Identification of bioactive ingredients in Chenopodium murale L Chenopodiaceae by HPLC and GC/MS

 

Rasha M.M. Mohasib1, A.Nagib2, A.Abdel Samad2, Zeinab A. Salama1*, Alaa A.Gaafar1 ,

Hanan A.A.Taie1, Sameh R. Hussein3

1Plant Biochemistry Department, National Research Centre, Dokki, Giza, Egypt.

2Biochemistry Departement, Faculty of Agricultural, Cairo university, Dokki, Giza, Egypt.

3Department of Phytochemistry and Plant Systematics, National Research Centre,

33 El Bohouth St., Dokki, Giza, Egypt.

*Corresponding Author E-mail: dr.zeinabsalama70@gmail.com

 

ABSTRACT:

This study aims to evaluate the successive extraction of the active ingredients and their antioxidant activity, anti-arthritic as well as anticancer activity of aerial parts (stem, leaves, and flowers) of Chenopodium murale L. Therefore, C. murale plants were extracted using four solvents with a wide range of polarities: n-hexane, ethyl acetate, methanolic and aqueous extracts. Chemical analysis proved it to be a potential source of protein, fat, carbohydrates, the results showed the percentages were: moisture content (92.45%), ash content (18.19%), crude protein (30.42%), crude lipid (2.86%), and carbohydrate (48.3%) respectively of the C.murale. Methanol extracts showed the highest content of total phenolic (TP), total flavonoid (TF), and total tannin (TT). The active ingredients were assessed as well employing gas chromatography coupled to mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). The HPLC analysis of phenolic compounds confirmed that the methanol extract of C.murale detected high amounts of coumarin, 3, 4, 5 methoxy-cinnamic, and pyrogallol respectively. The ethyl acetate extract of C.murale herb displayed a rise cytotoxic effects on MCF7 (89.30 %), aqueous HCT116 (81%), methanol (60.70%) as well as n-hexane (39.80) respectively at 37°C for 48 h of exposure and concentration 100 µg/ml. In anti-arthritic activity at a dose-dependent, the Ethyl acetate successive fraction proved to be the most significant where it produced a percentage of inhibition ranging from 51.73 to 95.77 % followed by methanol fraction 47.70 to 90.02 % at (P 0.05), compared to Diclofenac as standard 91.22 to 96.44 %. Thus, our findings highlight the potential of this plant for its possible clinical use to oppose malignancy development against especially breast and colon cell lines with anti-arthritic effects as a bioagent in pharmaceutical industries.

 

KEYWORDS: Chenopodium murale; HPLC; GC/MS; antioxidant; anti-proliferative, anti-arthritic activities.

 

 

 

INTRODUCTION:

The Chenopodiaceae is a family compromised from 102 genera and 1400 species1. In general, the genus Chenopodium contains collections of weedy herbs nearly more than 200 species native to Europe, America, and Asia2,3. Chenopodium murale L. commonly called nettle leaf goosefoot is an essential annual herbaceous weed that grows in waste places4,5. It is green, alternate, broad lobed ad stalked long or short leaves with forked stem6.

 

From the phytochemical point of view, the plants of Chenopodium L were reported to possess numerous medicinal activities used in popular medicine according to its variety of structural patterns of phytochemicals, theses chemical compounds are primary metabolites such as amino acids and proteins and secondary metabolites7, such as terpenoids8,9, sterols10, saponins11, alkaloids12, vitamins13, Flavonoids and cumarine14,15.

 

A wide range of applications is well-known in folk medicine for the treatment of cough, abdominal pain, pulmonary obstruction, and have diuretic ad laxative activity16, as well as it is reputed to be good pharmacological activities of Chenopodium species such as anthelmintic, anti-inflammatory activity17, antimicrobial18, antioxidant19,20. The Chenopodium extracts could be used as a readily accessible source of natural antioxidants and may be used in the pharmaceutical industry and for food supplements production21,22, antiviral23 and hypotension effect16, analgesic and spasmolytic.24,25.

 

THE AIM OF STUDY:

1.     Using some cheap medicinal plants as a source of bioactive ingredients and their use as natural antioxidants, anti-arthritic and antitumor agents.

2.     2-Biogided fractionation of plant and investigate the biological importance of each fraction.

 

MATERIALS AND METHODS:

1. Plant Material:

The plants of Chenopodium murale were collected from the farm, faculty of Agriculture, Cairo University in November 2016. The harps were identified by Prof. Dr. Mohamed El Khateeb, Ornamental Department faculty of Agriculture, Cairo University. The leaves were used for the extraction of active constituents which was followed by isolation, purification, and identification of each compound present in the extract.

 

2. Solvents:

n-Hexane, ethyl acetate, methanol, Dimethyl sulfoxide (DMSO), Hexa deutro dimethyl sulphoxide (DMSO-d6), n-Butanol, Acetic acid, Aluminum chloride, Benzene. All solvents were of analytical grade (from Sigma-Aldrich), purified and distilled.

