Impact factor: 0.3397/ICV: 4.

10 ISSN: 0976-7908 48

Pharma Science Monitor 11(1), Jan-Mar 2020

PHARMA SCIENCE MONITOR

AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES
Journal home page: http://www.pharmasm.com

STUDY OF IN-VITRO ANTI-OXIDANT ACTIVITY OF STEM OF SAMANEA SAMAN

Nidhi N Chauhan*1, Mukesh B. Jadeja2, B. N. Shah3, A K Saluja4
1
Department of Pharmacognosy, Laxminarayan Dev college of Pharmacy, Bholav, Bharuch, Gujarat, India.
2
Department of Pharmacognosy, Smt.S.M.Shah Pharmacy College, Ahemdabad, Gujarat, India.
3
Department of Pharmacognosy, Shree N. L. Patel College of Pharmacy, Umrakh, Gujarat, India.
4
Ex-Principal, A R College of Pharmacy, Anand, Gujarat, India.

ABSTRACT
Polyphenolic Compounds is potential antioxidant. An antioxidant defined as a substance that
when present at low concentrations, lower than the oxidizable compound to be protected,
significantly delays or inhibits its oxidation. The polyphenolic content and antioxidant activity
of stem of Samanea saman(Jacq) Merr. in methanol extract were studied. The Total Phenolic
Content (TPC) was determined using DPPH free scavenging activity, Nitric Oxide free radical
scavenging activity, Super Oxide free radical scavenging activity, reducing power by FeCl3.The
study showed that methanol better solvents for the extraction of phenols of stem of Samanea
saman(Jacq) Merr. It also showed that phenols contributed the antioxidant activity. This study
was aimed to determine polyphenolic content and antioxidant effect of stem of Samanea
saman(Jacq) Merr. The present study shows the antioxidant activity of methanolic extract of
Samanea saman(Jacq) Merr which is compared with standard sample of ascorbic acid and
curcumin by In-vitro evaluation methods.
KEYWORDS: Samanea saman(Jacq) Merr., antioxidant activity, herbal agents.

INTRODUCTION
Plant profile:
Samanea saman(Jacq) Merr. (Fabaceae), commonly called Ratosiris in Gujrati,VilaitiSiris,
Gulabisiris in Hindi and Saman, Rain Tree, Monkey Pod in English, is a native of tropical Africa
and Asia. The synonym of the Samanea saman is “Monkey pod” which is a fast growing tree
that is been introduced to many tropical countries throughout the world from its native habitats in
Central America and Northern South America. Even though, it has been planted as an
ornamental tree, it has a great value of pastures as shade for the cattle. Rain tree exhibits several
bioactive compounds which produce different medicinal properties such as antioxidants, anti-
ulcer, antibacterial, analgesic, antifungal, insecticidal and cytotoxic activities.The crude drug is
always been cheaper and readily available in abundance with negligible side effects and it is
suitable for the patients of all age groups.

