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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 68  |  Page : 93-98  

Isolation and biological evaluation of some secondary metabolites from seeds of Silybum marianum


1 Centre for Genetics and Inherited Diseases, Taibah University Al-Madinah Al-Munawwarah, Kingdom of Saudi Arabia
2 Department of Pharmacy, COMSATS University Islamabad, Lahore Campus 54000, Lahore, Pakistan
3 Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus Tobe Camp 22060, Abbottabad, Khyber Pakhtunkhwa, Pakistan
4 EMAN Biodiscoveries Sdn. Bhd., A1-4, Lot 5, Persiaran 2/1, Kedah Halal Park, Kawasan Perindustrian Sungai Petani, 08000 Sungai Petani, Kedah, Malaysia

Date of Submission23-Jul-2019
Date of Decision30-Sep-2019
Date of Web Publication31-Mar-2020

Correspondence Address:
Abdul Mannan
Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus Tobe Camp, Abbottabad, Khyber Pakhtunkhwa 22060
Pakistan
Mohamed Khadeer Ahamed Basheer
Biodiscoveries Sdn. Bhd.,A1-4, Lot 5, Persiaran 2/1, Kedah Halal Park, Kawasan Perindustrian Sungai Petani, 08000 Sungai Petani, Kedah
Malaysia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_315_19

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   Abstract 


Background: Despite the widespread ethnopharmacological use and significance of Silybum marianum (SM) seeds, most of its phytochemical and biological properties are still yet to be confirmed using validated scientific methods. Objectives: The current study was designed to investigate the phytochemical and biological properties of SM seed extract. Materials and Methods: Methanolic extract of SM (MESM) dried seeds was fractionated by column chromatography, and fractions (SA1–SA10) were evaluated for antioxidant, antimicrobial, and cytotoxic activities (brine shrimp lethality assay and antileishmanial assay). Results: All fractions showed considerable level of antioxidant potential. Free radical scavenging activity of fraction (SA9) was maximum at 80.7%. Fraction SA4 exhibited substantial total antioxidant capacity (101.81 μg/mg). In ferric-based reducing antioxidant power assay, fraction SA4 showed the highest antioxidant power (258.93 μg/mg). Phytochemical screening of the fractions (SA1–SA10) inferred that total phenolic contents were maximum in fraction SA7 (85.13 μg/mg) and total flavonoid contents were found to be highest in fraction SA1 (58.24 μgQE/mg). However, mild antibacterial and antifungal activities were shown by different fractions. To evaluate cytotoxic potential, brine shrimp lethality bioassay was performed. Among all the fractions, the fraction SA9 revealed the lowest LD50of 49.99 μg/mL, whereas all the other fractions tested demonstrated significant cytotoxic property. The results of antileishmanial assay showed that the fraction SA6 possesses the highest mortality percent (84%) compared to the other fractions. Conclusion: These findings revealed that MESM can be an important source of natural antileishmanial herb that can be used as a therapeutic alternative for leishmaniasis.

Keywords: Antileishmanial assay, antimicrobial assay, antioxidant assay, cytotoxic assay, milk thistle, phytochemical analysis


How to cite this article:
Latif M, Umar MI, Rahim A, Khan SA, Fatima N, Hussain I, Mannan A, Ahamed Basheer MK. Isolation and biological evaluation of some secondary metabolites from seeds of Silybum marianum. Phcog Mag 2020;16, Suppl S1:93-8

How to cite this URL:
Latif M, Umar MI, Rahim A, Khan SA, Fatima N, Hussain I, Mannan A, Ahamed Basheer MK. Isolation and biological evaluation of some secondary metabolites from seeds of Silybum marianum. Phcog Mag [serial online] 2020 [cited 2020 Jun 2];16, Suppl S1:93-8. Available from: http://www.phcog.com/text.asp?2020/16/68/93/281697



