The Pharma Innovation Journal 2019; 8(11): 212-216

ISSN (E): 2277- 7695

ISSN (P): 2349-8242
NAAS Rating: 5.03 Phytochemical and in- vitro antioxidant activities of
TPI 2019; 8(11): 212-216
© 2019 TPI methanolic extract of whole plant of Cynodon dactylon
www.thepharmajournal.com
Received: 16-09-2019 and Brassica rapa M27 seed
Accepted: 20-10-2019

Manoj Kumar Kakati Manoj Kumar Kakati, Dr. Ramakanta Sharma, Dr. Naba Kumar
PhD Scholar, Srimanta
Sankaradeva University of Hazarika and Dr. Satyendra Deka
Health Sciences, Guwahati,
Assam, India Abstract
Nowadays, the need to use natural products became of great importance, as because resistance of micro-
Dr. Ramakanta Sharma
organisms to antibiotics is increase day by day and also to decrease the side effects on human beings. A
Dept. Of RS& VK Govt.
Ayurvedic College, Guwahati,
wide range of medicinal plant parts are used to treat numbers of diseases from ancient time. Traditionally
Assam, India Cynodon dactylon is used in skin trouble, treatment of wounds, as laxative, brain and heart tonic, emetic,
expectorant, carminative, and for pains, inflammations and toothache etc. Brassica rapa M27 seed is
Dr. Naba kumar Hazarika used as rubefacient and a very good folk remedy for cancer. This work was done to investigate the
Dept. Of Microbiology phytochemical properties and the antioxidant activity of methanolic extracts of whole plant of Cynodon
Guwahati Medical College and dactylon and seed of Brassica rapa M27. In this study free radical scavenging activity was determined by
Hospital, Guwahati, Assam, in-vitro assay models such as DPPH free radical, reducing ability. Quercetin was used as reference
India standard. The findings indicated that it contains different phytochemical constituents and promising
antioxidant activity of crude extracts of the above mentioned plants in a dose dependent manner and
Dr. Satyendra Deka needs further exploration for their effective use in both modern and traditional system of medicine which
Dept. Of Pharmacy can be used to treat different types of diseases.
Pratiksha Institute of
Pharmaceutical Sciences,
Keywords: Whole plant and seed, Cynodon dactylon, Brassica rapa M27, antioxidant activity, DPPH
Guwahati, Assam, India

1. Introduction
The use of medicinal plants as a source for relief from illness can be traced back over five
millennia to written documents of the early civilization in China, India. Bacteria, in general,
possess the genetic ability to acquire and transmit resistance to therapeutic agent. Not today,
but from the ancient days itself, our ancestors depended on the plants for cure and medication.
Medicinal plants represent a rich source of antimicrobial agents. According to World health
organization (WHO) more than 80% of the world population relies on traditional medicine for
their primary health care needs. Now a days most of the synthetic antibiotics are resistance to
several bacteria. So there is a need to sift our study towards Plants. The major merits of herbal
medicine are low incidence of serious adverse effects and cost 1.
Cynodon dactylon (family-Graminae/Poaceae) has some common name as Bermuda grass,
dhoob. Brassica rapa M27 (family- Brassicaceae) has some common name oil seed, turnip,
rapeseed mustrd oil.

Methods and materials

2.1 Plant material
a. Collection and authentication of sample
Cynodon dactylon plant specimens were collected from Nalbari District of Assam and Seed of
Brassica rapa (Toria-variety M27) specimens were collected from Regional Agricultural
Research Station (Krishi Vigyan Kendra), Shillongani, Nagaon, Assam. The Cynodon dactylon
plant and Seed of Brassica rapa (Toria-variety M27) was collected in the month of February,
2016. The Cynodon dactylon plant was kindly authenticated with the standard Herbarium
specimen in Botany Department of Guwahati University, Assam(Acct.no:18125 on dated 29th
March 2016) and breeder Seed of Brassica rapa (Toria-variety M27) was kindly authenticated
Corresponding Author:
Manoj Kumar Kakati in Regional Agricultural Research Station (Krishi Vigyan Kendra), Shillongani, Nagaon,
PhD Scholar, Srimanta Assam (No:2091 on dated 10th march 2016).
Sankaradeva University of
Health Sciences, Guwahati,
Assam, India
~ 212 ~
The Pharma Innovation Journal http://www.thepharmajournal.com

b. Preparation of extract 3 indicates the presence of Tannin.