 

3. Chemicals:

All chemicals in the present study were of high analytical grade. 2,2′-azinobis (3-ethylebenzothiozoline-6-sulphonic acid) (ABTS·+), Potassium persulfate, (Folin–Ciocalteau reagent, Gallic acid, Quercetin,2,2-diphenyl-1-picrylhydrazyl (DPPH), 3-(2-Pyridyl)-5,6-bis(4-phenol sulfonic acid)-,1,2-triazine (Ferrozine), Butylated hydroxytoluene (BHT), 6-hydroxy-2,5,7,8-tetramethylchroman 2-carboxylic acid (Trolox), potassium ferricyanide, Trichloroacetic acid, concentrated sulfuric acid , MTT 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide. (MTT) were purchased from Sigma Chemical Co.( St. Louis, MO, USA).

 

4. Preparation of Plant Material:

The basic steps include drying of plant materials and grinding the plant sample to obtain a homogenous sample.

 

5. Chemical studies:

The moisture, ash, total nitrogen, crude protein, total lipid, and total carbohydrate were determined according to AOAC 26.

6. Extraction of bioactive compounds:

The freshly Chenopodium murale leaves were dried in the air and protected from direct sunlight and mechanically grounded to powder (2.6212 kg and 1.397 kg, respectively). The powder of leaves was macerated with organic solvents, namely n-hexane solvent (non-polar), followed by ethyl acetate (semi-polar), methanol (polar), and ended with water (polar). The leaves were macerated with n-hexane solvent, filtered and the filtrate obtained was concentrated in a rotary evaporator vacuum to obtain concentrated n-hexane extracts. The pulp portion was macerated with ethyl acetate solvents, filtered and the filtrate was concentrated to obtain concentrated ethyl acetate extracts. The pulp portion was re-macerated with methanol solvents, filtered and the filtrate was concentrated to obtain concentrated methanol extracts. Furthermore, the pulp portion was re-macerated with water, filtered and the filtrate was concentrated to obtain concentrated methanol extracts. The maceration processes lasted for 48 hours, with three times solvent additions. Finally, n-hexane, ethyl acetate, methanol, and water extracts concentrated on the Chenopodium murale leaves were prepared, respectively according to27

 

7. Secondary metabolites analysis:

a. Determination of total phenolic compound content:

The total phenolic (TP) was determined by Folin Ciocalteu reagent assay at 750 nm by spectrophotometer (Unicum UV 300), using Gallic acid as a standard. Total phenolics were expressed as mg Gallic acid equivalents (GAE)/g dry weight. Samples were analyzed in triplicates according to28

 

b. Determination of total tannin content:

Total tannin (TT) of chenopodium murale extracts was measured by Folin Ciocalteu`s reagent at 775 nm by spectrophotometer (Unicum UV 300), using tannic acid as a standard (13). Total tannins were expressed as mg tannic acid equivalent (TAE)/g dry weight as described by29,30

 

c. Determination of total flavonoid content:

Total flavonoid (TF) of chenopodium murale extracts was determined by the aluminum chloride method at 510 nm by spectrophotometer (Unicum UV 300), using quercetin as a standard. Total flavonoids were expressed as mg quercetin equivalents (QE)/ g dry weight according to31.

 

d. Identification and quantification of phenolic and flavonoid compounds:

Identification and quantitation of phenolic compounds by HPLC The dried crude methanolic extract (10 mg) of Brassica vegetable residues were dissolved in 2 ml methanol. HPLC spectral grade by vortex mixing for 30 min. The HPLC system is Agilent 1100 series coupled with a DAD detector following the method of 32. Sample injections of 5 μl were made from auto-sampler. The chromatographic separations were performed on a C18 column (4.6×250 mm, particle size 5 μm). A constant flow rate of 1 ml/min was used with mobile phases: (A) 0.5% acetic acid in distilled water at pH 2.65; and solvent (B) 0.5% acetic acid in 99.5% acetonitrile. The elution gradient was linear starting with A and ending with B over 50 min, using a DAD detector set at wavelength 280 nm. The results expressed as mg phenolic/100 g dry weight.

 

e. Identification of non-polar compound (methylated hexane fraction) by GC/MS (Gas Chromatography/ Mass Spectrometry) Analysis

The bioactive fractions were subjected to GC/MS analysis to identify the non-polar compounds present in the non-polar fraction.

 

Identification of the constituents was carried out by comparison of their retention times and fragmentation patterns of mass with those of published data 33,34and/or with those of the Wiley 9 and NIST 08 mass spectra libraries.

 

8. Biological studies:

a. In vitro antioxidant study:

1. DPPH Scavenging Activity:

Antioxidant activity Preparation of extracts:

The dried crude extracts (10mg) were dissolved in 10 ml methanol by vortex mixing for 30 min. for all assays. DPPH· Free radical scavenging activity of vegetable residue extracts at different concentrations (50, 100, 150, 200µg/ml) was measured spectrophotometrically (Unicum UV 300) at 515 nm according to35.

 

The capacity to scavenge the DPPH∙ radical was calculated using the following equation: DPPH∙ scavenging activity % = [(Ac – As (/ Ac] × 100 where: (Ac) was the absorbance of the control reaction and (As) the absorbance in the presence of the extracts. The results were expressed as IC50 (the concentration µg/ml of the C.murale extracts that scavenge 50 % of DPPH∙ radical).

