium supplemented with different concentrations of proline after 30d of inoculation. Proline (mg/l)
Mature nodal explants Area
Psoralen Quantity
Juvenile nodal explants Area
(µg/g fresh wt.)
Psoralen Quantity
Cotyledonary callus Area
Psoralen Quantity (µg/g fresh wt.)
(µg/g fresh wt.)
0
83189
1554.4
83192
474.8
83191
1931.6
1
79355
1484.7
39827
744.3
113747
2129.1
5
80378
1503.1
36900
694.6
117049
2197.6
25
113633
2125.5
50616
948.3
122950
2300.1
50
120502
2254.9
56604
1058.3
130329
2437.2
100
133344
2493.3
53351
997.5
120157
2247.2
200
112924
2113.7
53553
1001.2
119466
2235.9
300
112723
2106.6
46771
911.7
121581
2274.4
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the psoralen quantity was observed and1961.78, 2033, 2139.31and 2221.03 µg/g fresh wt. of psoralen was on 25, 50, 100 & 200 of yeast extract induced (Table 2 Fig 5). The maximum quantity of psoralen (2761.8µg/g fresh wt.) was estimated at higher concentration of yeast extract, i. e. 300mg/l (Table 2 Figs 2b). It is noteworthy here that quantity of the psoralen was highest at this level of yeast extract among all the organic elicitor tried. However, higher levels of yeast extract (100300mg/1) developed better for containing the significant amount of psoralen in the cultures (Table 2). The cultures were reared on B5 + 5µM BA medium that supplemented with various concentration of proline from mature and juvenile nodal explants. Lower concentrations of proline (1 & 5mg/l) did not raise better for increasing the psoralen in the cultures of mature nodal explants. A minimum of 1484.7 µg/g fresh wt. and 1503.1 µg/g fresh wt. of psoralen was assessed on 1 and 5mg/l of proline (Table 3 Figs 4). It was 1554.4µg/g fresh wt. in the culture raised on control medium (B5 +5µM BA) (Table 3 Figs 3). However, on higher concentrations of proline (25- 300mg/1) a significant amount of psoralen has been shown in the cultures. Then we saw higher amount of psoralen (2493.3µg/g fresh wt.) even among all of the organic elicitors tried was found at 100mg/l proline (Table 3 Figs 3). In case of juvenile nodal cultures the amount of psoralen increased markedly on different concentrations in comparison to control. among all the these elicitors tried, proline at 50mg/l induced maximum amount of psoralen (1058.3µg/g fresh wt.) in the cultures (Table 3 Figs 3). Therefore, it shifted from 696.7µg/g fresh wt. being minimum, at 5mg/l to 1001.2µg/g fresh wt. at 200mg/l of proline (Table 3 Figs 3). Psoralen content increased when MS medium along with 10µM + 5µM IBA was adjuvanted with 1, 5, 25, 50, 100, 200 and 300mg/l proline in cotyledonary callus cultures. 50mg/l proline contained the maximum psoralen content (2493.3µg/g fresh wt.) followed by 25mg/l of proline (92300.16µg/g fresh wt.) (Table 3 Figs 1c & 5). However, lower concentrations (2129.1µg/g fresh wt. at 1mg/l and 2197.6µg/g fresh wt. at 5mg/l) and higher concentrations (2247.2µg/g fresh wt. at 100mg/l, 2235.9µg/g fresh wt. at 200mg/l and 2274.4µg/g fresh wt. at 300mg/l) of proline too proved beneficial for increasing the psoralen quantity in