Powdered (200 g) were extracted with methanol using Soxhlet
apparatus. The methanol was added up to 2 siphons that is up 2.2.5 Test for flavonoids
to 500ml. The temperature was set to 700C.The extract was a. Lead acetate test: To the alcoholic solution of the extract
concentrated at 700C to dryness under reduced pressure to few drops of lead acetate solution (10%) was added,
yield a dried crude methanol extract. Similarly, the extracts appearance of yellow precipitate indicates the presence of
were then stored at 4 ºC till the time of use. The% yield of flavonoids.
methanolic extract of Cynodon dactylon and Brassica rapa
M27 was found to be 12.6% w/w and 11.5% w/w b. Ferric chloride test: Alcoholic solution of the extract and
respectively. a few drops of ferric chloride was added, appearance of green
colour indicates the presence of flavonoids.
2.2 Preliminary phytochemical Test 3,4,5
2.2.1 Test for Alkaloids: Solvent free extract, 50mg was 2.2.6 Test for Saponin glycosides
stirred with few ml of dilute hydrochloric acid & filtered. The Foam test – 5ml of the extract was diluted with 10ml of
filtrate was tested carefully with various alkaloidal reagents as distilled water; the solutions were then vigorously shaken and
follows: observed on standing to obtain persistent of foam.

a. Mayer’s test: To a few ml of filtrate, a drop or two of 2.3 In-vitro antioxidant activity 6-21
Mayer’s reagent are added by the side of the test tube. A An antioxidant can be broadly defined as any substance that
white or creamy precipitate indicates test as positive. delays or inhibits oxidative damage to a target molecule. The
main characteristic of an antioxidant is its ability to trap free
b. Dragendorff’s test: To a few ml of filtrate, 1 or 2ml of radicals. Antioxidant compounds like phenolic acids,
Dragendorff’s reagent was added by the side of the test tube. flavonoids scavenge free radicals such as peroxide,
A prominent yellow precipitate indicates test as positive. hydroperoxide inhibit the oxidative mechanism. Herbal plants
considered as good antioxidant since ancient times. Scientific
2.2.2 Test for Carbohydrates: The extract (100mg) evidence suggests that antioxidants reduce the risk for chronic
was dissolved in 5ml of water & filtered. The filtrate diseases including cancer and heart disease. Primary sources
was subjected to the following test- of naturally occurring antioxidants are whole grains, fruits
and vegetables. In this Experiment free radical scavenging
a. Molish’s test: To 2 ml of filtrate, 2 drops of alcoholic activity of whole plant of Cynodon dactylon and seed of
solution of alpha-naphthol was added, the mixture was shaken Brassica rapa M27 were determined by different in-vitro
well & 1ml of conc. H2SO4 was added slowly along the side assay methods such as DPPH free radical, reducing ability.
of the test tube & allowed to stand. A violet ring indicates the Quercetin was used as reference standard.
presence of carbohydrate.
2.3.1 DPPH radical scavenging activity Principle
b. Barfoed’s test: To 1 ml of filtrate, 1 ml of Barfoed’s A rapid, simple and inexpensive method to measure
reagent was added & heated on a water bath for 2 min. Red antioxidant capacity involves the use of the free radical, 2,2-
ppt. indicates presence of sugar. Diphenyl-1-picrylhydrazyl (DPPH) which is widely used to
test the ability of compounds to act as free radical scavengers
2.2.3 Test for Glycoside or hydrogen donors and to evaluate antioxidant activity. The
50gm of extract was hydrolysed with concentrated DPPH assay method is based on the reduction of DPPH, a
hydrochloric acid for 2hr on a water bath, filtered & the stable free radical. The free radical DPPH with an odd
hydrolysate was subjected to the following test- electron gives a maximum absorption at 517 nm (purple
colour). When Antioxidants react with DPPH, which is a
a. Borntrager’s test: To 2ml of filtered hydrolysate, 3ml of stable free radical becomes paired off in the presence of a
CHCl3 was added & shaken, CHCl3 layer was separated & hydrogen donor (e.g., a free radical scavenging antioxidant)
10% NH3 solution was added to it; pink color indicates the and is reduced to the DPPH-H and as consequence the
presence of glycosides. absorbance’s decreased from the DPPH radical to the DPPH-
H form, results in decolourization (yellow colour) with
b. Keller Killiani Test: Test solution was treated with few respect to the number of electrons captured. More the
drops of glacial acetic acid and Ferric chloride solution and decolourization more is the reducing ability. Radical
mixed. Concentrated sulphuric acid was added, and observed scavenging activity increased with increasing percentage of
for the formation of two layers. Lower reddish brown layer the free radical inhibition.
and upper acetic acid layer which turns bluish green would
indicate a positive test for glycosides. Procedure
Free radical scavenging activity of different extracts of whole
2.2.4 Test for Tannins plant of Cynodon dactylon and seed of Brassica rapa M27
a. Ferric chloride test: About 0.5 g of the extract was boiled were measured by (DPPH). In brief, 0.1 mM solution of
in 10 ml of water in a test tube and then filtered. A few drops DPPH in methanol was prepared. This solution (1 ml) was
of 0.1% FeCl3 were added. brownish colour indicates the added to 3 ml. of different extracts in methanol at different
presence of Tannins. concentration (20, 40, 60, 80, 100 μg/ml). Here, only those
extracts are used which are solubilise in methanol and their
b. Gelatin Test: The test solution was treated with 1% various concentrations were prepared by dilution method. The
Gelatin solution containing 10% NaCl, a white precipitate mixture was shaken vigorously and allowed to stand at room