 

2. ABTS·+ Scavenging Activity:

To estimate the total antioxidant activity of plant extracts by assay of radical cation 2,2′-azinobis (3-ethylebenzothiozoline-6-sulphonic acid) (ABTS·+) according to36

 

3. Reducing power assay:

The reducing power of chenopodium murale extracts at different concentrations (25, 50, 75, 100 µg/ml) was assayed spectrophotometrically (Unicum UV 300)) as described by37. The results were expressed as EC50 (the concentration µg/ml of the chenopodium murale extracts that provided the reading of 0.5 absorbances at 700 nm.

 

4. Chelating activity on Fe2+:

Chelating activities on ferrous ions of extracts or EDTA solution as a positive control at different concentrations (50, 100, 150, 200 µg/ml) was carried out spectrophotometrically (Unicum UV 300) at 562 nm according to38

 

The chelating ability was calculated using the following equation: Chelating activity (Inhibition %) = [(Ac – As) / Ac] × 100 where: (Ac) was the absorbance of the control reaction and (As) the absorbance in the presence of the plant extracts. The results were expressed as IC50 (the concentration µg/ml of the C. murale extracts that.

 

b. In vitro antitumor activity of Chenopodium murale by MTT Cell Viability Assay:

To find out the long-term cytotoxicity of the plant extracts on various cancer cell lines using the colorimetric MTT assay.

 

Cell viability was investigated using MTT 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide. (MTT) is a water-soluble tetrazolium salt yielding a yellowish solution when prepared in media or salt solutions lacking phenol red Dissolved MTT is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by mitochondrial dehydrogenase enzymes in metabolically active cells Dead or non-viable cells do not cause this change? This water-insoluble formazan can be solubilized using isopropanol or other solvents and the dissolved material is measured spectrophotometrically yielding absorbance as a function of the concentration of converted dye39,40.

 

MTT is converted by the succinate dehydrogenase enzyme in the cell, into an insoluble color formazan product. It is solubilized in DMSO and the cell viability depends on its absorbance value. The absorbance of untreated cells was taken as the 100 % viable cells.

 

c. In Vitro Anti-arthritic Activity:

Arthritis is developed as a symptom of chronic inflammation especially in the body joints. Rheumatoid arthritis is one of the most important rheumatic diseases. It is relatively common, and its serious forms can cause severe disability. The in vitro anti-arthritic activity was determined by the inhibition of the protein denaturation method by41

 

9. Statistical analysis:

Data were statistically analyzed using Costat statistical package data according to42

 

Table 1: Proximate analysis of C. murale

Plant

Moisture (F Wt)

Ash

Crude Protein

Crude Fat

Total carbohydrate

C. murale

g/100 g

92.45±0.69

18.19±4.05

30.42±0.31

2.86±0.80

48.53±3.56

 

 

RESULTS AND DISCUSSION:

1. Proximate analysis:

The results of the proximate analysis in (%) of C. murale are displayed in (Table 1). The moisture content of C. murale (92.45%) was higher than that found43,44 (82.02%) in C. murale and (86.7 %) of Chenopodium album based on fresh weight which may be advantageous given increasing the sample’s shelf life.

 

The moisture content of foods or their processed products indicates its freshness and shelf life, and high moisture content subjects food items to increased microbial spoilage, deterioration, and short shelf life45. The ash content is a reflection of the number of mineral elements present in the samples; the ash content of C. murale was (18.19%) which was higher than the values obtained for the leaves C. ambrosiodes (17%)46. On the other hand, our results were lower 21% than that found by47 based on the dry weight. Protein is an essential component of the diet needed for survival for animals and human beings to supply adequate amounts of essential amino acids. The crude protein content in C. murale (30.42%) was in the range that was obtained was obtained by44 (29.30%, 28.55%, 26.50%, and 31.20%) which characterized by different techniques Sun-dried, Solar dried, Shade dried and Cabinet dried and was similar that was found by46 for Chenopodium ambrosiodes. The fat content in C. murale (2.86%) was lower than that found by47,48 based on the dry weight. The total carbohydrates content in C. murale (48.53%) Similar results (46.94 %, 49.19 %, 54.43 %, and 44.26 %) were obtained by44 in dried leaves of Chenopodium album by different techniques Sun-dried, Solar dried, Shade dried and Cabinet dried.

 

The yield of different fractions of C.murale:

The percent yield from different fractions of C.murale is depicted in (Table 2). Since methanol and aqueous which has the polarity index of around 5.1 and 9.0 yielded a much higher yield 6.35% and 4.93%. They are the best solvent system for enhanced the solubility of both the methoxylated and hydroxylated compounds and hence improved the overall extraction yield of an entire group of compounds, this could be due to the higher polarity. While, ethyl acetate yielded 1.90% due to having a moderate polarity which holds semi-polar compounds such as terpenes, phenolics, flavonoids, glycosides compounds. Partitioning with n-hexane which yielded 1.50% separated non-polar molecules, however, methanol fraction holds polar molecules like sugars, glycosides, alkaloids. It has been validated through the current assessment that extraction yield and recovery of bioactive constituents are depending on the solvent polarity, extraction technique, and extraction time.49

 

 

2. Phytochemical screening of successive extracts of C. mura:

Table 2: Phenolic compounds, antioxidant and anticancer activities of different C. murale fractions

