Antihelmenthic activity and Antioxident activity of shuli

1. Evaluation of Antioxidant and Anthelminthic activity of the extract
of the leaves of Nyctanthes arbor-tristis Linn.
NAME – ARKENDU MUKHERJEE
ROLL NO - 19301916127
REGISTRATION NO - 161930210012
4TH YEAR, B. PHARM
SUPERVISED BY MR. ATANU CHATTERJEE
ASSISTANT PROF ESSOR OF
BENGAL SCHOOL OF TECHNOLOGY
SUGANDHA, DELHI ROAD, CHUCHURA , HOOGHLY 1

2. 2
CONTENTS
1. Introduction
2. Plant profile
3. Objective of the work
4. Methodology
5. Observation and Result
6. Discussion and Conclusion
7. Reference

3. INTRODUCTION
3
Nyctanthes arbortristis is a subcontinental plant of India as well as south Asian part of earth
also known as shiuli in West Bengal belonging to the family Oleaceae[1]. It is having
efficient pharmacological actions in-vivo i.e. inside the body due to presence of different
phytochemicals like Flavonoids, Phenols, Tannins, Alkaloids etc[2]. In this experiment we
have performed the phytochemical screening of leaf extract of N. abortritis followed by
determination of anthelminthic and antioxidant activity.

4. PLANT PROFILE
Synonyms : Parijat, Shiuli, Harsinger, Shefali.
Family : Oleaceae.
Geographical source: It is domestic in southern Asia, northern
Pakistan and Nepal, India(specially West Bengal).
Biological source : our sample consists of dried crushed leaves of the
plant Nyctanthes arbor-trsitis.
Therapeutic Uses : These leaves are used as anti-allergic, purgative,
anti-histaminic and antibacterial, antimalarial, anti-inflammatory, anti-
diabetic and so on[3].
4

5. 5
PLANT PROFILE
Macroscopic characters[4] Microscopic characters[5]
 Simple, petiolate leaves with
unicostate and reticulate venation.
 The flowers are of white petals and
orange stem with sweet fragrance.
 The fruit is flat, brown and heart-
shaped to rounded-capsule, around 2
cm in diameter with two sections.
 The cells of upper epidermis of
lamina were thick walled somewhat
straight and devoid of stomata.
 The cells of lower epidermis are
smaller in size than that of upper one
with sinuous walls at places, cuticle
striated and transverse by numerous
anomocytic stomata.
Microscopy of leaves
Macroscopy of leaves
Shiuli flower Shiuli fruit
Dorsal and ventral view of
shiuli leaf

6. 6
OBJECTIVE OF WORK
 The efficiency of synthetic drugs is becoming less popular due to their adverse
effects and contraindications. It is well to use indigenous natural medicinal plants
which are safe and less expensive also having same or more potency than the
established one.
 Here we will try to find the secret treasures those we can get easily from the
plants to make revolution in pharmaceutical field.
 The drugs may be with less adverse effects but with high potential to treat
disease.
 Here we will examine if Nyctanthes abor-tristis is having any anthelminthic and
antioxidant property or not.

7. METHODOLOGY
7
 Collection and identification of plant:
The fresh leaves of the plant N. arbor-tristis were taken and washed with pure water in the
month of September-October.
 Preparation of samples before extraction[6]:
Samples were prepared by washing the leaves with soft and pure water and drying them
under the shade for 5-7 days.
Half-dried leaves were crushed in the grinder and then they were kept for 24 hours
before starting the extraction.
 Extraction procedure[6]:
The dried leaves or the coarse powders were taken for extraction in the Soxhlet apparatus.
Extraction was done for 3 days by addition of ethanol as solvent at 40-60 C
After three days the extract was filtered and distillated to get a concentrated product

8. 8
METHODOLOGY
Extraction procedure[6]:
Now the distillated product was taken and dried for 10-15 minutes under a gentle
care.
After this, dry sticky product was kept in the refrigerator for further use.
Extraction procedure (soxhlation) of shiuli leaf