SUMMARY

  • With the background of the popular ethnopharmacological use of Silybum marianum (SM) seeds as traditional medicine against several human ailments, an attempt is made to investigate the phytochemical and biological properties of SM seed extract. Methanolic extract of SM dried seeds was subjected to column chromatography and based on thin-layer chromatography analysis, fractions (SA1–SA10) were pooled up. The collected fractions were tested for antioxidant, antimicrobial, and cytotoxic activities (brine shrimp lethality assay and antileishmanial assay). All the tested fractions demonstrated significant antioxidant activities. Fraction SA9 displayed pronounced free radical scavenging activity of 80.7%. Fraction SA4 exhibited significant total antioxidant capacity (101.81 μg/mg). In ferric-based reducing antioxidant power assay, fraction SA4 showed the highest antioxidant power (258.93 μg/mg). Phytochemical screening of the fractions (SA1–SA10) revealed that the total phenolic contents were abundant in fraction SA7 (85.13 μg/mg) and total flavonoid contents were found to be highest in fraction SA1 (58.24 μgQE/mg). On the other hand, mild antibacterial and antifungal activities were shown by the fractions. Brine shrimp lethality bioassay was performed to test the cytotoxic potentials of the fractions. Among all the fractions, fraction SA9 revealed the lowest LD50 of 49.99 μg/mL, whereas all the other fractions tested demonstrated significant cytotoxic property. The results of antileishmanial assay showed that the fraction (SA6) possessed the highest mortality percent (84%) compared to the other fractions. It can be concluded that based on the current study, SM seed extract has shown potential antioxidant and antileishmanic activities.




Abbreviations used: MESM: Methanolic extract of S. marianum; SA = Subfraction; LD50: Median lethal dose; SM: Silybum marianum; MeOH: Methanol; CHCl3: Chloroform; TLC: Thin-layer chromatography; DPPH: 2, 2-diphenyl-1-picrylhydrazyl; DMSO: Dimethyl sulfoxide; TDP: Total phenolic content; FCR: Folin–Ciocalteu reagent; TFC: Total flavonoid content.


   Introduction Top


Silybum marianum (SM), previously known as Cardus marianus, is a member of Asteraceae family frequently recognized as Daisy family. It is a biennial or an annual plant indigenous to North Africa, North America, Mediterranean region, Europe, Middle East, and Australia.[1] It is also common in India at a height of 1800–

2400 m.[2] Milk thistle is the common name of the plant, which is given due to the presence of “milky white” veins on the surface of leaves. The seeds of SM have been used as a therapeutic source from thousands of years, and Theophrastus was the first who reported this plant as a source of remedy and cure. The medicinal value of SM is well known for over 2000 years, and frequent use of its seeds has been reported in the West (European countries) as a therapeutic agent in the treatment of several diseases such as hepatic ailments (to remove gall stones), for pregnant women (as a bitter tonic), anxiolytic issues, stomach acidity, varicose veins, splenic congestions, uterine hemorrhage, amenorrhea, and menstrual disorders.[3],[4],[5] It has shown promising results in ameliorating pesticide-induced hepatotoxicity.[6] Similarly, its extract has exhibited tremendous cardioprotective potential by improving healing after a myocardial infarction.[7] When used in combination, SM extract has potentiated the antidiabetic and antibacterial activities of zinc oxide nanoparticles.[8]

SM has a variety of natural products with promising biological potential. Among them, antioxidants are those compounds which can stop or slow down the oxidation of biomolecules such as lipid oxidation by hindering the chain reactions of oxidation and have the ability to give protection or reconstruct cellular damage that can occur in the body because of oxygen.[9] The present study was designed to investigate the phytochemical and cytotoxic potential of SM seed extract with special focus on its antibacterial, antifungal, and antileishmanial effects.


   Materials and Methods Top


Collection and extraction of plant seeds

Plant material of SM was collected from Haripur district of Khyber Pakhtunkhwa (610 m above the see level), Pakistan, followed by identification by a taxonomist at Quaid-i-Azam University, Islamabad, Pakistan (herbarium voucher no. QAU/Bot-Herb-14328). The seeds were dried, crushed, pulverized into powdered form by using a heavy-duty blander, and weighed (~2.5 kg). The powdered material was macerated in methanol for 9 days with occasional shaking to achieve maximized extraction of the seed constituents and kept in a dry shaded place at room temperature. The filtrate was evaporated under reduced pressure by using a rotary evaporator at 30°C to complete dryness to yield crude seed extract. The dried seed extract was preserved till further experimentation.