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Samanea saman(Jacq) Merr. Stem shows present of polyphenols.Phenolics can act as pro-
oxidants by chelating metals in a manner that maintains or increases their catalytic activity or by
reducing metals, thus increasing their ability to form free radicals So the present study deals with
the antioxidant activity by In-vitro evaluation methods and confirms the activity of the different
extracts of the plant stem [1-6].
MATERIALS AND METHODS
The stem of Samanea saman Linn were collected fromAnand Agriculture University, near
Borsad road, Anand, Gujarat, India for studies in the month of May, June and dry stem of plant
was procured from Mac ayurcare, Khambhat. The plant was authenticated and confirmed by Dr.
A. S. Reddy, Prof. and Head of Botany Dept., Sardar Patel University, Vallabh Vidyanagar. The
stems were dried in shade and stored at 250°C. It was powdered, passed through 60# and stored
in air tight bottles. Studies of antioxidant activity was carried out using standard methodology [4]
STUDY OF IN-VITRO ANTI-OXIDANT ACTIVITY [7-18]:
1. Determination of Total Phenolic:
The total soluble phenolic contents were determined with FolinCiocalteau reagent.To each of
methanolic extracts of Samanea saman(1 mg/mL), 1 mL of FolinCiocalteau reagent (1:10 v/v)
was added and incubated at room temperature for 5 minutes. 1 mL of 7% sodium carbonate
solution was added and incubated at room temperature for 90 minutes. The absorbance was
measured at 720 nm using UV spectrophotometer (Shimadzu UV 1600). The same procedure
was carried out for Gallic acid (50-250 µg/mL) standard. The total phenol content of the extracts
was obtained by using the standard curve. The total phenol content was expressed as Gallic acid
equivalent in %, w/w of the extracts.
2. DPPH free scavenging activity Assay:
Samanea samanmethanolic extract and Ascorbic acid (std.)were a liquated into series of
concentrations 160-225µg/mL and 10-60 µg/mL respectively. 1mL of freshly prepared 0.1
mMMethanolic DPPH solution was added and incubated in the dark for 20 minutes. The
absorbance was measured at 517 nm. A similar procedure was repeated with distilled water
instead of the extract, which served as a control while Ascorbic acidwas used as a standard. All
the tests were performed in triplicates. The percentage of free radical scavenging was calculated
using the formula below.
Free Radical Scavenging (%) = [(Control OD-Sample OD)/Control OD]/100
3. NITRIC OXIDE FREE RADICAL SCAVENGING ACTIVITY:

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Samanea samanmethanolic extract(200-800µg/mL) was treated with 3 mL of 10 mM sodium

nitroprusside in phosphate buffer. The resulting solution was then incubated at 25˚C for 150
minutes. From the above solution, 0.5 mL was taken and 1 mL of 0.33% Sulphanilic acid was
added and incubated at room temperature for 5 minutes. 1 mL of 0.1%
Napthylethylenediaminedihydrochloride was added and incubated at 25˚C for 30 minutes. The
absorbance of pink chromophore formed during diazotization was determined by using a UV
spectrophotometer at 546 nm. Blank solutions were prepared without adding sodium
nitroprusside in the mixture. Experiments were repeated with distilled water without the plant
extract, which acts as a control. All the tests were performed in triplicates and a standard graph
was plotted by using curcumin (10-150 μg/mL). The percentage of scavenging activity was
calculated by using the standard graph.
4. SUPER OXIDE FREE RADICAL SCAVENGING ACTIVITY
The scavenging activity against chemically generated superoxide radicals of the crude extracts
and flavonoids was measured by means of spectrophotometric measurement of the product on
reduction of nitro blue tetrazolium (NBT) (Robak&Gryglewski, 1988). Test samples were
dissolved in DMSO and diluted in water to give a final concentration of 12% (v/v) for DMSO
(Hatano et al., 1991). Superoxide anions were generated in a nonenzymatic
[phenazinemethosulfate (PMS)/NADH] system. The reaction mixture contained 1 ml of test
solution, 1.9 ml 0.1 M phosphate buffer, pH 7.4, 1 ml of 20mM PMS, 156mM NADH, and
25mM of NBT in phosphate buffer, pH 7.4. After 2 min of incubation at 25 °C, the color was
read on a Hitachi U-2000 spectrophotometer at 560 nm against blank samples, which contained
no PMS. The percentage of scavenging activities (%) was calculated as follows: Figure 1.
Structure of flavonoids isolated from the stem of Samanea samanmethanolic extractstudied for
their scavenging capacity on nonenzymatically (phenazinemethosulfate/NADH) generated
superoxide radicals.
Scavenging activities % (capacity to scavenge the superoxide radical) = [1 - (absorbance of
sample at 560 nm)/ (absorbance of control at 560 nm)] × 100.
5. REDUCING POWER BY FeCl3
Different concentrations of Samanea samanmethanolic extract and its various fractions (10-3000
μg/mL) was added to 2.5 mL of 0.2 M sodium phosphate buffer (pH 6.6) and 2.5 mL of 1%
potassium ferricyanide [K3Fe(CN)6] solution. The reaction mixture was vortexed well and then
incubated at 50°C for 20 min using vortex shaker. At the end of the incubation, 2.5 mL of 10%
trichloroacetic acid was added to the mixture and centrifuged at 3,000 rpm for 10 min. The