~ 213 ~
The Pharma Innovation Journal http://www.thepharmajournal.com

temperature for 30 min. then, absorbance was measured at 3. Result and Discussion
517 nm. by using spectrophotometer (UV-VIS Shimadzu).6 The investigation details is mentioned below
Reference standard compound being used was Quercetin and
experiment was done in triplicate. The percentage inhibition Table 1: Phytochemical Screening result of Cynodon dactylon
of DPPH radical was calculated by comparing the results of methanolic extracts
the test with those of the control (not treated with extract) Test Methanol
using the following equation; Percentage inhibition = (1– Alkaloids +
absorbance of test/absorbance of control) × 100. Carbohydrates +
Glycoside -
2.3.2 Reducing power assay Principle Flavanoids +
This method is based on the principle of increase in the Tannins +
absorbance of the reaction mixtures. Increase in the Saponins +
absorbance indicates an increase in the antioxidant activity. In “(+)” means present and “(-)” means absent
this method, antioxidant compound forms a colored complex
Table 2: Phytochemical Screening result of Brassica rapa M27
with potassium ferricyanide, trichloro acetic acid and ferric methanolic extracts
chloride, ferrous chloride are found as a product. Which is
measured at 700 nm. Increase in absorbance of the reaction Test Methanol
mixture indicates the reducing power of the samples. Alkaloids +
Carbohydrates -
Glycoside -
Procedure Flavanoids +
In this case 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 Tannins +
ml of potassium ferricyanide (1% w/v) were added to 1.0 ml Saponins +
of different concentrations (200 to 2000 μg/ml) of the extract “(+)” means present and “(-)” means absent
fractions .The mixture was shaken thoroughly and incubated
at 50 °C for 20 min at room temperature followed by the Table 1 shows the results of Phytochemical analysis of the
addition of 2.5 mL of Trichloro acetic acid (10% w/v) to it methanolic extract of Cynodon dactylon of the family
and centrifuged at 3000 rpm for 10 min to collect the upper Poaceae showed that all critically important secondary
supernatant layer of the solution. Now 2.5 ml distilled water metabolites were present in the methanolic extract.
and 0.5 ml FeCl3 (0.1%) were added to it .Finally the Table 2 shows the results of Phytochemical analysis of the
absorbance of mixture was tested spectrophotometrically at methanolic extract of Brassica rapa M27 of family
700 nm using an appropriate blank of 3.5 ml of potassium Brassicaceae showed that all critically important secondary
ferrocyanide solution sample. metabolites were present in the methanolic extract.