callus cultures even over control (Table 3 Figs 3). However, the content of psoralen increased significantly on different concentrations of proline in comparison to control. The best response in terms of psoralen content (1917.4µg/g fresh wt.) was found at 25mg/l myoinositol in mature nodal explants (Table 4 Fig 3). With the gradual enhancement in the level of myoinositol from 1 mg/l to 25mg/l there was a gradual enhancement in the psoralen amount. At 1 and 5mg/l myo- inositol, 1894.5and 1915.4/µg/g fresh wt. of psoralen content, respectively was achieved that was more than control (1555.8µg/g fresh wt.) (Table 4, Fig 3). At concentration higher than 25mg/l of myo- inositol, the psoralen content gradually decreased with enhancement of the concentration (Table 4 Fig 3). Like as mature nodal cultures the best response of psoralen amount (750µg/g fresh wt.) was shown at 25mg/l of myoinositol in juvenile nodal cultures (Table 4 Fig 4). With the gradual enhancement of concentration in myo- inositol from 1mg/l to 25mg/l there was a gradual enhance in the psoralen content. At 1 and 5mg/l of myo- inositol, 318.5and 703.7µg/g fresh wt. psoralen content, respectively was assessed (Table 4 Fig 4). At concentrations higher than 25mg/l of myo- inositol, the psoralen content reduced gradually but the content of psoralen was always higher than that on controls. At higher level of myo- inositol, i.e. 150mg/1 relatively less content of psoralen, i.e. 454.4µg/g fresh wt. was induced (Table 4 Fig 4). Though, all the levels of myo- inositol tried (1-300mg/l) increased the psoralen content in the callus cultures but lower concentrations (at 25 and 50mg/l) proved better over higher concentrations (200 and 300mg/l) (Table 4 Fig 5). The optimum quantity of psoralen (2291.7µg/g fresh wt.) was found at 25mg/l of myo- inositol followed by 5mg/l myo- inositol (2230.20µg/g fresh wt.) (Table 4 Figs 2d & 5). Besides this, 2162.3, 2245.4, 2264.03, 1963.4and 1956.1µg/g fresh wt. psoralen was assessed on 1, 50, 100, 200 and 300mg/l, respectively (Table 4 Fig 5). The mature, juvenile nodal explants and cotyledonary callus cultured were elicited on medium augmented with 1.5, 3, 4.5, 6, 7.5 and 9% sucrose to increase the psoralen. A shifted response in terms of psoralen content has been assessed from the mature nodal cultures raised on B5 + 5µM BA medium along with various concentration of sucrose.
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Table 4: Assessment of psoralen in Mature nodal explants, Juvenile nodal explants and cotyledonary callus cultures of P. corylifolia elicited on medium supplemented with different concentrations of Myo- inositol (mg/l) after 30d of inoculation. Experiment was repeated twice. Myoinositol (mg/l
Mature nodal explants Psoralen Quantity
Area
(µg/g fresh wt.)
Juvenile nodal explants Area
Cotyledonary callus
Psoralen Quantity (µg/g fresh wt.)
Area
Psoralen Quantity (µg/g fresh wt.)
0
83186
1555.8
83182
473.3
83191
1931.7
1
101012
1894.5
17064
318.5
115529
2162.3
5
102474
1915.4
37622.7
703.7
121292
2270.1
25
102468
1917.4
40167
750
122459
2291.7
50
98774
1846.3
32177
601.3
120009
2245.4
100
74246
1387.2
32011.4
588.8
121057
2264.3
200
65039
1216.3
27063
504.7
104906
1963.4
300
64821
1200.7
24024
454.4
104447
1956.1
Table 5: Assessment of psoralen in Mature nodal explants, Juvenile nodal explants and cotyledonary callus cultures of P. corylifolia elicited on medium supplemented with different concentrations of Sucrose (%) after 30d of inoculation. Experiment was repeated twice. Sucrose (%)
Mature nodal explants Area
Psoralen Quantity
Juvenile nodal explants Area
(µg/g freshwt.)
Psoralen Quantity
Cotyledonary callus Area
Psoralen Quantity (µg/g fresh wt.)
(µg/g fresh wt.)