Fractionation via column chromatography

A slurry of crude methanolic extract (20 g) was prepared by dissolving the extract in minimum volume of 10% (MeOH in CHCl3) and adsorbed on silica gel by keeping the sample to adsorbent (1:1.5) proportion. The sample was loaded on the top of a chromatographic column packed with silica gel as stationary phase and eluted with 100% (CHCl3) to 10:1 (CHCl3: MeOH) followed by 50% (CHCl3: MeOH) in gradient manner. Forty fractions were collected and further subjected to normal-phase thin-layer chromatography (TLC) analysis. Based on TLC investigation, the fractions with similar Rf values were pooled up to obtain a total of ten fractions from SA1 to SA10, having % yield of 12.5, 18, 11.25, 5, 7.5, 10, 12.5, 12.75, 5, and 4, respectively. The fractions were used for further phytochemical and biological evaluation.

Antioxidant assays

2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity of fractions (SA1 SA10) was determined by a previously reported method with slight modification.[10],[11],[12] Briefly, 3.2% of DPPH reagent, 4% of ascorbic acid, and 1% of each sample fraction was prepared as stock solution. A volume of 20 μL of each fraction to be tested was taken in 96- well plate followed by the addition of 180 μL of DPPH reagent in each well to make the final volume of 200 μL and incubated for 1 h at 37°C. Ascorbic acid and ethanol were used as positive and negative controls, respectively. The DPPH reduction activity was measured by reading the absorbance at 517 nm. The experiment was performed in triplicate, and the percentage of final scavenging was calculated by the following formula:

DPPH scavenging activity (%) = (A0 – A1/A1) ×100.

The antioxidant capacity of all fractions (SA1 SA10) was determined using reported methodology with slight modifications.[13] Premeasured 1.63 mL H2 SO4 (conc.), NaH2 SO4(1.679 g), and ammonium-molybdate (0.247 g) were dissolved in a 100-mL volumetric flask and finally, the volume was made up to 100 mL. In 1 mL of dimethyl sulfoxide (DMSO), 4 mg of ascorbic acid was dissolved to prepare a stock solution of ascorbic acid. First, 1 mL of the reagent was taken in an Eppendorf tube, in which 0.1-mL sample was added and mixed properly. The mixture was then placed in an incubator at about 95°C for 1.5 h. The mixture was cooled to 28°C, and the absorbance of the mixture was measured at 695 nm with the help of a microplate reader. For calibration curve, ascorbic acid was tested at various concentrations (125, 100, 75, 50, and 25 μg/mL).

The reducing power of the plant extracts was determined by using the method reported previously with slight modifications.[10] Phosphate buffers (0.2 M), potassium ferric cyanide (1%), trichloroacetic acid (10%), and ferric chloride (0.1%) were used as stock solutions. Each fraction (200 μL) was taken in the Eppendorf tube and was added with buffer (500 μL). Then, potassium ferric cyanide (500 μL) was added and incubated for 20 min at 50°C. After incubation, trichloroacetic acid (500 μL) was added, and the mixture was then subjected to centrifugation for 10 min at 3000 rpm. A volume of 100 μL of the upper layer was removed and carefully poured into the assigned well. Ferric chloride (1%) was further added to each well followed by the addition of distilled water (20 μL) in each well. The absorbance was measured by a microplate reader at 630 nm wavelength.

Phytochemical analysis

The total phenolic content (TPC) was determined by using Folin–Ciocalteu assay.[14] Folin–Ciocalteu reagent (FCR) and distilled water (in 1:10 v/v ratio), 6% sodium carbonate, and 4% gallic acid in methanol were used as stock solutions. In 96-well plate, the sample (20 μL) was taken followed by the addition of FCR (90 μL) and incubated at 40°C for 5 min. After an interval of 5 min, 6% sodium carbonate solution (90 μL) was added and the reaction mixture was incubated at 40°C for 60 min. Gallic acid and DMSO were used as positive control and negative control, respectively. The absorbance was measured at 630 nm on a microplate reader.

The total flavonoid content (TFC) was measured by aluminum chloride colorimetric assay protocols as described earlier.[14] The stock solutions of aluminum chloride (10%), potassium acetate (1.0 M), and quercetin (4 mg/mL) in DMSO were prepared. To a sample (20 μL) placed in 96-well plate, aluminum chloride (10 μL), potassium acetate (10 μL), and distilled water (160 μL) were added to make a final volume to 200 μL and incubated for 30 min at room temperature. Quercetin and DMSO were used as positive control and negative control, respectively. The absorbance was measured at 405 nm using a microplate reader.

