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REGULAR ARTICLE
Extraction and Preparation of Psoralen from different Plant Part of Psoralea
Corylifolia and Psoralen Increasing with some Elicitors
1 Department of Plant Biology, Faculty of Science, Urmia University, Iran. 2 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 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
specific DNA damage and control gene
expression (Ping et al., 2005). Seed extract of P. *Corresponding author: Ebrahim Alinia Ahandani
Corresponding author e-mail [email protected]
Journal of Plant Biology Research 2013, 2(2): 25-37 eISSN: 2233-0275
pISSN: 2233-1980
http://www.inast.org/jpbr.html
J. Plant Bio. Res. 2013, 2(1): 25-37
26
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 (non-
small 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 reversed-
phase 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.
J. Plant Bio. Res. 2013, 2(1): 25-37
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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 myo-
inositol. 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.
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.
plant derived from
different callus
Callus derived from
different plant parts
In vitro plant parts In vivo plant parts plant parts
Psoralen
Quantity
(µg/g fresh
wt.)
Area Psoralen
Quantity
(µg/g fresh
wt.)
Area Psoralen
Quantity
(µg/g fresh
wt.)
Area Psoralen
Quantity
(µg/g fresh
wt.
Area
3047 163374 Brown seed
2272.2 121387 Flower
2251 120145 Bud
1449 77888 Bracket
746.13 39747.3 1103.3 59169 Node
422 22794 1042.7 56134 Leaf
319 17837 985 52877 Root
2501.7 139008 Cotyledon
callus
1852.2 100262 Node callus
1447 78429 Leaf callus
1058 56729 Root callus
537.19 28534 Leaf Callus
562.298 30578.6 Node Callus
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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. Cotyledonary callus Juvenile nodal explants Mature nodal explants
Yeast
extract
(mg/l)
Psoralen
Quantity
(µg/g fresh wt.)
Area Psoralen Quantity
(µg/g fresh wt.)
Area Psoralen Quantity
(µg/g fresh wt.)
Area
1928.8 83181 470.18 83183 1546.1 83292 0
1815 95719.7 549.53 29669 1719.5 92108 1
1870.49 104920 549.3 29823 1456.9 78057 5
1961.78 99955 533.4 28489 1412 76562 25
2033 109147 529.4 28393 1893.18 101210 50
2139.31 114614 476.6 25669 2081.19 103210 100
2221.03 118819 555.3 29668 2271.37 121318 200
2761.8 153969 639.58 34269 2033.34 10877 300
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. Cotyledonary callus Juvenile nodal explants Mature nodal explants
Proline
(mg/l) Psoralen Quantity
(µg/g fresh wt.)
Area Psoralen Quantity
(µg/g fresh wt.)
Area Psoralen Quantity
(µg/g fresh wt.)
Area
1931.6 83191 474.8 83192 1554.4 83189 0
2129.1 113747 744.3 39827 1484.7 79355 1
2197.6 117049 694.6 36900 1503.1 80378 5
2300.1 122950 948.3 50616 2125.5 113633 25
2437.2 130329 1058.3 56604 2254.9 120502 50
2247.2 120157 997.5 53351 2493.3 133344 100
2235.9 119466 1001.2 53553 2113.7 112924 200
2274.4 121581 911.7 46771 2106.6 112723 300
J. Plant Bio. Res. 2013, 2(1): 25-37
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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 (100-
300mg/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 myo- inositol in mature nodal explants (Table 4 Fig 3).
With the gradual enhancement in the level of myo-
inositol 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 myo- inositol 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. Cotyledonary callus Juvenile nodal explants Mature nodal explants
Myo-
inositol
(mg/l
Psoralen Quantity
(µg/g fresh wt.) Area
Psoralen Quantity
(µg/g fresh wt.) Area
Psoralen Quantity
(µg/g fresh wt.) Area
1931.7 83191 473.3 83182 1555.8 83186 0
2162.3 115529 318.5 17064 1894.5 101012 1
2270.1 121292 703.7 37622.7 1915.4 102474 5
2291.7 122459 750 40167 1917.4 102468 25
2245.4 120009 601.3 32177 1846.3 98774 50
2264.3 121057 588.8 32011.4 1387.2 74246 100
1963.4 104906 504.7 27063 1216.3 65039 200
1956.1 104447 454.4 24024 1200.7 64821 300
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. Cotyledonary callus Juvenile nodal explants Mature nodal explants Sucrose
(%) Psoralen Quantity
(µg/g fresh wt.)
Area Psoralen Quantity
(µg/g fresh wt.)
Area Psoralen Quantity
(µg/g freshwt.)
Area
1931.83 83190 474.8 83182 1556.2 83197 0
2031.2 108513 437.7 23437.4 1591.7 85104 1.5
2032.7 114084 495.4 26525 2081.7 111191 3
2072.7 110802 472.7 25304 1893.2 101117 4.5
2810.17 150201 486.4 26024 1691.1 90318 6
2331.4 124669 561.6 30063.6 1967.6 105212 7.5
2191.1 117180 568.2 30072 1627.7 87047 9
J. Plant Bio. Res. 2013, 2(1): 25-37
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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-4-
monophosphate and phosphatidylinositol-4,5-
bisphosphate 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
J. Plant Bio. Res. 2013, 2(1): 25-37
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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.
J. Plant Bio. Res. 2013, 2(1): 25-37
34
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.
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.
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
Sucrose 1627.7 1967.6 1691.1 1893.2 2081.7 1591.7 1556.2
0
500
1000
1500
2000
2500
3000
Psoralen QuantityYeast extract Proline Myo-inositole Sucrose
300 mg/l200 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
Sucrose 568.2 561.6 486.4 472.7 495.4 437.7 474.8
0
200
400
600
800
1000
1200
Psoralen QuantityYeast extract Proline Myo-inositole Sucrose
J. Plant Bio. Res. 2013, 2(1): 25-37
35
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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