ASSESSMENT OF NUTRITIONAL CONSTITUENTS AND IN VITRO ANTIOXIDANT CAPACITY OF METHANOLIC STEM EXTRACT OF CARALLUMA ATTENUATAHTML Full Text
ASSESSMENT OF NUTRITIONAL CONSTITUENTS AND IN VITRO ANTIOXIDANT CAPACITY OF METHANOLIC STEM EXTRACT OF CARALLUMA ATTENUATA
P. Mounika 1, M. Vijaya Jyothi *2, Y. Rajendra Prasad 3, C. Naresh Babu 2 and S. Triveni 2
Department of Pharmacology 1, Department of Pharmaceutical Chemistry 2, Raghavendra Institute of Pharmaceutical Education and Research, K.R. Palli cross, Ananthapuramu, Andhra Pradesh, India.
Department of Pharmaceutical Chemistry 3, College of Pharmaceutical sciences, Andhra University, Vishakapatanam, Andhra Pradesh, India.
ABSTRACT: The main objective of the present study includes the assessment of nutritional constituents and anti-oxidant activity of methanolic stem extract of Caralluma attenuata by in-vitro methods. In the present investigation the author has identified the phytochemical constituents which have nutritional importance by specific chemical tests, thin layer chromatography, atomic absorption spectroscopy for Calcium, Zinc and Magnesium. In vitro antioxidant property was also performed by Free Radical Scavenging Assay, Reductive Ability and Nitric Oxide Method. Carbohydrates, proteins and amino acids, flavonoids and inorganic essential trace elements were identified and the results were tabulated. On the basis of the results observed the methanolic stem extract of Caralluma attenuata found to possess considerable amounts of nutritional constituents and anti-oxidant property.
Caralluma attenuata, Atomic absorption spectroscopy, Free radical scavenging activity, Reductive Ability, Nitric oxide method
INTRODUCTION: Nutritional problems such as Vitamin deficiencies, anaemia, and malnutrition affect cognitive ability and intellectual development. Free radicals are highly reactive and harmful which may also lead to above specified problems. These are capable of damaging almost all types of bio molecules such as proteins, lipids, carbohydrates and nucleic acids. The fact is that the free radicals would be formed as a chain reaction. Oxidative stress can arise when cell cannot adequately destroy the excess free radicals formed 1, 2.
Plants are the primitive source for medicinal principles. Various kinds of herbs possess distinct and specific therapeutic uses. Among them Caralluma attenuate 3, 4 is a perennial herb belongs to the family Apocynaceae and sub-family is Asclepiadaceae. According to the literature the folkal information reveals that the tribes generally chew the whole plant to treat peptic ulcer, diabetes, in weight reduction 5, to suppress hunger and also to produce endurance.
It has been reported for the presence of steroids, flavanoids, saponins, triterpenes and pregnane glycosides etc., several biological activities like antidiabetic 6, antihyperlipidemic 7 and anti ulcerogenic activities was reported. The present study is aimed to identify nutritional chemical constituents and antioxidant potential 8 of methanolic stem extract of Caralluma attenuata.
MATERIAL AND METHODS:
Collection of plant material: The whole stem parts of Caralluma attenuata was collected in Ananthapuramu district, Andhra Pradesh. The plant was authenticated by Taxonomist Professor. J. Sreeramulu, Sri Krishna Devaraya University, Anantapuramu.
Preparation of extract: The dried and powdered stem part of Caralluma attenuata was passed through a sieve no.22 and each kilo gram was extracted successively by cold maceration with 2.5 litres of aqueous methanol (50:50). The extract was concentrated to dryness under reduced pressure using rotary vacuum evaporator.
Experimental Procedure: The extract was subjected for the nutritional constituent’s investigations by chemical tests and TLC. The essential trace elements like calcium magnesium and zinc were quantified by AAS 9. The extract was also utilised to evaluate in vitro antioxidant activity by Free Radical Scavenging Assay 10, Reductive Ability and Nitric Oxide Method. The results obtained were tabulated.
Identification of flavanoids by using TLC: TLC technique became an important analytical tool for micro-analytical separation and determination of natural products. In the present study the methanolic stem extracts of Caralluma attenuata was subjected to TLC.
In this analysis pre coated aluminium plates (10 cm length) coated with silica gel were used and different solvent systems were employed depending upon the nature of the analyte. For saturation of TLC chamber a sheet of filter paper was laid so as to cover internal part of three sides of the chamber which is soaked in the solvent system.
The chamber was left undisturbed to ensure saturation. A solution of stem extract of Caralluma attenuata plant was prepared in methanol. The spots of identical volume were applied 2cm away from the lower edge of plate with the help of microcapillary tube. The solvent was allowed to evaporate after each application by air drying. The spotted plate was then placed vertically in the chamber with the bottom edge immersed in developing medium. The solvent system was allowed to run approximately up to 8cm then the plates were taken out and solvent front was marked. The resolution of components of all extracts of plant of Caralluma attenuata was studied by locating the spots on the chromatogram. The spots were identified first by visual observation and then under UV lamp at 365 nm. Then the plates were developed in an Iodine chamber and the spots were located.