0
83197
1556.2
83182
474.8
83190
1931.83
1.5
85104
1591.7
23437.4
437.7
108513
2031.2
3
111191
2081.7
26525
495.4
114084
2032.7
4.5
101117
1893.2
25304
472.7
110802
2072.7
6
90318
1691.1
26024
486.4
150201
2810.17
7.5
105212
1967.6
30063.6
561.6
124669
2331.4
9
87047
1627.7
30072
568.2
117180
2191.1
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Though, all the levels of sucrose tried, developed the psoralen content in the cultures but a maximum 2080.8µg/g fresh wt. of psoralen was detected at 3% sucrose (Table 5 Fig 3). Besides this, 1591.7, 1893.2, 1691.1, 1967.6 and 1627.7µg/g fresh wt. of psoralen were shown in the cultures reared on 1.5%, 4.5%, 6%, 7.5% and 9% sucrose, respectively (Table 4 Fig 3). The juvenile nodal explants cultured were elicited on B5 + 5µM BA medium augmented with 1.5, 3, 4.5, 6, 7.5 and 9% sucrose to enhance the psoralen. Addition of sucrose (1.5- 9%) did not develop much beneficial for increasing the psoralen quantity in the cultures (Table 5 Fig 4). However, higher concentrations of sucrose (6-9%) could slightly improved amount of psoralen. Beyond 4.5% sucrose, it enhanced from 486.4µg/g fresh wt. at 6% to 568.2µg/g fresh wt., being maximum, at 9% of sucrose (Table 5 Fig 4). Despite this subject, relatively higher amount, i.e. 495.4µg/g fresh wt. of psoralen was also shown at 3% sucrose compared to control 474.8µg/g fresh wt. of sucrose and lower level i.e. 1.5 % failed to raise the amount of psoralen in juvenile nodal cultures (Table 5 Fig 4). 1.5%, 3%, 4.5%, 6%, 7.5% and 9% sucrose were incorporated to the MS + 10µM BA + 5µM IBA medium to elicit the callus cultures. An increasing in the psoralen content has been analyzed on addition of different concentrations of sucrose in the medium. With the gradual increase in concentration of sucrose from 1.5% to 6% there was a gradual increase in the psoralen content. At 1.5, 3 and 4.5% sucrose 2031.2, 2032.7 and 2072.7µg/g fresh wt. of psoralen content was assessed, respectively (Table 5 Fig 5). A sudden increase in the psoralen amount was analyzed at 6% sucrose (Table 5 Figs2e & 5). However, 7.5 and 9% sucrose also induced 2331.4 and 2191.1µg/g fresh wt. of psoralen content, respectively in the callus cultures (Table 5 Fig 5). Thus beyond 3% of sucrose psoralen significantly increased.
The response observed was explant and dose dependent and a higher amount of psoralen was detected almost on all the levels and explant used. Our results determined that a variable answer in terms of psoralen content has been observed when yeast extract was feeded to P. corylifolia cultures. Cotyledonary callus cultures produced a maximum of 2761.8g/g of psoralen at 300mg/l of yeast extract level as we had not any proof to accept it before. Proline is a non important amino acid (Berg, 2001). It is distinguished by the most beneficial elicitor. Though, all the levels of proline improved the psoralen content but in 25mg/l it enhanced significantly in P. corylifolia cultures. Inositol material has a role in signal transduction pathways. Inositol of 1, 4, 5 triphosphate must bind to sites on the cytosolic side of the membrane protein to open the channel and release Ca2+ (Berg, 2001). It is capable to enhance Ca2+ concentration by associating with a membrane protein called IP3-gated channel or IP3 receptor. In the present study, inositol operate as elicitor and increased the psoralen content until 25mg/l inositol. After that the psoralen production gradually reduced in both mature and juvenile explants in P .corylifolia. Memon et al. (1989) showed that phosphatidyl inositol-4monophosphate and phosphatidylinositol-4,5bisphosphate enhanced the activity of ATPase associated with plasma membranes extracted from both sunflower hypocotyls and carrot suspension culture cells. The data suggest that operation of the inositol phospholipid kinases could be a critical step in signal transduction in plants. Lower levels, i.e 5 and 25mg/l of inositol was found to be optimum dose in both the plants, while relatively higher amount of psoralen was detected. Higher levels of inositol did not determine better for the increasing of metaboilites (psoralen and asiatic acid) in all the cultures of P. corylifolia and C. asiatica. Sucrose operates as an ATP generation material that is needed for various biological reactions (Berg, 2001). Zhang et al., 2004
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Figure 2 A-E: HPLC determination of psoralen of cotyledonary callus cultures of P. corylifolia reared on MS + 10 µM BA + 5 µM IBA medium supplemented with different concentrations organic elicitors after 30 d of inoculation. Chromatograms showing psoralen peaks* at retention time 5.4 min.: A= Control B= 300 mg/l Yeast extract, C= 50 mg/l Proline, D= 50 mg/l Myo-inositol, E= 6% Sucrose.
