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 ebi.alinia@gmail.com

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

J. Plant Bio. Res. 2013, 2(1): 25-37

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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

J. Plant Bio. Res. 2013, 2(1): 25-37

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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.

J. Plant Bio. Res. 2013, 2(1): 25-37

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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

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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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