Flavanoids were detected using n-butanol: acetic acid: water (4: 1: 5 V/V) as mobile phase which has produced prominent fluorescent yellow colour spot. The TLC was shown in Fig. 1.
FIG. 1: TLC OF FLAVONOIDS
Identification of nutritional constituents by chemical tests: The following chemical tests were performed to identify various constituents present in plant Caralluma attenuata. The results were tabulated in Table 1.
TABLE 1: IDENTIFICATION OF NUTRITIONAL CONSTITUENTS
|Tests for fibres|
(advance solely towards flame or heat in a crucible)
|Rapid burning with flame.
No foul odour or fumes, white ash.
|Carbohydrate fibres are present|
|2||Boiling with aqueous picric acid solution.||Permanent yellow stain||Protein fibres are present|
(boil with millions reagent)
|Red colour||Protein fibres are present|
|Test for carbohydrates:|
|1||Molisch’ test (general test)
To 2-3 ml aqueous extract, add few drops of α-napthol solution in alcohol shake and add concentrated H2SO4 from sides of the test tube.
|Violet ring is formed at the junction of two liquids.||Carbohydrates are present.|
|Tests for reducing sugars:|
Mix 1ml of fehling’s A and 2ml of fehling’s B solutions, boil for 1 min, and add equal volume of test solution. Heat in boiling water bath for 5-10 min
|First yellow, then brick red ppt is observed||Reducing sugars are present|
Mix equal volume of Benedict’s reagent and test solution in test tube. Heat in boiling water bath for 5 min.
|Solution appears green, yellow or red depending on amount of reducing sugar present in test solution||Reducing sugars are present|
|Test for hexose sugars|
|1||Tollen’s fluoroglucinol test for galactose:
Mix 2.5 ml of conc.HCl and 4ml 0.5% fluoroglucinol. Add 1-2 ml of test solution heat.
|Light green colour appears||Sugars are present|
|Test for amino acids:|
|1||Ninhydrin test :
Heat 3ml test solution and 3 drops 5% ninhydrin solution in boiling water bath for 10 min
|Purple or bluish colour appears||Amino acids are presents|
|Tests for Vitamins|
|1||Test for Vitamin A:
Dissolve a quantity equivalent 10 -15 units in 1ml of chloroform and 5ml of Antimony trichloride solution.
|Violet colour appeared||Vitamin A is present|
|2||Test for Vitamin D:
Dissolve a quantity equivalent to about 1000 units of vitamin D activity in chloroform and add 10ml of antimony trichloride solution
|Pinkish red colour appeared at once||Vitamin D is present|
|Tests for Inorganic ions:|
|1||Tests for Iron:
i. NH4OH test:
Sample solution + NH4OH solution
|Reddish brown colour appeared||Iron is present|
|ii. Potassium ferrocyanide test:
sample solution + K4[Fe2+(CN)6] solution
|Intense blue ppt formed and insoluble in dil.HCl||Iron is present|
|2||Tests for Phosphates:
i. Ammonium molybdate test:
sample solution + conc. HNO3 solution
|Yellow ppt was formed||Phosphate is present|
|ii. AgNO3 test:
sample solution + AgNO3 solution
|Light Yellow ppt was formed||Phosphate is present|
|3||Tests for Sulphates:
i. BaCl2 test:
sample solution + BaCl2 solution
|White ppt insoluble in hot water and hot conc. HNO3||Sulphates present|
|ii. Lead acetate test:
sample solution + lead acetate solution
|White ppt||Sulphates present|
Estimation of Calcium, Zinc and Magnesium by AAS: Sample preparation: Weighed a 200mg of sample in to Teflon beaker then added 5ml of HNO3 then the contents were mixed with spatula and heated the contents on hot plate at 200°C for 15min then cooled. Transferred the contents in to 50ml of volumetric flask, rinsed the beaker with few ml of water and transferred to volumetric flask and made up to the mark with water.
Sample blank: The blank is prepared in the same manner as for the sample preparation but without addition of sample.
Standard preparation for Calcium, Zinc & Magnesium: 0.5ml of 1000ppm element 11 (Calcium / Zinc / Magnesium) standard solution was transferred to 10ml volumetric flask and made up to the volume with water to contain 50 ppm of calcium / Zinc / Magnesium.
Calibration standards for Calcium:
Calibration standards for Zinc:
Calibration standards for Magnesium:
Standard blank: Water
AAS Procedure: Aspirated the calibration blank (standard blank), calibration standard, sample blank, (resultant blank) and sample solution. Then concentration of Ca, Zn & Mg in mg/g was calculated. The standard graphs obtained were shown in Fig. 2, 3 and 4.
FIG. 2: AAS OF Ca 2+
FIG. 3: AAS OF Zn 2+
FIG. 4: AAS OF Mg 2+
Reductive Ability: The reductive ability 13 of medicinal plants was determined according to the Oyaizu method. Methanolic stem extract (0.08-0.4 mg) of Caralluma attenuata was dissolved in 1ml of distilled water and then 2.5ml of phosphate buffer (0.2M, pH 6.6) and 2.5ml of potassium ferric cyanide [K3Fe(CN)6] (1%) were added. The mixture was added to the mixture, which was then centrifuged at 3000rpm for 10min. 2.5ml of upper layer of the solution was mixed with 2.5ml of distilled water and 0.5ml of FeCl3 (0.1%). Absorbance was measured at 700nm. Butylated hydroxyl toluene (BHT) was used as reference compound. All the analysis was performed in triplicate. The UV absorption spectrum was shown in Fig. 5 and 6.