For preliminary phytochemical screening and identification of bioactive components in MESM, several phytochemical investigations were carried out by using the standard procedures described previously with slight modifications.[15],[16],[17]

For alkaloids, the following tests were performed:

  1. Mayer test: Methanolic extract (5 mL) was added with 2–3 drops of Mayer's reagent (potassium mercuric iodide solution). The appearance of cream yellow or white brown color indicated the presence of alkaloids
  2. Wagner's test: Methanolic extract (5 mL) was added with 2–3 drops of Wagner's reagent (iodine potassium iodide solution). Formation of reddish brown precipitates also indicated the presence of alkaloids
  3. Dragendroff's reagent test: Methanolic extract (5 mL) was added with dilute HCl (2 mL) followed by the addition of Dragendroff's reagent (1 mL) in the mixture. Instantaneous formation of orange or red precipitates showed the presence of alkaloids.


Test for saponins

Methanolic extract (5 mL) was stirred vigorously with distilled water (15 mL) in a test tube and warmed gently with continuous stirring for 10 min. The formation of a stable layer of foam was taken as an indication of the presence of saponins.

Test for terpenoids

Methanolic extract (~10 mg) was taken and dissolved in chloroform (1 mL). Acetic anhydride (1 mL) was added slowly followed by the addition of concentrated H2 SO4 (2 mL). Appearance of reddish violet color showed the presence of terpenoids.

Test for tannins

Methanolic extract (5 mL) was added with 2–3 drops of 1% lead acetate. Formation of yellow or pale yellow precipitates indicated the existence of tannins.

Antimicrobial assays

The antibacterial effect of the extracts was studied against four bacterial strains, i.e. Staphylococcus aureus (American Type Culture Collection [ATCC] 6538), Escherichia coli (ATCC 15224), Bacillus subtilis (ATCC 6633), and Enterobacter aerogenes (ATCC 13048), by following the standard disc diffusion method as advised earlier.[18] The stock solution of methanolic extract of SM (MESM) (20 mg/mL) was prepared in DMSO. Initially, nutrient agar was prepared and pH was adjusted (pH = 7) and autoclaved for 20 min at 121°C. About 25 mL was poured in each glass Petri plate separately. The freshly made inoculum for each bacterium was swabbed on a solid media. Then, the sample solution was poured on filter paper discs and carefully placed on their assigned positions in Petri plates. One disc was used as a positive control. The plates were placed in the incubator for 24 h at 37°C. The zones of inhibition caused by the test samples in the bacterial cultures were measured. The test was performed in triplicate for each sample. Cefotaxime solution in DMSO (4 mg/mL; 20 μg/disc) was used as the positive control, whereas DMSO was taken as the negative control.

The antifungal effect of the extracts was evaluated against four fungal strains, i.e., Fusarium solani (FCBP 0064), Mucor species (FCBP 0300), Aspergillus flavus (FCBP 066), and Aspergillus niger (FCBP 0198) by disc diffusion method.[19] In brief, the sample (20 mg/mL) in DMSO was prepared as a stock solution. Dextrose agar media was prepared and pH was adjusted (pH = 6.5) and autoclaved. The media was poured in each Petri plate and once solidified, the fungal strains were swabbed cautiously on the media surface of each plate. Then, 5 μL of each sample solution was poured on the surface of filter paper disc and the discs were placed carefully on its assigned place. The Petri plates were placed in an incubator for 24 h at 28°C. Then, the zone of inhibition was measured using a Vernier caliper. Terbinafine (4 mg/mL; 20 μg/disc) was used as the positive control, whereas the solvent (DMSO) was taken as the negative control. The test was performed in triplicate for each sample.

