CYTOTOXICITY AND PHYYTOCHEMICAL INVESTIGATION OF Selaginella

Bryopteris

SUBMITTED BY

Dikchha Agrawal
2015A5TS939P
B .Pharmacy
Prepared in partial fulfillment of the
Course No.
BITS F421
Under the guidance of Dr.Atish T.Paul
AT

BIRLA INSTITUTE OF TECHNOLOGY & SCIENCE, PILANI

 ACKNOWLEDGEMENT

I wish to express my sincere gratitude to Dr.Atish T.Paul, HOD for providing the opportunity to
do the
training and project work in pilani campus..
I sincerely thank Pracheta Sengupta for providing me guidance and supporting me throughout in
my thesis work.
I sincerely thank Anupama Mittal for her guidance throughout the thesis work.
I also thank Ginson George and S.N.C. Shridhar for their support in my thesis work.
I also thank BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCES, PILANI for providing
the opportunity to embark on this Project Report .
Table of Content

Table of Contents

1 – Abstact......................................................................................................................1

2 – Introduction.............................................................................................................2

2.1 – Cytotoxicity ......................................................................................................................2

2.1.1-- Introduction To Cytotoxicity

2.1.2-- How To Evaluate Cytotoxicity

2.1.3-- Significance Of Cytotoxicity Assay

2.2 – Experiment With Brine Shrimp..........................................................................................

2.2.1-- Brief Details On How It Is Done And Why Is It Done

2.3 – Selaginella bryopteris.....................................................................................

3-- Aim....................................................................................................................................4

4—Material And Methods..............................................................................................................

4.1 Material
 4.2 Methods

4.2.1 Collection of plants

4.2.2--Preparation of plant extracts...............................................................................

4.2.3--Hatching the brine shrimp..................................................................................

4.2.4--Bioassay................................................................................................................

5-Result...................................................................................................................................

6-- Discussion..........................................................................................................................

7-- Conclusion..........................................................................................................................

References

List of Tables

Table no 1

Table no 2

Table no 3

Table no 4
Table no 5
 Chapter 1 Abstract

Chapter 1
Abstract
Selaginella bryopteris L. (family: Selaginaceae), is often used in traditional Indian
systems of medicine for the prevention and cure of several disorders and for the
treatment of patient with spermatorrhoea, venereal disease, constipation, colitis, urinary
tract infections, fever, epilepsy, leucorrhoea, beri-beri and cancer. It is also used as a
strength tonic. This study aimed to evaluate the cytotoxic effect of selaginella bryopteris
on brine shrimps using brine shrimp assay.and then identifying its chemical constituents
using tlc and hptlc.methods. Cytotoxicty study was performed using brine shrimp assay
and results obtained in dilutions of 10mg/ml,1mg/ml,100ug/ml;,10ug/ml,1 ug/ml was
100%,43%,46%,40%,36% respectively.
Chapter 2 Introduction

Chapter 2
Introduction
2.1 Cytotoxicity
2.1.1 Introduction To Cytotoxicity
cell cytotoxicity refers to the ability of certain chemicals or mediator cells to destroy living
cells.Some drugs are toxic to cells, cell-toxic, cell-killing.They kill cells. The prefix cyto-
denotes a cell. It comes from the Greek kytos meaning hollow, as a cell or container. Toxic is
from the Greek toxikon = arrow poison.cytotoxicity is the degree to whch an agent has a specific
destructive acion on certain cells.
Cells exposed to a cytotoxic compound can respond in a number of ways. If the insult is lethal,
the cells may undergo necrosis, during which they lose membrane integrity and die rapidly, or
the cells may follow another pathway of cell death, such as apoptosis or autophagy.
2.1.2-- How To Evaluate Cytotoxicity

Cytotoxicity can be measured by the following assays:

1.MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole): MTT assay
is named after the enzyme ,meaning the dye that we use for this assay that is the MTT or the
tetrazolium.we use ths assay to check the cytotoxicity ,to check he number of viable cells present
qualitatively as well as quantitatively .viable cells contain living mitochondria which contain
NADPH oxido redustase enzyme which convert tetrazolium dye to purple colour formazon.

