Paste your esIn Vitro Study Of Antioxidant Property Of Ethnolic Extract Of Asparagus Racemosus Root And Its Component Steroidal Saponins
M.Vani, * D.H.Gopalan,* S.Manikandan** and V.Vijayakumar#
*Mrs.M.Vani Assistant professor, Department of Anatomy, Tagore Medical College and Hospital, Rathinamangalam. The TN Dr. MGR Medical University and PhD scholar at Saveetha university.
*Dr.D.H.Gopalan Associate Professor, Department of Anatomy, Tagore Medical College and Hospital, Rathinamangalam. The TN Dr. MGR Medical University and PhD scholar at Saveetha university.
**Dr.S.Manikandan Associate Professor, Department of Physiology, Tagore Medical College and Hospital, Rathinamangalam, The TN Dr. MGR Medical University.
# Dr.V.Vijayakumar Professor, Department of Anatomy, Saveetha Medical College and Hospital, Saveetha University.
ABSTRACT
Asparagus racemosus is an important medicinal plant of tropical and subtropical countries like India. The genus Asparagus is considered to be of medicinal importance because of the presence of steroidal saponins and sapogenins in various parts of the plant. Roots of Asparagus racemosus (Family – Asparagaceae) is commonly known as than neervittankizhangu in Tamil and Shatavari in Sanskrit. It is recommended in traditional medicine for the prevention and treatment of gastric ulcers, dyspepsia, diarrhoea and nervous disorders. Very few studies have been reported regarding the antioxidant activities of ethanolic extract of Asparagus racemosus (EEAR) but antioxidant activity of its component steroidal saponins, to our knowledge, has not been previously described in the available literature.. In the present study the antioxidant activity steroidal saponins and EEAR were assessed using DPPH & Nitric oxide radicals.The radical scavenging activity of tested samples were expressed as an inhibition percentage. Also Physio-chemical parameter, Preliminary phytochemical analysis and TLC Photo documentation of EEAR were done and documented. The data obtained in this study showed both EEAR and steroidal saponins has high oxidant activity but EEAR.showed more antioxidant activity when compared to its component steroidal saponins.
Introduction :
Asparagus racemosus is an important medicinal plant of tropical and subtropical countries like India(Gomase). The genus Asparagus is considered to be of medicinal importance because of the presence of steroidal saponins and sapogenins in various parts of the plant3.Roots of Asparagus racemosus (Family – Asparagaceae) is commonly known as than neervittankizhangu in Tamil and Shatavari in Sanskrit. The name "Shatavari" translates to "a woman who possesses 100 husbands", referring to the Shatavar rejuvenation effect in female reproductive organs. Shatavari has been mentioned in Ayurvedic Shatavari is known for its phytoestrogenic properties and is extensively used in combating menopausal symptoms and increasing lactation(Sabnis PB) .The medicinal benefits of this plant has been reported in the Indian and British Pharmacopoeia and also in traditional system of medicine, such as Ayurveda, Siddha and Unani. It is recommended in traditional medicine for the prevention and treatment of gastric ulcers, dyspepsia, diarrhoea and nervous disorders(Goyal). The plants also possess antioxidant, immunostimulant, antidyspepsia and antitussive properly(Bopana).
Oxidation mechanisms and role of free radicals have attracted lots of attentions for the past few decades [Halliwell]. In cellular metabolism, during uptake of oxygen there will be production of reactive oxygen species (ROS) [Chaillou]. ROS contain free radicals such as superoxide anion radicals, hydroxyl radicals, and also non-free-radicals such as hydrogen peroxide and singlet oxygen, which are nothing but various forms of activated oxygen [BüyükokuroğluME]. They are dangerous and results in various alteration in nucleic acids, protein, and lipid molecules. ROS are very harmful, and they have the capacity to produce damage on the crucial biomolecules if they are not scavenged by the cellular constituents [Gülçin İ].The antioxidants has the potential to stop the free radical’s harmful action. The antioxidants will scavenge the free radicals and detoxify the organism [Kumaran A]. Thereby it protects the body from free radicals and also ROS effects. The antioxidants also checks the progress of many chronic diseases along with lipid peroxidation [Lai LS].
The major active constituents of Asparagus racemosus are a group of steroidal saponins. This plant also contains many vitamins and minerals like A, B1, B2, C, E, Mg, P, Ca, Fe, and folic acid. Other primary chemical constituents of Asparagus racemosus are essential oils, asparagine, arginine, tyrosine, flavonoids (kaempferol, quercetin, and rutin), resin, and tanninsteroidal glycosides (asparagosides), bitter glycosides, asparagines and flavonoids. In addition to these, it contains diosgenin and other saponins such as shatavarins I and IV in leaves and roots of A. racemosus [Ravikumar PR]. Also Shatavarin V and Shatavarin VI-X were reported in roots of A. racemosus by Hayes et al [Hayes PY(2006) , Hayes PY(2007)]. Racemofuran, (α, α-diphenyl-β- picrylhydrazyl) was reported from roots of A. racemosus by Wiboonpun et al. [Wiboonpun NP]. Sekine et al also reported racemosol and asparagamine from roots of A. racemosus [Sekine TN(1997), Sekine T (1994. Recently few reports are available demonstrating effects of steroidal saponins in some clinical conditions e.g. immunomodulatory effect, antihepatotoxic anti- HIV activity, Antiabortifacient activity, anticancer activity.The articles demonstrating the antioxidant effect of steroidal saponins and its comparison with ethanolic extract of asparagus racemosus (EEAR) was not available.The present article includes detailed analysis of antioxidant property of steroidal saponins and phytochemical analysis of EEAR .