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supernatant (2.5 mL) was mixed with 2.5 mL of deionised water and 0.5 mL of 0.1% ferric
chloride. The colored solution was read at 700 nm against the blank with reference to standard
using UV Spectrophotometer. Here, ascorbic acid was used as a reference standard, the reducing
power of the samples were comparable with the reference standard.
RESULT AND DISCUSSION:
1. TOTAL PHENOLIC CONTENT:

Figure.1. Total Phenolic content of StandardGallic acid andSamanea samanmethanolic

extract (SSME)
Total Phenolic content of methanolic extracts of Samanea saman(1 mg/ml)was found to be 98.4
± 5.24 μg /mg of Gallic acid equivalent to phenol. It has been reported that phenolic compound
associated with antioxidant activity and play important role in stabilizing lipid peroxidation.
2. DPPH FREE SCAVENGING ACTIVITY:
The DPPH free radical is a stable free radical, which has been widely accepted as a tool for
estimating free radical-scavenging activities of antioxidants. The percentages of remaining
DPPH in the presence of the SSME and STD Ascorbic acid are shown in figure.2.

Figure.2. DPPH scavenging effect of standard Ascorbic acid andSamanea samanmethanolic

extract (SSME)
Ascorbic acid (std.) showed IC50= 31.339 ± 2.87 µg/ml
Samanea samanmethanolic extract showed IC50=148.49 ± 6.78 µg/ml

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The methanolic extract exhibited the highest radical scavenging activity and ferric reducing
power with the greatest amount of phenolic content. The presence of polyphenolic compounds in
methanolic extract of SSME might be responsible for this high antioxidant activity.
3. NITRIC OXIDE FREE RADICAL SCAVENGING ACTIVITY:
The nitrite radical scavenging assay was carried out on the water extracts from a concentration
range of 100 to 1000 μg/mL. The percentage radical scavenging of the nitrite radical by the water
extracts is shown in Figure 3. The water extracts exhibited less free radical scavenging capacity
than the ethanol extracts. The methanolic extracts results were used to determine potency and
maximum percentage scavenging of the plants since they exhibited the greatest scavenging
activity. The potency of the extracts was interpolated from the graph in Figure 3. The summary
of the results is shown below.

Figure.3. NITRIC OXIDE free radical scavenging activity of standard Curcumin

andSamanea samanmethanolic extract (SSME)
Curcumin (std.) showed IC50 = 40.401 ± 5.05 µg/ml
Samanea samanmethanolic extract showed IC50 = 553.889 ± 15.64µg/ml
4. SUPER OXIDE FREE RADICAL SCAVENGING ACTIVITY

Figure.4. Super oxide scavenging effect of standard Ascorbic acid and Samanea
samanmethanolic extract (SSME)

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Ascorbic acid (std.) showed IC50 = 14.231 ± 2.33 µg/ml

SSME showed IC50 = 74.251 ± 4.92 µg/ml
5. REDUCING POWER BY FeCl3

Figure.5.Reducing power of standard Ascorbic acid and Samanea samanmethanolic extract

(SSME)
Table 1 Comparisons of Anti-oxidant effect of SSME with STD by following methods:
SR METHODS STD USED STD IC50 SSME IC50
NO
1 DPPH FREE Ascorbic acid 31.339 ± 2.87 148.49 ± 6.78
SCAVENGING ACTIVITY µg/ml µg/ml
2 NITRIC OXIDE FREE Curcumin 40.401 ± 5.05 553.889 ±
RADICAL SCAVENGING µg/ml 15.64µg/ml
ACTIVITY
3 SUPER OXIDE FREE Ascorbic acid 14.231 ± 2.33 74.251 ± 4.92
RADICAL SCAVENGING µg/ml µg/ml
ACTIVITY

SUMMARY AND CONCLUSION

The methanolic extract of Samanea saman (SSME) have higher IC50 value compared to Ascorbic
acid (Std.) which indicates that extract scavenge free radicals like Superoxide in higher
concentration so concentration required to produce significant antioxidant activity is high.