FIG. 5: UV SPECTRA OF SAMPLE
FIG. 6: UV SPECTRA OF CONTROL
Free Radical Scavenging Activity (FRSA) using hydrogen peroxide: The hydrogen peroxide FRSA14 of the methanolic extracts was done as suggested by Czochra and Widwnsk. 2ml of hydrogen peroxide (43milli mole) and 1.0ml of methanolic sample [20-100 μl of methanolic extract (4mg/ml) in methanol] followed by 2.4ml of 0.1 M phosphate buffer (pH 7.4) were added. The resulting solution was kept for 10min and the absorbance was recorded at 230nm. All readings were repeated three times. Blank was prepared without adding hydrogen peroxide and control was prepared without sample. Ascorbic acid was used as a standard compound. Free radical scavenging activity of hydrogen peroxide percentage was calculated as FRSA (%) = [(V0-V1)/ V0] ×100, V0= absorbance of control and V1= absorbance of sample. The UV absorption spectrum was shown in Fig. 7 and 8.
FIG. 7: UV SPECTRA OF SAMPLE
FIG. 8: UV SPECTRA OF CONTROL
Nitric oxide scavenging assay: Nitric oxide was generated from Nitroprusside and measured by the Greiss reaction sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide which interacts with oxygen produce nitric ions that can be estimated by use of Greiss reagent. Scavengers of nitric oxide 15 compete with oxygen leading to reduced production of nitric oxide. Sodium nitroprusside (5 milli moles) in phosphate buffered saline (PBS) was mixed with 3.0ml of different concentrations of the drugs dissolved in the methanol and incubated at 25 °C for 150 min. The samples from the above were reacted with Greiss reagent (1% sulphanilamide, 2% H3PO4 and 0.1% napthylethylenediamine dihydrochloride). The absorbance of the chromophore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediamine was read at 546 nm. The experiment was done in triplets.
Standard sodium nitrite (10mg/100ml) standard was processed in the same way and the inhibition percentage was calculated.
% inhibition = (absorbance of control- absorbance of sample) ×100/ absorbance of control
RESULTS AND DISCUSSION: The methanolic stem extract of Caralluma attenuata found to posses flavanoids its Rf value is 0.73 by using specific mobile phase which is nearer to the standard 0.72. Various nutrients like carbohydrates, proteins, Vitamins were identified by qualitative tests. Inorganic essential trace elements 16, 17 were notified by AAS and the percentage mg/g was identified as 0.324, 0.009, and 0.56 for calcium, zinc and magnesium respectively. The antioxidant 18, 19 capacity of the extract was performed by Reductive ability, FRSA and Nitric oxide scavenging assay methods and the results observed as 94.18%, 89.6%, and 92.4% respectively. From the results obtained indicates that the methanolic stem extract of Caralluma attenuata contains considerable amount of nutrients and essential trace elements. The methods which were used for evaluation of anti-oxidant activity had shown appreciable percentage of activity.
CONCLUSION: The methanolic stem extract of Caralluma attenuata as it contains essential nutrients, flavanoids and anti-oxidants; it is useful to reduce malnutrition, anaemia, calcium deficiency and to enhance immunity. It also prevents the process of biological oxidation, it is the unchecked problem associated with free radical formation - oxidative stress. Severe oxidative stress can cause cell death and moderate oxidation triggers apoptosis & necrosis and cardiovascular diseases. Several medicinal herbs have been known as good nutritive and remedy for prevention of disorders and ailments. The present study reveals that methanolic stem extract of Caralluma attenuata enhances endurance, enzyme production and hepatoprotection. So the present research can be extended further to develop a new herbal nutrient for prevention of various diseases by proceeding in-vivo tests.
ACKNOWLEDGMENT: The researchers are thankful to Dr. Y. Padmanabha Reddy, principal of RIPER, S. Nagarjuna Department of Pharmacology and A. Sanjeev Kumar Department of Pharmacognosy, RIPER for their support.
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How to cite this article:
Mounika P, Jyothi MP, Prasad YR, Babu CN and Triveni S: Assessment of nutritional constituents and in vitro antioxidant capacity of methanolic stem extract of Caralluma attenuata. Int J Pharm Sci Res 2017; 8(7): 2910-15.doi: 10.13040/IJPSR.0975-8232.8(7).2910-15.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
P. Mounika, M. Vijaya Jyothi *, Y. Rajendra Prasad, C. Naresh Babu and S. Triveni
Department of Pharmaceutical Chemistry, Raghavendra Institute of Pharmaceutical Education and Research, K.R. Palli cross, Ananthapuramu, Andhra Pradesh, India
17 December, 2016
08 February, 2017
17 February, 2017
01 July, 2017