MATERIALS AND METHODS
Collection of plant material
The fresh roots of A. Racemosus were obtained from kolapakkam, Vandalur, Chennai, Tamilnadu in the month of July – September authenticated by Department Of Botany, National Institute of Siddha, Chennai. (Voucher no: NISMB1492014).
Source of Chemicals
All the chemicals were of analytical reagent grade and purchased from Southern India Corporation limited, Chennai.
Preparation of plant material
Asparagus racemosus roots were shade dried for a week. The dried roots were powdered using electric blender, further subjected to soxhlet extraction with 99 % ethanol for 48 hours. The resulted mixture was evaporated to dryness using a rotary flash evaporator and the condensed extract was stored in refrigerator which was then used for preliminary phytochemical screening following standard procedure (Harborne’s method, 1973).
Column chromatography
Glass column was cleaned and dried vertically; a small plug of cotton was pushed at the bottom of the glass column. The column was packed with silica mixed in hexane to about half the length of the column and then the extract was mixed with silica gel and solvent which was packed in the column to one-third length. The solvent was then added and the column was allowed to settle without cracks or air bubbles. The fractions were eluted using solvents like hexane (low polar), chloroform (middle polar), ethyl acetate (polar) and ethanol (highly polar) in different ratio in the order of increasing polarity were used. The collected fractions were then concentrated to be used in thin layer chromatography
Thin layer chromatography
Thin-layer chromatography (TLC) is the simplest and cheapest method of detecting plant constituents since the method is easy to run, reproducible and requires little equipment (Marston et al., 1997). TLC is an important method for the isolation, purification and confirmation of natural products. Compared with other chromatographic methods, TLC is often considered to be deficient in reproducibility and accuracy, but some distinctive attributes of this tool should be considered: low cost analysis, high-throughput screening of samples, minimal sample preparation, whole sample integrity, disposable stationary phase. Thin Layer Chromatography (TLC) is a solid-liquid type in which the two phases are a solid (stationary phase) and a liquid (moving phase). Solids most commonly used in chromatography are silica gel (SiO2 x H2O) and alumina (AL2O3xH2O). In our experiments thin layer chromatography (usually 5 μl of a 100 mg extract/ml solution) is loaded on Merck TLC F254 or manual silica gel glass plates using hexane; ethyl acetate mixture as eluents. Measure the distance from the starting point to the center of the spot on the TLC plate (Distance- a).
Measure the distance from the starting point to the solvent front (distance -b).
Calculate the Retention Factor as:
Rf=a/b
Invitro antioxidant evaluation
DPPH free radical scavenging assay:
The ability of the extracts to annihilate the DPPH radical (1,1-diphenil-2-picrylhydrazyl) was investigated by the method described by (Blois 1958). Stock solution of compound was prepared to the concentration of 10 mg/ml. Different concentrations (25, 50, 75, 100, 200 & 250 µg) of sample were added, at an equal volume to ethanolic solution of DPPH (0.1mM). The reaction mixture is incubated for 30min at room temperature; the absorbance was recorded at 517 nm. The experiment was repeated for three times. Ascorbic acid was used as standard control. The annihilation activity of free radicals was calculated in % inhibition according to the following formula
% of Inhibition = (A of control – A of Test)/A of control * 100
Nitric oxide scavenging activity:
The nitric oxide assay was performed as described previously with slight modification (Garrat, 1964). Sodium nitroprusside (1.5 mL, 10mM) in Phosphate buffered saline was mixed with the samples (concentrations – 250, 500 & 1000 µg). The amount of nitrite, a stable metabolite of NO, was measured using Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediaminedihydrochloride in 2.5% phosphoric acid). The sample with sodium nitroprusside was mixed with 1.5 mL of Griess reagent. Subsequently, the mixture was incubated at room temperature for 10 min and the absorbance at 540 nm was measured in a UV spectrophotometer.
The annihilation activity of free radicals was calculated in % inhibition according to the following relation:
Inhibition % = (Absorbance of control – Absorbance of sample)
Absorbance of control
Statistical analysis: The obtained experimental data were expressed as Mean±SD of three measurements.
Results and Discussion:
Phytochemical analysis:
EEAR in Table 1 showed a strong presence of saponins, tannins, flavonoids, alkaloid, quinones, cardiac glycosides, phenols, coumarins and steroids. Carbohydrates,terpenois,phlobatannins and anthraquinones were absent. Basically, if a reaction takes place, the changes in the solution will be positive, by means, there is the presence of a compound in the solution.