Fractions

Phytochemicals

Antioxidant activities

Anti-proliferative activities

TP mg/g DW

TF mg/g DW

TT mg/g DW

Yield %

DPPH

IC50 µg/ml

ABTS•+

IC50 µg/ml

Fe-Chelating

IC50 µg/ml

Reducing power

EC50 µg/ml

Remarks % at 100 µg/ml

MCF-7

HCT-116

HepG2

Hexane

1.10a

 ± 0.01

1.51a

 ± 0.03

0.69a

 ± 0.00

1.50

371.47d

 ± 12.28

479.52d

 ± 16.35

395.65d

± 1.65

321.50e

 ± 0.50

39.80

0.00

34.03

Ethyl acetate

3.15b

± 0.08

3.11b

 ± 0.09

1.58b

 ± 0.07

1.90

111.72b ± 1.69

183.31d

± 7.02

269.86b

± 0.13

179.95b

 ± 6.91

89.30

71.00

2.70

Methanol

16.66d

 ± 0.30

8.78d

 ± 0.18

9.19d

± 0.43

6.35

1636.84e ± 53.81

103.36b

± 0.42

445.14e

± 1.21

201.56c

 ± 1.25

60.70

8.30

19.90

Aqueous

6.38c

± 0.10

3.44c

 ± 0.05

3.36c

± 0.05

4.93

236.34c

± 8.14

175.53c

 ± 1.34

349.04c

 ± 0.25

262.33d

 ± 2.52

81.00

68.30

18.10

BHT Standard

-

-

-

-

15.22a

± 0.67

-

-

21.84a

± 0.79

-

-

-

Trolox standard

-

-

-

-

-

16.66a

 ± 1.39

-

-

-

-

-

EDTA standard

-

-

-

-

-

-

30.19 a

± 0.37

-

-

-

-

LSD at 0.05

0.26

0.23

0.44

-

49.36

16.09

3.74

6.37

-

-

-

All values are the mean of three replicates ± S.D. Values with different letters are significantly different at p ≤ 0.05.

 

 

Solvent polarity plays a key role in the extraction of the plant bioactive compounds and increasing their contents solubility. As shown in (Table 2) n-hexane, ethyl acetate, methanol, and aqueous extracts are different solvents that are mainly used for phenolic and flavonoid and tannins extraction from plants, food or vegetables50,51. This result was confirmed by52 who proved that the leaves extracts of the C.murale plant contain a high percentage of phenolic compounds, especially phenolic and flavonoids. Preliminary phytochemical analysis of different fractions of C. murale revealed the presence of phenolic, flavonoids, and tannins. These results revealed that most of the medicinal plants have quite a several chemical constituents, which may be responsible for pharmacological actions like hypolipidemic53 antipruritic54, anthelmintic activity,55 and antimicrobial56, anti-inflammatory57. Among the various solvent used methanol fraction showed the highest values of total phenolic 16.66mg G/g DW, flavonoid 8.78 mg Q E /g DW, and Tannin 9.19mg TE/g DW compared to hexane which showed the lowest values total phenolic 1.10 mg G/gDW, flavonoids 1.51 mg QE/g DW and Tannins 0.69 mg TE/gDW. These results are in a good agreement with58,59 they found that the extract of C. murale is effective as an antioxidant and possess many pharmacological actions antioxidant properties, anti-inflammatory, and anticancer. Even for flavonoids and tannin, the patterns are the same. Methanol extraction efficiency was the best for the extraction of phenolic compounds which could be due to the polarity that is suitable for phenolic compounds as well as the ability to dissolve phenolic compounds from within plant cells60. Previous studies confirmed our results, which found that the total phenolic contents in Chenopodium album varied considerably and ranged from 3.71 to 32.14 mg GAE/g and the total flavonoid contents in Chenopodium album varied considerably and ranged from 0.06 to 0.50 mg of quercetin equivalent/ g61. Phenolics and Flavonoids show a wide range, induction of apoptosis, inhibition of enzymes, and other antibacterial and antioxidant effects62 and, provide the plants with defense mechanisms to neutralize reactive oxygen species ROS) to survive and prevent molecular damage and damage by microorganisms, insects, and herbivores63. It is also known that phenolic compounds of high molecular weight (> 500) are known as tannin compounds64. Results from Table 2 showed that the greatest amount of tannins was obtained from methanol extract higher than those of n-hexane, ethyl acetate and aqueous respectively. The total content of phenols, flavonoids, and tannins varies depending on the different parts of the plant, plant age, and the polarity of the solvent used in the extraction, and their total content also affects their properties as antioxidants and their medicinal and therapeutic properties65.

 

3. HPLC identification and quantification of phenolic and flavonoid compounds of C. murale:

Natural products have always been a preferred choice of all as it plays a great role in discovering new medicines. During extraction, solvents diffuse into the solid plant material and solubilize compounds with similar polarity. Extraction and separation of chemical constituents of plants depend on selective solvents through standard procedure. Fractionation is the best method to separate each group of constituents alone when the plant contains several groups of constituents15.