9. 9
Detecting groups Procedure Observation
Test for alkaloid Five ml of extract was added to 2ml of HCl. To
this acidic medium, 1ml of Dragendroff’s reagent
was added.
An orange color precipitate produced.
Test for amino acid 1ml of extract was treated with few drops of
Ninhydrin reagent.
Appearance of purple color.
Test for anthraquinones 5 ml of extract solution was hydrolyzed with
diluted conc. H2SO4 extracted with benzene. 1ml of
dilute ammonia was added to it.
No coloration occurs.
Test for flavonoids To 1 ml of extract, a few drops of dilute NaOH
was added.
An intense yellow color was produced which
became colorless on addition of few drops of dilute
acid.
Test for cardiac glycosides 0.5g of extract was diluted to 5 ml in water was
added 2 ml of glacial acetic acid containing one
drop of feCl3. This was underlaid with 1 ml of
conc. Sulphuric acid.
A brown ring at the interface. A violet ring was
appeared below the brown ring. Greenish ring may
form just above the brown ring.
Test for saponins The extract was diluted with 20ml of distilled
water and it was agitated in a graduated cylinder
for 15mins.
Formation of about 1cm layer foam.
Test for steroids 1ml of extracts was dissolved in 10ml of
chloroform and equal volume of concentrated
H2SO4 was added by sides of test tube.
No change in solution.
Test for carbohydrates
(Molisch’ test)
To 2ml of the extract, 1 ml of α-naphthol solution
was added, concentrated sulphuric acid was added
through the side of the test tube.
Purple or reddish violet colour at the junction of
the two liquids.
Test for phenol 2 ml of extract was taken and add 2 ml of Folin’s
reagent.
Appearance of violet or brown colour.
Test for tannins About 0.5 g of the extract was boiled in 10 ml of
water in a test tube and then filtered. A few drops
of 0.1 fecl3 was added.
Brownish green or blue- black coloration.
PHYTOCHEMICAL TEST[7]

10. 10
ANTHELMINTHIC ASSAY[8]
Samples of different concentration (100%; 50% and 25%) of extract solution were
prepared
Standard solution of albendazole was prepared from 400mg of drug
Earthworms are divided into four different groups (each groups containing five
earthworms which were previously washed with saline solution)
Different concentration of samples were given into the petri plates containing
worms
Times for paralysing and time for death of the worms were noted down
Mean paralysing time and death time were determined.
All the results were expressed as mean ± standard error mean (SEM).
Anthelminthic assay

11. 11
ANTIOXIDANT PROPERTY DETERMINATION
DPPH scavenging activity determination[9]
extract solution of different Concentrations were
prepared (50.100,150,200,250,300 mcg/ml)
1ml of 0.1mM DPPH solution was added to each
concentrations
Absorbance of these solution was measured at
517nm against blank solution
Hydrogen peroxide free radical scavenging
activity Determination[10]
extract solution of different Concentrations
were prepared (50.100,150,200,250,300
mcg/ml)
0.6 ml of 40mM Hydrogen Peroxide solution
(in phosphate buffer) was added to each
concentration.
Absorbance was measured at 230 nm against
blank solution
NO radical scavenging activity
determination[11]
3ml of extract solutions of different
Concentrations(50.100,150,200,250,300
mcg/ml) were taken in different test tubes
5mM Sodium nitroprusside solution in
phosphate buffer was added to each test tube
Incubated for 150 mins at 25 C and reacted with
Griess reagent
Absorbance was measured at 546nm
Determination of Total Flavonoid
Content[12]
1mg of extract was dissolved in 80% Ethanol
0.5 ml of aliquot of 0.1ml (10%) aluminium
nitrate, 0.1ml (1M) potassium acetate and
4.3ml of 80% Ethanol was added to the above
solution
Absorbance was measured at 415nm

12. 12
Yield product:
Weight of the Petridis is which the collected extract was taken = 47.81 gm
Weight of the Petridis with the collected extract = 182.05 gm
Weight of the collected extract = (182.05 – 47.81) gm = 134.24 gm
OBSERVATION AND RESULT

13. PHYTOCHEMICAL SCREENING
Serial Number Phytochemicals Result
1 Alkaloids Present
2 Amino acids Present
3 Flavonoids Present
4 Cardiac glycosides Present
5 Saponins Present
6 Steroids Absent
7 Carbohydrates Present
8 Phenols Present
9 Tannins Present
10 Anthraquinones Absent
13
OBSERVATION AND RESULT

14. 14
ANTHELMINTHIC ASSAY
EXPERIMENTAL
OBSERVATION
CONCENTRATIONS OF
SAMPLES
TIME FOR SAMPLES TIME FOR STANDARD
(400mg)
PARALYSED TIME
100% 56.33±1.24
64.66±1.69*50% 67.66±1.24
25% 98.28±0.81
DEATH TIME
100% 67.33±1.24
76.0±1.63*50% 71.33±1.24
25% 120.66±1.24
OBSERVATION AND RESULT

15. 15
Concentrati
ons (μg/ml)
Absorbanc
e
(control-
0.474)
%
scavenging
activity
IC50
(u/ml)
50 0.175 40.67
188.17
150 0.132 55.25
200 0.079 73.22
250 0.131 55.59
300 0.167 43.38
DPPH SCAVENGING
0
40.67
52.25
73.22
55.59
43.38
y = 0.1409x + 21.879
0
10
20
30
40
50
60
70
80
0 50 100 150 200 250 300 350
%SCAVENGING
CONCENTRATIONS
IC50 CALCULATION
sc. Linear (sc.)
OBSERVATION AND RESULT