The results of the preliminary phytochemical analysis of ethanolic extract of asparagus racemosus roots were shown below
Table: 1 Qualitative phytochemical analysis
S.No
Phytochemical Tests
Results
1 Carbohydrates test –
2 Tannins test +
3 Saponins test +
4 Flavonoids test +
5 Alkaloid test +
6 Quinones test +
7 Glycosides test +
8 Cardiac glycosides test +
9 Terpenoids test –
10 Phenols test +
11 Coumarins test +
12 Steroids &Phytosteroids +
13 Phlobatannins test –
14 Anthraquinones test –
‘+’ and ‘-’ indicates the presence and absence of phytochemicals respectively
PHYSIOCHEMICAL ANALYSIS
The physico-chemical data of the Asparagus racemosus root is given in Table No.2. The root contains 4.73 % moisture content. The root contains 4.74% of silicates as shown by the acid insoluble ash, but contains considerable amount of inorganic materials as shown by the total ash
value (9.73 %). The alcohol soluble extractive value 39.94 % shows the polar constituents of the plant and water soluble extractive values 64.38 % showed the polar and inorganic constituents of the plant.
Table: 2 PHYSIOCHEMICAL ANALYSIS
S.No: Parameters (% w/w) Asparagus racemosus
1 Total ash value 9.73
2 Acid insoluble ash 4.74
3 Water insoluble ash 8.43
4 Moisture content 4.73
5 Foreign organic matter 4.85
6 Crude fiber content 32.43
7 Alcohol solubility 39.94
8 Water solubility 64.38
Antioxidant activity:
DPPH Assay:
In nitric oxide scavenging assay, at a concentration of 100 µg, EEAR showed 61% and Steroidal saponins showed 51% inhibition, respectively. As the concentration of components and extracts increased percentage of inhibition also increased simultaneously. For a concentration of 250 µg, EEAR showed an inhibition of 70% and Steroidal saponins showed 58% inhibition. For a concentration 0f 500 µg EEAR showed an inhibition of 76% and Steroidal saponins showed 67% inhibition.Here, ascorbic acid was taken as a standard. EEAR showed high antioxidant activity than EEAR at all concentrations .(Table 2)(figure 1)
Table 1: In vitro antioxidant activity by DPPH radical scavenging activity
% inhibition by substances /extracts
Concentration
(µg/ml) EEAR Steroidal saponins Ascorbic acid
100 61.68±2.21 51.68±4.76 81.35±1.15
250 70.74±0.74 58.23±4.79 94.25±1.96
500 76.35±2.06 67.38±1.99 99.80±0.08
Values are means ±SD
Figure 1:
Nitric Oxide Scavenging Assay:
In nitric oxide scavenging assay, at a concentration of 250 µg, EEAR showed 44% and Steroidal saponins showed 35% inhibition, respectively. As the concentration of components and extracts increased percentage of inhibition also increased simultaneously. For a concentration of 500 µg, EEAR showed an inhibition of 59% and Steroidal saponins showed 51% inhibition. For a concentration 0f 1000 µg there is no much difference in percentage of inhibition.Here, ascorbic acid was taken as a standard. EEAR showed high antioxidant activity than Steroidal saponins at both 250 µg and 500 µg .(Table 2)(figure 2)
Table 2: In vitro antioxidant activity by nitric oxide scavenging method
% inhibition by substances /extracts
Concentration
(µg/ml) EEAR Steroidal saponins Ascorbic acid
250 44.53±2.32 35.44±1.50 79.75±0.67
500 59.69±1.48 51.09±2.67 81.25±1.56
1000 62.52±0.65 60.85±4.23 83.28±2.09
Values are means ±SD
Figure 2:
TLC ANALYSIS:
The Thin layer chromatography is used for characterising and isolating plant bioactive compounds from the crude extract. Here the crude extract after isolation from the column chromatography is run on a TLC plate. Figure 1a is the air dried TLC plate, figure 1b vanillin dyed TLC plate, figure 1c is the TLC plate visualized in the short UV, figure 1d is the TLC plate visualized in the long UV. The bands identified in the TLC plate represent the compound of interest , steroidal saponins.
Figure .1 show the visible light, short UV, long UV and vanillin dyed TLC sheet of the Crude, Fraction & Compound Asparagus racemosus respectively
Conclusion:
In our study, it was proven that steroidal saponins and EEAR possess antioxidants activity. They also displayed effective antioxidants activity when compared to the standard antioxidant compound in different antioxidant assays such as nitrogen peroxide and DPPH. According to data obtained, EEAR has more antioxidant activity than Steroidal saponins. Thus, because of their antioxidant activity, they have the ability to scavenge the free radicals, which are dangerous for the body. Hence, these components can be used in drug formulations, and more research can be carried out in future to explore the medicinal properties of these components.
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