 

 

Table 3: Identification and quantification of different phenolic and flavonoids compounds of C. murale fractions

Phenolic compounds

µg/g DW

Ethyl acetate

Methanol

Aqueous

Flavonoid compounds µg/g DW

Ethyl acetate

Methanol

Aqueous

Pyrogallol

3.06

57.19

127.69

Apigenin 6-arbinose 8- galactose

182.27

1103.65

332.25

Gallic acid

0.09

1.81

2.50

Narengin

8.24

85.64

10.39

3-OH tyrosol

4.21

17.98

5.97

Rosmarinic

-

-

2.73

Protocatchuic

0.66

4.62

4.22

Luteolin 7-glucose

20.32

580.59

60.02

Catechol

0.59

8.10

4.91

Hesperidin

486.28

4901.76

3381.37

4-Amino-benzoic

0.36

0.42

0.72

Rutin

7.08

123.11

22.17

Catechein

1.46

17.01

7.55

Apigenin-7-glucose

11.21

9.57

6.38

Chlorogenic

5.54

25.37

3.79

Apigenin 7-O-neohespiroside

-

604.53

86.71

p-OH- benzoic

1.30

-

12.14

Quercetrin

23.35

374.80

117.22

Benzoic

4.37

32.55

12.81

Naringenin

4.45

49.45

19.89

Caffeic

0.16

3.45

1.10

Quercetin

16.86

80.64

26.78

Vanillic

-

9.69

3.60

Hespirtin

6.10

14.20

5.03

Caffeine

1.52

12.16

9.29

Kampferol 3-2-p- comaroyl glucose

-

22.93

-

p-Coumaric

0.32

0.66

0.66

Acacetin 7 neo. Rutinoside

24.75

162.32

20.42

Ferulic

1.51

2.33

2.97

Kampferol

4.18

5.84

1.12

Iso-Ferulic

0.83

3.69

4.58

-

-

-

-

α-Coumaric

0.77

14.74

2.38

-

-

-

-

Coumarin

14.56

208.45

13.43

-

-

-

-

3,4,5-Methoxy-cinnamic

5.02

86.82

54.64

-

-

-

-

 

 

HPLC is frequently used for the quantification of polyphenols from plant extracts due to its accuracy, reliability, and repeatability. Quantification of phenolic compounds in plant extract is influenced by the chemical nature of the analytic, as well as assay method, selection of standards, and presence of interfering substances66. Results showed that all extracts contained different compositions and contents of phenolic and flavonoids compounds (Table 3). In the C. murale herb, it was observed that the major phenolic compounds were coumarin (208.45ug / g DW) followed by, 4,5-Methoxy-cinnamic (86.82 µg/gDW in methanol fraction. While, Pyrogallol (127 µg/Kg), 3,4,5 methoxycinnamic (54.64 µg/g DW), and Coumarin (13.43 µg/g DW) were detected in C.murale aqueous fraction (Table 3). Also, remarkable amounts of hesperidin, Apigenin 6 –arabinose 8 galactose, apigenin 7-O neohespiroside, Luteolin 7-glucose, Quercetrin, Acacetin 7 neo. Rutinoside, Rutin, and quercetin were highly detected and quantified in methanol fraction followed by aqueous and ethyl acetate fractions of C. murale (Table 3).

 

Previous studies confirmed our results, they studied the composition of selected phenolic compounds in three wild representatives of the Chenopodioideae which are rich in quercetin glycosides67. Based on our results, we can conclude that methanol and aqueous are more suitable than ethyl acetate for extracting most of the phenolics and flavonoids, in particular, coumarin, hesperidin, Apigenin 6-arbinose 8- galactose acid, and Apigenin 7-O-neohespiroside (Table 3). A wide variety of phenolic and flavonoids of aqueous methanolic extract of C. murale was isolated by HPLC analysis68 for example quercetin, gallic acid, chlorogenic acid, p-coumaric acid, sinapic acid, and kaempferol. (Ahmed et al., 2017)15 reported that the different chromatographic and spectroscopic results revealed the presence of gallic acid and coumarin in the ethyl acetate fraction of C. murale. The chromatographic profile of the phenolic compounds in the C. ambrosioides L., by HPLC–DAD revealed the presence of rutin in the crude extract (12.5 ± 0.20 mg/g), ethyl acetate (16.5 ± 0.37 mg/g), and n-butanol (8.85 ± 0.11 mg/g), whereas quercetin and chrysin were quantified in chloroform fraction (1.95 ± 0.04 and 1.04 ± 0.01 mg/g), respectively69. Therefore, the species of the family Chenopodiaceae are widely distributed weedy herbs often used commercially as spices or drugs because of the presence of useful secondary metabolites8

 