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Yeast extract
Proline
Myo-inositole
Psoralen Quantity
Sucrose
3000
2500
2000
1500
1000
500
0 300 mg/l
200 mg/l 10%
100 mg/l 8%
50 mg/l 6%
25 mg/l 4%
5 mg/l 2%
1mg/l 1%
0
Yeast extract
2033.34
2271.37
2081.19
1893.18
1412
1456.9
1719.5
1546.1
Proline
2106.6
2113.7
2493.3
2254.9
2125.5
1503.1
1484.7
1554.4
Myo-inositole
1200.7
1216.3
1387.2
1846.3
1917.4
1915.4
1894.5
1555.8
1627.7
1967.6
1691.1
1893.2
2081.7
1591.7
1556.2
Sucrose
Figure 3: Quantity of psoralen of mature nodal explants cultures of P. corylifolia reared on medium supplemented with different organic elicitors. Analysis was done after 30 d of inoculation.
Yeast extract
Proline
Myo-inositole
Sucrose
Psoralen Quantity
1200 1000
800 600 400 200 0 300 mg/l
200 mg/l 10%
100 mg/l 8%
50 mg/l 6%
25 mg/l 4%
5 mg/l 2%
1mg/l 1%
0
Yeast extract
639.58
555.3
476.6
529.4
533.4
549.3
549.53
470.18
Proline
911.7
1001.2
997.5
1058.3
948.3
694.6
744.3
474.8
Myo-inositole
454.4
504.7
588.8
601.3
750
703.7
318.5
473.3
568.2
561.6
486.4
472.7
495.4
437.7
474.8
Sucrose
Figure 4: Quantity of psoralen of Juvenile nodal cultures of P. corylifolia reared on medium supplemented with different organic elicitors. Analysis was done after 30 d of inoculation.
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J. Plant Bio. Res. 2013, 2(1): 25-37
Yeast extract
Proline
Myo-inositole
Psoralen Quantity
Sucrose
3000 2500 2000 1500 1000 500 0 300 mg/l
200 mg/l 10%
100 mg/l 8%
Yeast extract
2761.8
2221.03
Proline
2274.4
2235.9
Myo-inositole
1956.1
Sucrose
50 mg/l 6%
25 mg/l 4%
5 mg/l 2%
2139.31
2033
1961.78
2247.2
2437.2
2300.1
1963.4
2264.3
2245.4
2191.1
2331.4
2810.17
1mg/l 1%
0
1870.49
1815
1928.8
2197.6
2129.1
1931.6
2291.7
2270.1
2162.3
1931.7
2072.7
2032.7
2031.2
1931.83
Figure 5: Quantity of psoralen of cotyledonary callus cultures of P. corylifolia reared on medium supplemented with different organic elicitors. Analysis was done after 30 d of inoculation.
employed abiotic elicitors to stimulate the secondary metabolite production in hairy root culture of Salvia miltiorrhiza plant and concluded that sucrose feeding or medium renewal before the addition of Ag+ to the culture significantly prevented the growth inhibition and significantly enhanced the biomass concentration and volumetric tanshinone yield. In the present study all the levels of sucrose develop and progress the psoralen amount in P. corylifolia cultures. Therefore Psoralen increasing depends on part, elicitors and used location.
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