16. 16
H2O2 SCAVENGING
Concentrati
ons (μg/ml)
Absorbanc
e
(control-
0.91)
%
scavenging
activity
IC50
(u/ml)
50 0.502 44.8
138.0
100 0.432 52.2
150 0.337 62.9
200 0.302 66.8
250 0.315 65.3
300 0.229 74.8
0
44.8
52.2
62.9
66.8 65.3
74.8
0
y = 0.2x + 22.4
0
10
20
30
40
50
60
70
80
90
0 50 100 150 200 250 300 350
%SCAVENGING
CONCENTRATIONS
IC50 CALCULATION
Series1 Series2 Linear (Series1) Linear (Series1)
OBSERVATION AND RESULT

17. 17
Concentra
tions
(μg/ml)
Absorban
ce
(control-
0.098)
%
scaveng
ing
activity
IC50
(u/ml)
50 0.089 9.1
228.89
100 0.076 22.4
150 0.080 18.4
200 0.060 38.77
250 0.047 52.04
300 0.023 76.53
NO RADICAL SCAVENGING
0
9.1
22.4
18.4
38.77
53.04
76.53
0
y = 0.2385x - 4.5914
-10
0
10
20
30
40
50
60
70
80
90
0 50 100 150 200 250 300 350
%SCAVENGING
CONCENTRATIONS
IC50 CALCULATION
Series1 Series2 Linear (Series1) Linear (Series1)
OBSERVATION AND RESULT

18. 18
Calculation of percentage flavonoid content:
Absorbance of the test sample extract solution = 0.401
A = 0.0067 C + 0.0132
Or, 0.0067 C = A – 0.0132
Or, C = (0.401 – 0.0132) / 0.0067
Or, C = 57.89
Therefore,
the concentration of flavonoid present in the extract equivalent to quercetin residue =
57.89 μg/ml
OBSERVATION AND RESULT

19. DISCUSSIONAND CONCLUSION
19
 After performing the entire experiment on the leaf extract of plant Nyctanthes arbor-tristis it
can be concluded that therapeutic active phytochemicals like Alkaloid, Flavonoids, Tannins,
Saponins, Glycosides and a small amount of amino acid are present in the extract.
 These phytochemicals are responsible for the therapeutic and subtherapeutic activity of shiuli
leaves. In addition, the total flavonoid contain is about 57.89 μg/ml equivalent to the quercetin
residue.
 The extract possesses a good anthelminthic property, may be due the synergistic activity of
phytochemicals as compared to the standard drug Albendazole, commonly popular in the
pharmaceutical market.
 Beside that the extract is having also antioxidant property that may be used as protectives and
antiaging agent in cosmetic preparation.
 We can say that plant Nyctanthes arbor-tristis is enriched in phytochemicals. In near future, if
proper facilities and infrastructure are given, anti-inflammatory and other related
pharmacological activity of the plant extract could be studied.

20. REFERENCES
20
1. I. Biswas and A. Mukherjee: Pharmacognostic studies on the leaf of Nyctanthes abor-tristis,
Acta Botanica Hungarica, 30th May 2011.
2. Suresh et al. Pharmacognostical and Preliminary Phytochemical studies of bark of Nyctanthes
arbor-tristis Linn. Inter Nyctanthes arbortristisional. Journal of Pharmacy and Pharmaceutical
Sciences. 2012.
3. P.P. Gupta, R. Srimal, M. Srivastava, K.L. Singh & J. S Tandon. (2008). Antiallergic Activity
of Arbortristosides from Nyctanthus Arbortristis. Pharmaceutical Biology. 33. 70-72.
10.3109/13880209509088151.
4. A. Kumar, B. Rathi, V. Tyagi, Priyanka and Manisha; Department of Biotechnology and
Microbiology, Shri Ram College Muzaffarnagar, UP-251001, India; Systemic Review on Anti-
Sciatica Plant “Night Jasmine” (Nyctanthes arbortristis Linn.); International Journal of Current
Microbiology and Applied Sciences; 2017; (doi.org/10.20546/ijcmas.2017.606.118)
5. I. Siddiqui, M. Anis., A.A Jahan; 2006. Rapid multiplication of Nyctanthes arbortristis through
in-vitro auxillary shoots proliferation. World J. Agri.
6. Sandhya Kumari and Singara Charya; Phytochemistry, anticancer and anti-inflammatory
activities of solvent leaf extract of Nyctanthes abor-tristis.

21. 21
REFERENCES
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METHANOLIC AND AQUEOUS EXTRACT OF MEDICINAL PLANT-NYCTANTHES ARBOR-
TRISTIS LINN. 05. 1393-1401. 10.20959/wjpps201611-8091.
8. V. Suresh, G. Arunachalam, S. Kumar; In vitro anthelmintic activity of Nyctanthes arbor-tristis
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9. Kedare SB, Singh RP. Genesis and development of DPPH method of antioxidant assay. J Food Sci
Technol. 2011;48(4):412–422. doi:10.1007/s13197-011-0251-1
10. F. Ngonda; In- vitro Anti-oxidant Activity and Free Radical Scavenging Potential of roots of
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