Brine shrimp lethality assay

The cytotoxic potential of methanolic extract was evaluated by brine shrimp lethality bioassay using Artemia salina eggs (Ocean Star International Inc., Snowville, UT, USA) as described previously with slight modifications.[20],[21] From the stock solution, three dilutions of 1000, 100, and 10 μg/mL were prepared to obtain the concentrations of 10, 1, and 0.1 μg/mL, respectively. Doxorubicin was used as reference, and DMSO was used as a negative control. The eggs were hatched in continuous oxygen supply for 24 h. Ten shrimps were transferred to each well of 96- well plate by using a Pasteur pipette under a ×3 magnifying glass. The samples were applied in triplicate using 0.5, 1.5, and 3.0 μL. The 96- well plate was placed in an incubator for 24 h. Then, the shrimps were removed from the wells by using the Pasteur pipette, and survivors of each well were counted. Terbinafine (10 μg/mL, 1 μg/mL, and 0.1 μg/mL in DMSO) was used as the standard drug.

In-vitro antileishmanial assay

The in-vitro antileishmanial assay was performed by using protocols reported by Pulivarthi et al. with slight modifications.[22]Leishmania tropica ( KWH23) strain was incubated at 24°C for 7 days in 199 medium consisting of about 10% bovine serum. A stock solution of 4000 ppm was used. In the 96- well plate, the stock solution was serially diluted. Amphotericin B was used as a positive control, whereas DMSO was used as a negative control. The 96- well plate was incubated at 24°C for 72 h. After 3 days, the test culture (~15 μL) was then transferred to improve Nubauer counting chamber, and live promastigotes were counted under light microscope. Percentage mortality was calculated, and the experiment was performed in triplicate.


   Results and Discussion Top


Antioxidant assays

The crude methanolic extract showed 63% DPPH scavenging effect, however, the fractions demonstrated interestingly differential percentage of antioxidant effects. In DPPH free radical assay, all fractions were examined for scavenging activity. It was observed that contents in methanolic extract of the selected plants were observed to be potent DPPH reducing agents. The crude methanolic extract showed 63% DPPH scavenging effect, however, the maximum DPPH radical scavenging activity was exhibited by fraction SA9 (80.7%). The lowest DPPH radical scavenging activity was shown by fraction SA6 (23.7%). All the other fractions showed DPPH reducing activity in the range of 78.9%–

80.0% when compared with ascorbic acid. Our findings are in line with those of a previous study carried out for the antioxidant activity of fractions of methanolic extract of Silybum species having significant DPPH value.[23] The obtained fractions were having different proportions of phenolic contents, which might vary in the number and the position of hydroxyl groups. This variation might affect the DPPH % scavenging activity.[24] The unit for showing antioxidant potential was % scavenging/mg. The results of the analyzed samples are depicted in [Figure 1].
Figure 1: Antioxidant, total phenolic, and flavonoid contents of the fractions of Silybum marianum seed extract

Click here to view


In total antioxidant capacity assay, the crude methanolic extract presented statistically significant antioxidant capacity (P < 0.05) (73.47 μg/mg), whereas the fraction SA4 demonstrated maximum antioxidant capacity which was 101.81 μg/mg followed by other fractions SA2 (93.85 μg/mg), SA3 (89.76 μg/mg), and SA8 (89.12 μg/mg). SA5 exhibited the least total antioxidant capacity (34.91 μg/mg). These results are in line with those of a previous study where Silybum species showed significant antioxidant activity.[25] To the best of our knowledge, it is the first study to evaluate the different activities on various fractions of SM extract eluted with solutions of different polarities. The total antioxidant capacity was expressed in ascorbic acid equivalent/mg (AAE/mg). The results of the tested fractions are depicted in [Figure 1].

In reducing power assay, the crude extract showed statistically considerable activity (138.33 μg/mg), while the fraction SA4 (258.93 μg/mg) showed the maximum reducing power followed by fractions SA2 (250.90 μg/mg), SA5 (248.26 μg/mg), and SA7 (245.10 μg/mg). The lowest reducing power was shown by fractions SA5 (104.49 μg/mg) and SA6 (103.77 μg/mg). Our findings are relevant with those of a previous study, in which only a single extract with 4.77% of phenolic content was analyzed.[23] However in our study, we examined the reducing power assay of ten different fractions with different phenolic concentrations. The unit AAE/mg was used to express the reducing power. The tested sample results are depicted in [Figure 1].