4. The GC/MS analysis of the methylated n-hexane fraction of C. murale:

GC/MS analysis indicated that C. murale hexane fraction after methylation process consists of twenty-one compounds. The probabilities of the structures of the detected compounds are listed in Table (4), where the major peak areas were 9-Octadecenamide (15.97%); 2,6,10,14,18,22-Tetracosahexaene,2,6,10,15,19,23- hexamethyl (CAS) (12.96%);13-Docosenamide, (Z) (12.32%); 6,9, 12 Octadecatrienoic acid, methyl ester (8.12%); Pentadecanoic acid, 14methyl, methyl ester (CAS), (5.82%); 7, 10 Octadecadienoic acid, methyl ester (3.66%). GC/MS results show that C. murale hexane extract contains many bioactive compounds which may be the reason for its biological activity; 9-Octadecenamide and 13-Docosenamide were reported to exhibit anti-inflammatory and antimicrobial, strong antioxidant70, Squalene 2, 6, 10, 14, 18, 22 -Tetracosahexaene, 2, 6, 10, 15, 19, 23 hexamethyl (CAS) (12.96%) has anticancer effects against ovarian, breast, lung, and colon cancer71. Moreover, 6, 9, 12 Octadecatrienoic acid, methyl ester, and Pentadecanoic acid, 14-methyl, methyl ester (CAS), they are fatty acid methyl was reported to possess antifungal and antimicrobial activities72 . This may due to the chemical components of the C. murale fraction acting together and the sensitivity of cancer cells in medicinal herbs. The bioactive compounds have increased bio-functions when they are together and the type of solvent and extraction method play a role in the selective functions.

 

Previous studies mentioned that the most abundant compounds of a non-polar fraction of C. ambrosioides were characterized as vitamin E, squalene, and phytol73. In another studies74 elucidated 11 phytochemical components of C. murale by GC/MS. The highest peak occurred for compound 6 (phytol, acetate), followed by compounds 9 and 10, which belong to the same compound (hepatocosane). The analysis and identification of the chemical components of medicinal herb extracts could help with the discovery of target molecules and the evaluation of their biological functions to create a library for medicinal chemistry targets. The types of solvents (polar and non-polar) and extraction methods produce different potential natural molecules; also, GC/MS analysis is used to identify the available molecules in the extracts. They reported that the C. murale is a garden herb that grows fast, has biological functions, and produces various bioactive compounds that depend on the environmental changes, soil type, solvents, and extraction methods. The selective technique for extraction of bioactive compounds may accelerate the steps to isolate and identify active natural compounds from C. murale or other medicinal herbs.

 

 

Table 4: Main compounds identified in the methylated hexane fraction of C. murale by GC/MS.

S. N.

Rta

Compound name

Area %b

Molecular formula

1

8.35

4,13,20 Tri-O-methylphorbol 12 acetate

0.64

C25H36O7

2

8.64

1-Butanol-4-butoxy-(CAS)

0.74

C8H18O2

3

12.24

10,13 Dioxatricyclo [7.3.1.0 (4,9)] tridecan 5 ol 2 carboxylic acid, 4 methyl 11(1propenyl),methyl ester

0.72

C17H26O5

4

13.03

Lucenin 2

1.04

C27H30O16

5

13.56

Pentadecanoic acid, 14methyl, methyl ester (CAS)

5.82

C17H34O2

6

14.77

Hexadecanoic acid, octadecyl ester

0.37

C34H68O2

7

16.36

Colchifoleine

0.67

C21H23NO7

8

17.36

7, 10 Octadecadienoic acid, methyl ester

3.66

C19H34O2

9

17.50

6,9,12 Octadecatrienoic acid, methyl ester

8.12

C19H32O2

10

19.38

1-O- Retinoyl á- D-methyl-2', 3' 4'-tri- O-acetyl

glucopyranuronate

2.25

C33H44O11

11

21.58

Glycodeoxycholic acid

0.41

C26H43NO5

12

23.08

9-Octadecenamide

15.97

C18H35NO

13

23.52

Hexanedioic acid, dioctyl ester

0.49

C22H42O4

14

23.61

Agaricic acid

2.30

C22H40O7

15

26.50

1, 2-Benzene di carboxylic acid, di octyl ester

2.51

C24H38O4

16

26.61

3',8,8'-Trimethoxy-3-piperidyl-2,2'-binaphthalene-1,1',4,4' -tetrone

0.61

C28H25NO7

17

27.12

Dihydrositsirikine

0.63

C21H28N2O3

18

27.22

1',1'-Dicarboethoxy-1á, 2á- dihydro3'-Hcycloprop [1,2] cholesta-1,4,6- trien3one

0.90

C34H50O5

19

30.80

13-Docosenamide,(Z)

12.32

C22H43NO

20

31.52

2,6,10,14,18,22- Tetracosahexaene, 2,6,10,15,19,23-hexamethyl(CAS)

12.96

C30H50

21

36.01

Stearic acid, 3-(octadecyloxy) propyl ester (CAS)

0.56

C39H78O3

aRt: retention time (min).

be percentage composition was computed from the gas chromatography peak areas

 

 

 

5. Biological Studies:

5.1. Antioxidant assays:

5.1.1. DPPHscavenging activity:

The antioxidant ability and radical scavenging properties of C.murale herp is associated with its medicinal values. In this study, the antioxidant activities of Arial parts (stem, leaves and flowers) of C.murale L. in different four extracts were measured using four different assay methods including DPPH, ABTS•+, metal chelation effect in the Fe2+-ferrozine test system and reducing power activity (Table 2). Free radicals of 1, 1-diphenyl 1-2-picrylhydrazyl (DPPH) are widely used for screening medicinal plants to investigate their antioxidant potential. To evaluate the in-vitro antioxidant activity of C. murale, the DPPH radical scavenging activity of the C. murale fractions was measured and compared with BHT as a positive control.