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Like the antioxidant activity, the reducing power of Samanea samanmethanolic extract increase
with increasing concentration. The methanolic extract exhibited significant reducing power at
high concentration compared to Ascorbic acid (Std.).
In vitro antioxidant activity was carried out by DPPH free radical scavenging method, Super
oxide method,Nitric oxide and reducing power by FeCl3 methods. The IC50 value was
determined for each compound. From results of DPPH, Super oxide, Nitric oxide and reducing
power by FeCl3 methods, it found that Polyphenolic constituents displayed strong antioxidant
activity compared to the ascorbic acid and curcumin and it suggested that these compounds could
have great importance as therapeutic agents used as antioxidant.
REFERENCES
1. Mukherjee, P.K., “Quality Control of Herbal Drugs”. The Approach to Evaluation of
Botanicals, Pharmaceutical Publishers, 2002, 13-4.
2. Zaman, H. and Bannerman, R.H., “Traditional Medicine”. World Health Organization,
Geneva, 1974, 231-9.
3. Prabhuji, S.K., Rao, S.K. and Patil, S.K., “Recent Advances in Medicinal Plants Research”.
Satish serial Publishing House, 2005, 2.
4. Buenz, E. J., et. al., “Techniques: bioprospecting historical herbal texts by hunting for new
leads in old tomes”. Trends in Pharmacological Sciences, 2004, 25, 494–s8.
5. Park, E. J. &Pezzutto, J. M., “Botanicals in cancer chemoprevention”. Cancer and Metastasis
Reviews, 2002, 21, 231–55.
6. Khandelwal, K.R. “Practical Pharmacognosy”. NiraliPrakashan,Pune, 2003, 10, 146-60.
7. Anonymous, “Physical Constant: Indian Pharmacopoeia”, 1996, 2, 53-4.
8. Mukarji, P.K., “Quality Control of Herbal Drugs”. BusinessHorizons Pharmaceutical
Publishers, New Delhi, 2002, 1, 186-95.
9. Gupta, A.K., “Physical Constant: Evaluation of Crude Drug”. Quality Standard of Indian
Medicinal Plant, Indian Council of Medical Research, New Delhi, 2003, 1, 236-237.
10. Anonymous, “The Ayurvedic Pharmacopoeia of India”. Ministry of Health and Family
Welfare, Department of Health, Government of India, 1986,1(1), 143.
11. Anonymous, “British Pharmacopoeia”. HMSO, London, 1993, 1, 222.
12. Khandelwal, K.R. “Practical Pharmacognosy”. NiraliPrakashan,Pune, 2003, 10, 146-60.
13. Anonymous, “Physical Constant: Indian Pharmacopoeia”, 1996, 2, 53-4.
14. Mukarji, P.K., “Quality Control of Herbal Drugs”. BusinessHorizons Pharmaceutical
Publishers, New Delhi, 2002, 1, 186-95.

Nidhi et al. / Pharma Science Monitor 11(1), Jan-Mar 2020 , 48-55

Impact factor: 0.3397/ICV: 4.10 ISSN: 0976-7908 55

15. Richards, R.T., Sharma, H.M., “Free radicals in health and disease”. Indian Journal of
Clinical Practice, 1991, 2 (7), 15–26.
16. Gupta, A.K., “Physical Constant: Evaluation of Crude Drug”. Quality Standard of Indian
Medicinal Plant, Indian Council of Medical Research, New Delhi, 2003, 1, 236-237.
17. Anonymous, “The Ayurvedic Pharmacopoeia of India”. Ministry of Health and Family
Welfare, Department of Health, Government of India, 1986,1(1), 143.
18. Anonymous, “British Pharmacopoeia”. HMSO, London, 1993, 1, 222.

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