Phytochemical analysis

Total phenolic contents

In the following results, the TPC was found maximum in fraction SA7 (85.13 μg/mg) followed by fractions SA10 (68.37 μg/mg) and SA1 (68.14 μg/mg). Fraction SA3 (40.03 μg/mg) showed the least phenolic content. The findings of the current study are in support with those of a previous report showing varied phenolic contents in ethyl acetate, methanol, and ethanolic extracts in Iraqi species of SM.[26] TPCs were expressed in gallic acid equivalents (GAE). The results of the tested samples in species obtained from Haripur, Pakistan, are depicted in [Figure 1].

Total flavonoid contents

In this assay, TFCs were found maximum for fraction SA1 (58.24 μg/mg) followed by fractions SA3 (56.48 μg/mg), SA7 (55.16 μg/mg), and SA10 (53.92 μg/mg). The least TFC was found in fraction SA5 (40.61 μg/mg). Our results are in strong agreement with the findings obtained by Sun et al., who studied that higher phenolic content was present in seeds (17.10 mg/g dry weight) as compared to roots (15.134 mg/g dry weight).[27] The elevated flavonoid contents in our sample might be due to the species that was obtained from Haripur, Pakistan, and it is the first study to examine the properties of this species gathered from this locality. TFCs were expressed as QE/mg. The results of the analyzed samples are depicted in [Figure 1].

Phytochemical screening tests

In case of qualitative screening of phytochemicals, all methanolic extract fractions (SA1–SA10) showed significant result and indicated the presence of alkaloids, saponins, terpenoids, and tannins. Our findings are in correlation with a previous study that showed the presence of phytochemicals in MESM.[28] The results are summarized in [Table 1].
Table 1: Phytochemical screening of methanolic fractions from seeds of Silybum marianum

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Antibacterial assay

In antibacterial assay, disc diffusion method was applied. The result of crude extract demonstrated significant antibacterial activity with 6 mm, 9 mm, 5 mm, and 11 mm zone of inhibitions for S. aureus, E. coli, E. aerogens, and B. subtilus, respectively. Most of the fractions showed poor activity against bacterial strains which were used in this assay except the fractions SA9 (10 mm in case of S. aureus), SA7 (11 mm in case of E. coli), and SA7 (9 mm in case of E. aerogenes) which showed mild zone of inhibition. In case of Bacillus species, all fractions showed no activity against B. subtilis as shown in [Figure 2]. The results of our study are in partial agreement with a previous study that showed mild or non-significant activity against Gram-negative and Gram-positive bacteria.[28] The results of the tested samples on the four different bacteria are depicted in [Figure 2].
Figure 2: Antibacterial effect of the fractions of Silybum marianum seed extract. *The statistical difference between the treatment and the control groups at P < 0.05

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Antifungal assay

In antifungal activity assay, the extract displayed significantly poor antifungal effect on the tested fungal. Whereas, most of the fractions (SA1–SA10) showed no activity against selected fungal strains, but maximum activity was found against Mucor species (8 mm) and A. niger (8 mm). Our findings are in correlation with a previous study that showed non-significant zone of inhibition against fungal strains.[28] All results from sample fractions are depicted in [Figure 3].
Figure 3: Antifungal potential of the fractions of Silybum marianum seed extract. *The statistical difference between the treatment and the control groups at P < 0.05

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Cytotoxicity assay (brine shrimp lethality assay)

Results of the cytotoxic assay showed that the crude extract was found to be poorly toxic on brine shrimp with LD50311 μg/mL. Whereas, the lowest LD50 was found in fraction SA9 (49.99 μg/mL) followed by fraction SA5 (68.55 μg/mL). LD50 values were found highest in case of fraction SA6 (211.74 μg/mL). Our findings are in line with those of another study report on cytotoxic activity that showed significant results.[29] The findings of our study were presented as % cytotoxicity as depicted in [Figure 4].
Figure 4: Brine shrimp lethality bioassay of the fractions of Silybum marianum seed extract. *The statistical difference between the treatment and the control groups at P < 0.05

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In-vitro antileishmanial assay

In case of antileishmanial activity assay, the crude extract exhibited statistically significant (P < 0.05) activity with 68% inhibition. However, the fraction SA6 was found more significant and mortality rate was 84%, followed by fractions SA8 (80%), SA9 (75%), and SA7 (73%). The least mortality rate was found for fraction SA1 (12%). These findings correlated with those of a previous report that methanolic extract of Sonchous cornatus showed significant % mortality against antileishmanial parasite.[30] The tested sample results are depicted in [Figure 5].
Figure 5: Antileishmanial activity of the fractions of Silybum marianum seed extract. *The statistical difference between the treatment and the control groups at P < 0.05