 

Regarding the DPPH assay, the antioxidant capacity (IC50) is ranged from 111.72 to 1636.84 µg/ml. Ethyl acetate fraction had the highest DPPH scavenging capacity (111.72 µg/ml). While the lowest scavenging capacity (1636.84 µg/ml) was given in C. murale methanol fraction. These results are in good agreement with 71, 75, 76, who found that the extract of C. murale is effective as an antioxidant and its health-protective potential due to its capability of directly reacting and quenching DPPH radicals77.

 

5.1.2. ABTS•+ scavenging activity:

The ABTS•+ assay depends on the antioxidant ability to scavenge ABTS•+ radical77. According to the current findings in Table (2) the free radical scavenging ability of the different four fractions of C.murale was also determined. Various fractions of C. murale, as well as the positive control Trolox, revealed considerable differences in their ABTS•+ radical scavenging activities. All the C. murale fractions caused an inhibition in ABTS•+ activity as presented in Table (2). The highest ABTS•+ scavenging activity obtained corresponded to methanol fraction (103.36μg/ml), followed by aqueous, and ethyl acetate fractions (175.53 and183.31 μg/ml), respectively. While n-hexane showed the lowest activity (479.52μg/ml) compared to the standard Trolox (16.66a μg/ml). These results are higher than that previously reported by47 of methanolic and water extract C. album. It has been also shown that the methanol fraction had the highest ABTS•+ radical scavenging activity, being rich with phenolic and flavonoids48. In addition, the scavenging effects by the ABTS•+ radical were increased with the increasing the concentration of the samples to a certain extent strongly dependent on the extract concentration.

 

5.1.3. Fe2+-Chelating activity:

Some phenolic and flavonoid compounds exhibit antioxidant activity through the chelating of metal ions. The measurement of the metal chelating activity of an antioxidant is depending on the absorbance measurement of the Fe2+ferrozine complex after prior treatment of a ferrous ion solution with the test material. All fractions of the C. murale plant had an active chelating capacity in iron ions, however, in all cases; all fractions were less than EDTA at (P0.05). In the Fe2+-chelating assay, chelating capacity as IC50 of various fractions of C. murale was ranged 269.86 – 445.14 µg/ml. Ethyl acetate fraction had the highest Fe2+ chelating capacity (269.86 µg/ml) followed by an aqueous fraction (349.04 µg/ml), while methanol fraction had the lowest ones (445.14 µg/ml). This result indicates clearly that compounds with strong chelating activity in this herb were mid-polar.

 

5.1.4. Reducing power activity:

The reducing ability of the plant extracts reflects a significant effect of its activity as antioxidants78. The reduction of the (Fe3+) to (Fe2+) may be due to the donation of an electron and the creation of Perl’s Prussian blue complex37. EC50 (the concentration (μg/ml) of plant extract that provides the reading 0.5 absorbances at 700 nm)79. The EC50 of reducing power activity of various fractions of C. murale was ranged from 179.95 - 321.50 μg/ml. Ethyl acetate had the highest activity and hexane fraction exhibited the lowest ones (Table 2).

 

In previous studies, it has been found that the IC50 of methanol extract of Chenopodium botrys L. (Amaranthaceae) collected from six different locations in the Republic of Macedonia was 3.01-12.71 mg/mL80. Therefore, reducing power assay may be considered as one of the most important methods for the estimation of plant antioxidant activity. The scavenging activity strongly dependent on the extract concentration. Our results suggesting a potential antioxidant activity of the plant extract of C.murale and can be used for the treatment of ailment in which free radicals are implicated. Our results supported by the findings obtained by20,81

 

 

6. In vitro anti-arthritic activity of C. murale.

Table 5: In-vitro anti-arthritic activity of C. murale fractions on bovine serum protein denaturation

Concentrations

500 µg/ml

1000 µg/ml

1500 µg/ml

Fractions

Denaturation %

Inhibition %

Denaturation %

Inhibition %

Denaturation %

Inhibition %

Hexane

8.74d ± 2.43

41.26b± 2.43

45.78d±0.65

54.22b ± 0.65

14.01c ± 0.94

85.99b ± 0.94

Ethyl Acetate

48.27b ± 0.59

51.73d ± 0.59

33.17b ± 0.57

66.83d ± 0.57

4.23a ± 2.04

95.77d ± 2.04

Methanol

52.30c ± 0.29

47.70c ± 0.29

38.07c ± 0.49

61.93c ± 0.49

9.98b ± 1.80

90.02c ± 1.80

Aqueous

76.68e ± 0.12

23.32a ± 0.12

67.15e ± 0.24

32.85a ± 0.24

58.33d ± 0.39

41.67a ± 0.39

Control

100

0

100

0

100

0

Concentrations

100 µg/ml

250 µg/ml

500 µg/ml

Diclofenac as standard

8.78a ± 1.06

91.22e ±1.06

5.59a ± 1.15

94.41e ±1.15

3.56a ± 1.20

96.44d ±1.20

LSD at 0.05

2.10

2.10

1.28

1.28

2.53

2.53

All values are the mean of three replicates ± S.D. Values with different letters are significantly different at p ≤ 0.05

 

 

