INTRODUCTION:

The scientific researches on medicinal plant in Iraq is still in infancy and a lot of important plant in spite of their wide distribution are still in shade and unused, That millions of dollar spent every year to import supplement made from plants naturally grown in Iraq¹. One of these endogenous important medicinal plants is Silybum marianum².

Actually it's one of the top selling supplements in the united state that provides approximately 3000000 dollars every year, but the concentration of silymarin is greatly varied among these preparation³.

Figure 1: Silybum marianum image

Silymarin is an organic solvent extract of Silybum marianum, which is a mixture of about 75% flavolignan and trace content of flavonoid (mainly taxifolin)⁴, other ingredients involve fatty acids and polyphenolic compounds⁵. The major flavolignans are silybin, isosilybin, silydianin, and silychristin, quantitatively silybin is considered as the major compound followed by isosilybin, both are present as a mixture of 2 diasterioisomers (A and B) and represent approximately 60% of silymarin⁶.

Currently silymarin is used as a reference during the search of new hepatoprotective agents^7-9Silymarin hepatoprotective effect is benefit in different liver ailment, mainly cirrhosis, hepatocellular carcinoma¹⁰, growth promotant, alcoholic liver disease, chronic hepatitis C, chronic liver disease and jaundice^11,12 Also silymarin have the ability to regenerate liver tissue after hepatoctomy by accelerating the cell cycle and inhibiting apoptosis which leads to proliferate hepatocytes, regenerate the lost hepatic tissue and restorate the normal hepatic mass^13,14. This study was designed to determine the concentration of silybin in different parts of the plant and to study the effect of extraction methods, solvents and the time of collection on silybin concentration.

MATERIALS AND METHODS:

Plant material:

All the parts of the plants were collected from the College of Pharmacy, Al-Mustansiriyah University, The plant was authenticated by Dr. Sukaina Abbas, College of Science, Baghdad University, Baghdad, Iraq.

Leaves, flowers, stems, roots and seeds of the plant were collected at different seasons as summarized in table 1, then they were washed thoroughly by tap water, dried in shade, at room temperature from 2 weeks for flowers, seeds and roots up to 1 months for leaves and stems, then grinded to a powder and weight for further investigations¹⁵.

Table 1: time of plants collection

Part of the plant	Time of collection
Purple flower (PF)	At the end of February and the beginning of March before seed formation
White flower (WF)	At August and September after seeding completion
Seeds (S)	At the end of march to may
Stems (St)	At January before flower formation
Leaves (L1)	At January before flower formation
Leaves (L2)	At April during seeding
Roots (R)	At January

Extraction of the plant materials:

Extraction by hot method (continues extraction by soxhlet apparatus):

For defatting process 25grams of shade-dried pulverized purple flowers, white flowers, seeds, roots, stems and leaves (L1 and L2) were packed in the thimble of soxhlet apparatus respectively and extracted by 300ml of hexane until exhaustion¹⁶. The extract was filtered then solvent was evaporated by rotary evaporator at 45˚C. The residue was re-extracted with absolute ethanol 300ml in the soxhlet apparatus until the solution become clear in the soxhlet chamber, and then it was filtered, concentrated by rotary evaporator at 45˚C, the process was repeated by using 300ml of acetone once and by 300ml of distilled water in the second time, each process was repeated 4 times and the yields were collected and weights were recorded as a percent^17,18.

Extraction by cold method (maceration):

After defatting process 100gm of powdered material (PF, WF, S, St, L1, L2 and R) were soaked in ethanol in glass container tightly closed for 5 days, filtered and evaporated by rotary evaporator at 45˚, weight and labeled for further investigations¹⁹.

Calculation the percentage of extract obtained from plant materials:

Yield percentages of the various extracts obtained were defined as being the ratio between the mass of the extract and that of the dry plant matter subjected to the extraction²⁰. It is calculated by the following formula:

Percent yield = [W1-W2 / W3] × 100

W1: Weight of the flask after evaporation, W2: Weight of the empty flask, W3: Weight of the starting dry plant matter.

Identification of Silybin in Silybum marianum extract

Qualitative identification by TLC:

A readymade TLC Gf254 plates, UV detector at 254 nm are used for the identification of silybin in different plant parts, three solvent systems (S1, S2 and S3)are used for development and the Rf value was recorded as compared with standard^21-23

S1: toluene: ethyl acetate: formic acid 3.6: 1.2: 0.5

S2: chloroform: acetone: formic acid 4:1:0.8

S3: ethyl acetate: pyridine: methanol: water 8: 2:1:0.5

Quantitative evaluation of silybin by HPLC:

Silybin concentration was determined using Shimadzu HPLC instrument at the College of Pharmacy, Al-Mustansiriyah University with a UV-Visible detector.

Silybin standard were supplied from Hyperchem China 99.8% and methanol used was HPLC grade (BDH/UK). The environments used in HPLC are listed in table 2.