Table 3: Reducing ability of Methanolic Extract of whole plant of Cynodon dactylon and seed of Brassica rapa M27 With respect to standard
Quercetin at 700 nm
Absorbance at 700 nm
Sl. Concentration
Whole plant of Seed of Brassica Rapa
No (μg/ml) Quercetin
Cynodon dactylon M27
1 200 0.352 0.254 2.041
2 400 0.705 0.602 2.521
3 600 1.050 0.932 2.736
4 800 1.402 1.423 2.814
5 1000 1.73 1.871 2.896
6 2000 2.82 2.617 2.974

Table 4: DPPH Radical Scavenging Activity of Methanolic Extract of whole plant of Cynodon dactylon and seed of Brassica rapa M27
Absorbance at 517 nm
Sl. Concentration
Whole plant of Seed of Brassica Rapa
No (μg/ml) Quercetin
Cynodon dactylon M27
1 20 1.761 1.662 2.889
2 40 1.423 1.324 2.542
3 60 1.021 0.876 1.924
4 80 0.872 0.753 1.821
5 100 0.467 0.439 1.252

Table 5: Percentage (%) inhibition of Methanolic Extract of whole plant of Cynodon dactylon and seed of Brassica rapa M27.
Percentage (%) inhibition of Methanolic Extract
Sl. No Concentration (μg/ml)
Whole plant of Cynodon dactylon Seed of Brassica Rapa M27
1 20 39.1 42.5
2 40 44.1 48.0
3 60 47.0 54.5
4 80 52.2 58.7
5 100 62.7 64.9

~ 214 ~
The Pharma Innovation Journal http://www.thepharmajournal.com

Fig 1: Percentage (%) inhibition of methanolic extract of whole plant of Cynodon dactylon and seed of Brassica rapa M27.