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   Conclusion Top


In this study, phytochemical and pharmacological investigation of SM seed extract was undertaken. The results demonstrated an interesting phenomenon between the crude extract and its constituent fractions. In general, the extract has lower effect in the tested biological assays than compared to the fractions. In all the biological tests, the fractions were more pronounced than the extract. In addition, the results of the present study demonstrated that fractions of MESM seeds, prepared using column chromatography, demonstrated abundant levels of total phenols and flavonoids. This could probably be attributed to the potential antioxidant property of the extract. However, due to the difference in the polarity, different fractions revealed differential antioxidant properties. Moreover, the fractions exhibited remarkable antibacterial and cytotoxic activities. Generally, all the tested fractions display strong antileishmanial effect. The results revealed that all the tested fractions showed differential potentials in the biological assays tested, which indicates the presence of different types of bioactive phytoconstituents in extracts. With this background, it can be concluded that the extract of SM seeds can be a good source of therapeutic agents. However, further study is required to confirm the effect of the extract using suitable preclinical and clinical models.

Acknowledgements

The authors are thankful to Higher Education Commission of Pakistan and COMSATS University Islamabad for providing the research facilities and technical support for the successful accomplishment of the project.

Financial support and sponsorship

This study was funded by the Deanship of Scientific Research (DSR), Taibah University, Kingdom of Saudi Arabia, under Research Project Grant No. 438-30191.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Duan L, Carrier DJ, Clausen EC. Silymarin extraction from milk thistle using hot water. Appl Biochem Biotechnol 2004;113-116:559-68.  Back to cited text no. 1
    
2.
Das S, Mukherjee S, Vasudevan D. Medicinal properties of milk thistle with special reference to silymarin – An overview. Indian J Nat Prod Resour 2008;7:182-92.  Back to cited text no. 2
    
3.
Flora K, Hahn M, Rosen H, Benner K. Milk thistle (Silybum marianum) for the therapy of liver disease. Am J Gastroenterol 1998;93:139-43.  Back to cited text no. 3
    
4.
Abenavoli L, Capasso R, Milic N, Capasso F. Milk thistle in liver diseases: Past, present, future. Phytother Res 2010;24:1423-32.  Back to cited text no. 4
    
5.
Bhattacharya S. Phytotherapeutic properties of milk thistle seeds: An overview. J Adv Pharm Educ Res 2011;1:69-79.  Back to cited text no. 5
    
6.
Jindal R, Sinha R, Brar P. Evaluating the protective efficacy of Silybum marianum against deltamethrin induced hepatotoxicity in piscine model. Environ Toxicol Pharmacol 2019;66:62-8.  Back to cited text no. 6
    
7.
Vilahur G, Casaní L, Peña E, Crespo J, Juan-Babot O, Ben-Aicha S, et al. Silybum marianum provides cardioprotection and limits adverse remodeling post-myocardial infarction by mitigating oxidative stress and reactive fibrosis. Int J Cardiol 2018;270:28-35.  Back to cited text no. 7
    
8.
Mohammadi Arvanag F, Bayrami A, Habibi-Yangjeh A, Rahim Pouran S. A comprehensive study on antidiabetic and antibacterial activities of ZnO nanoparticles biosynthesized using Silybum marianum L seed extract. Mater Sci Eng C Mater Biol Appl 2019;97:397-405.  Back to cited text no. 8
    
9.
Tachakittirungrod S, Okonogi S, Chowwanapoonpohn S. Study on antioxidant activity of certain plants in Thailand: Mechanism of antioxidant action of guava leaf extract. Food Chem 2007;103:381-8.  Back to cited text no. 9
    
10.
Padmanabhan P, Jangle SN. Evaluation of DPPH radical scavenging activity and reducing power of four selected medicinal plants and their combinations. Int J Pharm Sci Drug Res 2012;4:143-6.  Back to cited text no. 10
    
11.
Shaikh RU, Pund MM, Gacche RN. Evaluation of anti-inflammatory activity of selected medicinal plants used in Indian traditional medication system in vitro as well as in vivo. J Tradit Complement Med 2016;6:355-61.  Back to cited text no. 11
    