Arthritis is a type of joint disorder that involves inflammation of one or more joints, responsible for pain, swelling, stiffness, and loss of function in joints. One of the main causes of arthritis is the production of autoantigen in certain arthritic diseases which may be due to the denaturation of protein. The mechanism of denaturation probably involves alteration in electrostatic hydrogen, hydrophobic and desulphated bonding82. The anti-denaturation method was performed by using bovine serum albumin (BSA) to evaluate the anti-arthritic potential of aerial parts (stem, leaves, and flowers) of C. murale L. different four fractions. Denaturation probably involves alteration in electrostatic hydrogen, hydrophobic and desulphated bonding83. The anti-denaturation method was performed by using bovine serum albumin (BSA) to evaluate the anti-arthritic potential of aerial parts (stem, leaves, and flowers) of C. murale L. results of the anti-inflammatory activity of C. murale are summarized in (Table 5) and provide the biological importance of developing suitable substitutes in the prevention and treatment of inflammatory diseases. It can be noticed that the different successive fractions of C. murale, produced variable anti-inflammatory activity. The inhibition percentage increase with increasing the concentration of plant extract. The Ethyl acetate successive fraction proved to be the most significant where it produced a percentage of inhibition ranged from 51.73 to 95.77 % followed by methanol fraction 47.70 to 90.02 % at (P ≤ 0.05), compared to Diclofenac as standard 91.22 to 96.44 %. Diclofenac Sodium, a standard anti-inflammation drug showed the maximum inhibition (96.44%) at 500 µg/ml. Hence, the presence of bioactive compounds in the fractions of C. murale may contribute to its anti-inflammatory activity. On the other hand, a comparison of the anti-inflammatory activity of different solvents in the aqueous fraction shows the lowest anti-arthritic activity (41.67%). This is the first report concerning the in vitro anti-denaturation effects on bovine serum albumin for these species. From the result, it can be stated that these extracts are capable of controlling the production of autoantigen to inhibit the denaturation of protein84. Acute inflammation is associated with the release of histamine, serotonin, prostaglandins, and leukotrienes, and chronic inflammation, Chenopodium ambrosiodes was speculated to inhibit inflammation by regulating the biosynthesis of these inflammatory molecules85,86. Based on the preliminary phytochemical analysis, it can be stated that the anti-inflammatory action of C. murale depends at least in part, on the synergistic interaction of the phenolic and flavonoids identified by the HPLC technique. The results of our study proved that these compounds exerted a significant anti-inflammatory activity. The anti-inflammatory activity of apigenin might be due to the inhibition of histamine release87 and quercetin might be through inhibition of lipooxygenase leading to blockage of the production of leukotrienes. Quercetin also blocked histamine release87,88. The in-vitro results provided for the popular use of the C. murale in inflammation.

 

7. Anti-proliferative activity of the C. murale:

C To find out the long-term cytotoxicity of the plant extracts on various cancer cell lines using the colorimetric MTT assay. The anti-cancer method was performed by MTT [3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium] bioassay to evaluate the anti-cancer potential activity of aerial parts of Chenopodium murale L. The effects of hexane, ethyl acetate methanol, and water fractions of C.murale on human cancer cell lines: Caucasian breast adenocarcinoma (MCF7), colon carcinoma (HCT116), and hepatocellular carcinoma cell line (HePG 2) are given in Table (2) which showed that the ethyl acetate of C.murale had the highest percentage of cytotoxicity against breast (MCF7) and Colon Carcinoma (HCT116) (89.30% and 71%) respectively followed by aqueous (extract (81% and 68.30%). Hexane showed moderate effects against MCF7 and HePG2 (39.80% and 34.03%). Also, methanol extract shows moderate cytotoxicity effects for MCF7 (60.70%). This anticancer activity may be due to the presence of major chemical constituents in the extracts of C.murale such as volatile compounds which were reported for their anticancer activity89,90. Besides, it can be revealed that C.murale herbe has promising anti-cancer bio-efficacy and must have to contain some chemical moiety that might be responsible for observed beneficial effects. The anticancer activity may be attributed to the presence of major chemical constituents in the extract such as 9 Octadecanoids and 13Docosenamide, (Z) which reported for their anticancer activity89,90. According to recent studies polyphenol compounds have anticancer activity. It has been found that the consumption of polyphenols such as rutin (a kind of flavonoids) and b-sitosterol (a kind of sterols) the major components which modulate multiple biological functions with anticancer and antioxidant activities due to its appreciable free radical-scavenging and antioxidant capacities91,92. Also, it was shown that the presence of quercetin in C. murale reduced the risk of cancer by induces apoptosis of cancer cells and inhibition of proliferation21,68.

 

CONCLUSION:

The present study confirmed that the high availability and valuable bioactive compounds in C.murale with a wide range of medicinal and biological effects as an antioxidant, Anti-arthritic and anticancer activities. Thus, the ethyl acetate fraction of C.murale may be useful in designing new protocols for inflammation and cancer diseases. Further in vivo studies are required to investigate their active component's mechanism of action.

 

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Received on 12.01.2021            Modified on 29.03.2021

Accepted on 15.06.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(1):177-187.

DOI: 10.52711/0974-360X.2022.00029