In the present study, we have investigated the phytochemical healing activity, European Journal of Experimental
constituents and in-vitro Antioxidant activity of Methanolic Biology. 2011; 1(2):33-40.
extract of whole plant of Cynodon dactylon and Brassica rapa 3. Subhash S. Phytochemical analysis and Evaluation of
M27 seed. In the phytochemical investigation, we have found Antimicrobial activity in Ethnomedicinal herb
that, the Methanolic extract of Cynodon dactylon contains Corynandra chelidonii Var. pallae (Cleomaceae), Journal
carbohydrates, alkaloids, flavonoids, tannins, saponin whish is of Pharmacognosy and Phytochemistry. 2017; 6(6):1565-
listed in Table 1 and Brassica rapa M27 contains alkaloids, 1569.
flavonoids, tannins, saponin as constituents which is listed in 4. Deka S, Sharma R, Lahkar M. Phytochemical and in vitro
Table 2.The study of antioxidant activity by using two models antioxidant activities of methanolic leaves extract of
ystems i.e. DPPH method and reductive ability which is Trichosanthes dioica Roxb., The Pharma Innovation
shown in tables. Journal. 2015; 4(2):19-22
In Table 3 shows the Reducing ability With respect to 5. Okwute SK, Phytochemical analysis and cytotoxic
standard Quercetin at 700 nm. activity of the root extract of Commiphora africana
In Table 4 shows the DPPH Radical Scavenging Activity and (Caesalpiniaceae), Journal of Pharmacognosy and
Table 5 shows the Percentage (%) inhibition of Methanolic Phytochemistry 2017; 6(6):451-454.
Extract of whole plant of Cynodon dactylon and seed of 6. Shekhar TC. Antioxidant Activity by DPPH Radical
Brassica rapa M27. It shows that increase in the Scavenging Method of Ageratum conyzoides Linn.
concentration of the extracts increase the percentage (%) of Leaves, American Journal of Ethnomedicine. 2014;
inhibition which is graphically represent in Fig 1. 1(4):244-9.
7. Kokate CK, Purohit AP, Gokhale SB. Textbook of
4. Conclusion Pharmacognosy, the Phytochemical study of the extract
By this we can conclude that the methanolic extract of whole 46th edition 01-06.46, 2010; I, II,
plant of Cynodon dactylon (Family-poacea) and Brassica 8. The Phytochemical study of the extract was carried out
rapa M27 seed (Family-Brasicacea) had shown the presence by referring the book, Practical Pharmacognosy by Dr.
of different type of phytochemical constituents. From the Khandelwal K.R, nineteenth edition.
result of antioxidant activity it can be concluded that the 9. Alam MN. Review on in vivo and in vitro methods
Methanolic extracts shows in-vitro antioxidant activity in evaluation of antioxidant activity. Saudi Pharmaceutical
Dose dependant manner. Journal. 2012; 21:143-52.
10. Badakhshan. Mahdi-Pour1. Antioxidant activity of
5. Acknowledgement methanol extracts of different parts of Lantana camara.
The authors are grateful to the Ayurvedic College, Guwahati, Asian Pacific Journal of Tropical Biomedicine. 2012;
Assam, and India for providing the facilities in support to 2(12):960-5.
carry out he research work. 11. Proestos C. Antioxidant Capacity of Selected Plant
Extracts and Their Essential Oils. Antioxidants journal.
6. References 2013; 2:11-22.
1. Shaik G, Sujatha N, Mehar SK. Medicinal plants as 12. Ahmed D. Comparative Analysis of Phenolics,
source of antibacterial agents to counter Klebsiella Flavonoids, and Antioxidant and Antibacterial Potential
pneumonia, Journal of Applied Pharmaceutical Science, of Methanolic, Hexanic and Aqueous Extracts from
2014; 4(1):135-147. Adiantum caudatum Leaves. Antioxidants journal. 2015;
2. Malan R, Walia A, Saini V, Gupta S. Comparison of 4:394-409.
different extracts leaf of Brassica juncea Linn on wound 13. Patel R. DPPH free radical scavenging activity of

~ 215 ~
The Pharma Innovation Journal http://www.thepharmajournal.com

phenolics and flavonoids in some medicinal plants of

India. Int. J Curr. Microbiol App Sci. 2015; 4(1):773-80.
14. SZABO MR. Improved DPPH Determination for
Antioxidant Activity Spectrophotometric Assay. Chem
Pap. 2007; 61(3):214-6.
15. Kirtikar KR, Basu BD. Indian medicinal plants. New
Delhi: Bishen Singh Mahendra Pal Singh, 1935, 1110-
1111.
16. Preliminary Phytochemical Screening Of Various
Extracts of Punica Granatum Peel, Whole Fruit And
Seeds, Satheesh Kumar Bhandary, Suchetha Kumari N.,
Vadisha S. Bhat, Sharmila K.P., Mahesh Prasad Bekal,
NUJHS. 2012; 2(4).
17. Ansari AQ. Extraction and determination of antioxidant
activity of Withania somnifera Dunal. European Journal
of Experimental Biology. 2013; 3(5):502-7.
18. Gaikwad SA. In vitro evaluation of free radical
scavenging potential of Cassia auriculata L. Journal of
Chemical and Pharmaceutical Research. 2011; 3(4):766-
72.
19. Huyut Z. Antioxidant and Antiradical Properties of
Selected Flavonoids and Phenolic Compounds.
Biochemistry Research International, 2017.
20. Ryu JP. Antioxidant potential of ethanol extract of
Brassica rapa L. root. Journal of Medicinal Plants
Research. 2012; 6(9):1581-4.
21. Ismail A. Total antioxidant activity and phenolic content
in selected vegetables. Food Chemistry, science direct.
2004; 87(2004):581-6.

~ 216 ~