Table 2: HPLC conditions²⁴

Mobile phase	Methanol: water
Mobile phase	(50:50)
Column	C18 25cm
Flow rate	1 mL/min
detector	290 nm

Standard solution of silybin was dissolved in methanol and diluted to reach a concentration range of 0.1-2mg/ ml, triplicate 100μL injection were made of each concentration, the peak area values were plotted against corresponding concentrations and linear relationship was obtained, then each extract (1mg/1ml conc.) was injected three times in three different days, peak areas were recorded and mean was calculated for each sample²⁵.

RESULTS AND DISCUSSION:

Percentage of extract obtained from plant materials

Total extract was calculated by the following equation:

Percent yield = [W1-W2 / W3] × 100

W1: Weight of the flask after evaporation, W2: Weight of the empty flask, W3: Weight of the starting dry vegetable matter.

And the result is recorded in table 3.

Identification of silybin by TLC:

Silybin has been detected in different plants parts by TLC using three different solvent systems in the presence of standard silybin and UV detector (254 nm) as shown in figure 2, Rf value are calculated and recorded in table 4.

Figure 2: TLC of Silybum marianum extracts in three solvent systems

Sb: silybin standard, S: seed, F: flower, T: steam, L: leave

Table 4: Table of Rf values

Solvent system	silybin standard	Seed extract	Leave extract	Flower extract	Steam extract
S1	0.5	0.5	0.52	0.5	0.52
S2	0.9	0.9	0.91	0.92	0.9
S3	0.1	0.14	0.12	0.14	0.14

Quantitative estimation of silybin by HPLC:

The chromatogram of silybin standard are shown below in figure 3.

Figure 3: HPLC chromatogram of silybin standard

The peak of silybin standard appear at 2.658 retention time, which is used to identify silybin in the HPLC chromatogram of the different plant parts extracts which are given below in figure 4.

Figure 4: HPLC chromatogram of Silybum marianum extracts

For the determination of silybin concentration in different parts of the plant, a calibration curve were calculated from a series of silybin standard concentrations as shown in figure 5, concentration of silybin in each plant part were calculated and tabled in table 5, and the percent of silybin from the total extract are shown in figure 6.

Figure 5: Calibration curve of silybin standard measured by HPLC method

Table 5: Concentration of silybin in different parts of Silybum marianum in g/ 100 g of dried plant material

Plant parts	Hot extraction			Cold extraction
Plant parts	ethanol	water	acetone	By ethanol
White flowers	0.68	0.122	0.396	0.45
Purple flowers	0.348	0.177	0.202	0.266
Leaves (L1)	0.634	0.88	0.314	0.303
Leaves (L2)	0.27	0.04	0.185	0.204
seeds	1.06	0.344	0.459	0.868
Stems	0.622	0.1	0.314	0.41
Root	0.05	-	-	0.025

Figure 6: Percent of silybin concentration in different parts of the plant extracted by different solvents

WF: white flower, PF: purple flower, S: seed, L1: leave before flowering, L2: leave during flowering, T: stem, R: root.

HE: hot ethanol extract, HW: hot water extract, HA: hot acetone extract, CE: cold ethanol extract .

CONCLUSIONS:

From the results above we can conclude several points:

1. Using heat during extraction yield higher concentration of silybin than extraction by cold method.

2. Ethanol is a better solvent for silybin extraction than water and acetone.

3. Silybin is present in all plant parts in different concentration.

4. Although that 100g of the seeds yield only 3g of the extract which is lower than the yield of other parts, it contain the higher concentration of silybin (1.06g) than other parts, which represents 35.5% of total extract.

5. The concentration of silybin in the leaves, stems and flowers is high before seedling and decrease when the seeds are produced.

6. Since that the aerial parts of the plant grown in different seasons, and the plant produce them in huge amount with a good production of silybin when compared with seeds, So they can be used as an alternative source for silybin production

RECOMMENDATIONS:

1. Silybum marianum is grown naturally in Iraq in a large distance that it can be used for large scale production of silymarin that can cover local consuming and the excess can enter a worthwhile income to country from exporting.

2. Other methods for the plant extraction could be studied such as reflux, decoction, etc. also the use of other solvents for extraction such as methanol, ethyl acetate and chloroform could be tried for silybin extraction.

ACKNOWLEDGMENTS:

The authors would like to thank Mustansiriyah University (www.uomustansiriyah.edu.iq) Baghdad-Iraq for its support in the present work.

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Received on 05.11.2020 Modified on 27.11.2020

Research J. Pharm. and Tech. 2021; 14(9):4567-4572.

DOI: 10.52711/0974-360X.2021.00794

Plant parts	Hot extraction				Cold extraction
Plant parts	ethanol	water	hexane	acetone	By ethanol
White flowers	12	10	1	9.7	11.8
Purple flowers	12.7	11.3	1.2	10	12
Leaves( L1)	11	10.4	1.6	9	10.8
Leaves (L2)	10	9.6	1.5	9.5	9.5
seeds	3	2.5	0.2	1.8	2.8
Stems	10.2	8.6	1.5	8.9	8
Root	7.5	4	0.5	3.5	7