12.
Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. LWT Food Sci Technol 1995;28:25-30.  Back to cited text no. 12
    
13.
Tabart J, Kevers C, Pincemail J, Defraigne JO, Dommes J. Comparative antioxidant capacities of phenolic compounds measured by various tests. Food Chem 2009;113:1226-33.  Back to cited text no. 13
    
14.
Marinova D, Ribarova F, Atanassova M. Total phenolics and total flavonoids in Bulgarian fruits and vegetables. J Univ Chem Technol Metall 2005;40:255-60.  Back to cited text no. 14
    
15.
Evans WC. Trease and Evans' Pharmacognosy International Edition E-Book: Elsevier Health Sciences; 2009.  Back to cited text no. 15
    
16.
Odebiyi OO, Sofowora EA. Phytochemical screening of Nigerian medicinal plants II. Lloydia 1978;41:234-46.  Back to cited text no. 16
    
17.
Gracelin DH, Britto A, Kumar B. Qualitative and quantitative analysis of phytochemicals in fve Pteris species. Int J Pharm Pharm Sci 2013;5:105-7.  Back to cited text no. 17
    
18.
Zaidan MR, Noor Rain A, Badrul AR, Adlin A, Norazah A, Zakiah I. In vitro screening of five local medicinal plants for antibacterial activity using disc diffusion method. Trop Biomed 2005;22:165-70.  Back to cited text no. 18
    
19.
Karaca N, Koç AN. In vitro susceptibility testing of dermatophytes: Comparison of disk diffusion and reference broth dilution methods. Diagn Microbiol Infect Dis 2004;48:259-64.  Back to cited text no. 19
    
20.
Inayatullah S, Irum R, Rehman AU, Chaudhary FM, Mirza B. Biological evaluation of some selected plant species of Pakistan. Pharm Biol 2007;45:397-403.  Back to cited text no. 20
    
21.
Rehman AU, Mannan A, Inayatullah S, Akhtar MZ, Qayyum M, Mirza B. Biological evaluation of wild thyme (Thymus serpyllum). Pharm Biol 2009;47:628-33.  Back to cited text no. 21
    
22.
Pulivarthi D, Marie Steinberg K, Monzote L, Pinon A, Setzer W. Antileishmanial Activity of compounds isolated from Sassafras albidum. Nat Prod Commun 2015;10:1229-30.  Back to cited text no. 22
    
23.
Wojdylo A, Oszmianski J, Czemerys R. Antioxidant activity and phenolic compounds in 32 selected herbs. Food Chem 2007;105:940-9.  Back to cited text no. 23
    
24.
Okawa M, Kinjo J, Nohara T, Ono M. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of flavonoids obtained from some medicinal plants. Biol Pharm Bull 2001;24:1202-5.  Back to cited text no. 24
    
25.
Anthony KP, Saleh MA. Free radical scavenging and antioxidant activities of silymarin components. Antioxidants (Basel) 2013;2:398-407.  Back to cited text no. 25
    
26.
Noor HA, Wafaa MA. Preliminary phytochemical screening and in vitro evaluation of antioxidant activity of Iraqi species of Silybum marianum seeds. Int Res J Pharm 2014; 5:378-83.  Back to cited text no. 26
    
27.
Sun J, Li X, Yu X. Polysaccharides, total flavonoids content and antioxidant activities in different parts of Silybum marianum L. plants. In AIP Conference Proceedings. AIP Publishing; 2017.  Back to cited text no. 27
    
28.
Shah SM, Khan FA, Shah SM, Chishti KA, Pirzada S, Khan MA, et al. Evaluation of phytochemicals and antimicrobial activity of white and blue capitulum and whole plant of Silybum marianum. World Appl Sci J 2011;12:1139-44.  Back to cited text no. 28
    
29.
Alowonle OT. Phytochemical investigation and brine shrimp lethality assay of extracts of Picralima nitida (Apocynaceae) staph. seeds. Toxicology 2014;2:11-5.  Back to cited text no. 29
    
30.
Tahir AE, Ibrahim AM, Satti GM, Theander TG, Kharazmi A, Khalid SA. The potential antileishmanial activity of some Sudanese medicinal plants. Phytother Res 1998;12:576-9.  Back to cited text no. 30
    


    Figures

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