2.XTT (sodium 3-[1(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene

sulfonic acid hydrate):The assay is based on the cleavage of the tetrazolium salt XTT in the
presence of an electron-coupling reagent, producing a soluble formazan salt. This conversion
only occurs in viable cells. Cells grown in a 96-well tissue culture plate are incubated with the
XTT labeling mixture for 2 - 20 hours. After this incubation period, the formazan dye formed is
quantitated using a scanning multi-well spectrophotometer (ELISA reader). The measured
absorbance directly correlates to the number of viable cells.Tetrazolium salts are cleaved to
formazan by the succinate-tetrazolium reductase system (EC 1.3.99.1) which belongs to the
respiratory chain of the mitochondria, and is only active in metabolically intact cells.
3.Trypan blue (TB):The Trypan Blue dye exclusion test is used to determine the number of
viable cells present in a cell suspension. It is based on the principle that live cells possess intact
cell membranes that exclude certain dyes, such as trypan blue, Eosin, or propidium, whereas
dead cells do not. When a cell suspension is simply mixed with the dye and then visually
examined to determine whether cells take up or exclude dye. A viable cell will have a clear
cytoplasm whereas a nonviable cell will have a blue cytoplasm.Trypan Blue has a greater affinity
for serum proteins than for cellular protein. If the background is too dark, cells should be pelleted
and resuspended in protein-free medium or salt solution prior to counting.
4.Sulforhodamine B (SRB):The sulforhodamine B(SRB) assay was developed by Skehan and
colleagues to measure drug-induced cytotoxicity and cell proliferation for large-scale dug-
screening applications.Its principle is based on the ability of the protein dye sulforhodamine B to
bind electrostatically and pH dependent on protein basic amino acid residues of trichloroacetic
acid fixed cells.
5.WST :Water-soluble tetrazolium salts (WSTs)r. The stable tetrazolium salt WST-1 is cleaved to
a soluble formazan by a complex cellular mechanism that occurs primarily at the cell surface.
This bioreduction is largely dependent on the glycolytic production of NAD(P)H in viable cells.
Therefore, the amount of formazan dye formed directly correlates to the number of metabolically
active cells in the culture.

6.Colony Formation method: its a unit measure the viable cells.we can measure the amount of
vaable cells in growing culture medium.
7.Crystal Violet method:Crystal violet (CV) cell cytotoxicity assay is one of the common
methods used to detect cell viability or drug cytotoxicity. CV is a triarylmethane dye that can
bind to ribose type molecules such as DNA in nuclei. Normally, dead adherent cells will detach
from cell culture plates and will be removed from viable cell propulation during washing steps.
CV staining can be used to quantify the total DNA of the remaining population and thus
determine cell viability. The CV staining is directly proportional to the cell biomass and can be
measured at 570 nm.

2.1.3-- Significance Of Cytotoxicity Assay

1.Cytotoxicity studies are a useful initial step in determining the potential toxicity of a test
substance, including plant extracts or biologically active compounds isolated from plants.
Minimal to no toxicity is essential for the successful development of a pharmaceutical or
cosmetic preparation and in this regard, cellular toxicity studies play a crucial role.
2.In vitro testing of drugs on animals is expensive .This animal drug testing is ethically non-
acceptable.The animals testing of drugs is difficult to introduce as complexity of wide range of
effects seen in vivo.But with Cytotoxicity assay, researchers can look for Cytotoxic compounds
that targets rapidly dividing cancer cells.

2.2 – Experiment With Brine Shrimp

2.2.1-- Brief Details On How It Is Done And Why It Is Done

Artemia salina, brine shrimp, is an invertebrate of saline aquatic ecosystem. this experiment is
based on the killing ability of test compounds on a simple zoological organism-brine shrimp
(Artemia salina).the test compounds are extracts of plants material .Salt water is prepared and eggs
of brine shrimp are hatched by providing them conditions such as artificial seawater and an air pump to
supply oxygen to the organism.After the eggs are hatched , 10 brine shrmp are collected in a test tube
and solution of plant extract in artificial sea water is added. The concentration of solution added are 10
mg/ml ,1mg/ml,100ug/ml,10ug/ml.1ug/ml.after24 hrs ,the mortality rate is calculated which is given by
the formula:( number of brine shrimp dead/number of brine shrimp taken)*100

Many natural products could serve as the starting point in the development of modern medicines
because of their numerous biological and pharmacological activities. However, some of them are
known to carry toxicological properties as well. In order to achieve a safe treatment with plant
products, numerous research studies have recently been focused on both pharmacology and
toxicity of medicinal plants. Moreover, these studies employed efforts for alternative biological
assays. Brine Shrimp Lethality Assay is the most convenient system for monitoring biological
activities of various plant species. This method is very useful for preliminary assessment of
toxicity of the plant extracts. Rapidness, simplicity and low requirements are several advantages
of this assay.

2.3 –- Selaginella Bryopteris

Introduction- It is a lithophytic xerophyte that grows on the hills of tropical areas,

particularly the Arawali mountain terrains from east to west in India.It is popularly known as
Sanjeevani which translates as "One that infuses life" derives from the medicinal properties of
the herb. It was believed that medicines prepared from this herb could revive a dead person.

General characteristics – Selaginella bryopteris(L.) Bak. is also known as Spike-moss

plants that grow on rock crevices and feed off moss, nutrients in rain water, litter, and even
their own dead tissue. These usually have dichotomously branched stems, microphylls (small
leaves), alternate, opposite or whorled, one veined, sometimes dimorphic (two sizes), with
scale like structures called ligules, out growths near the base of the upper surface of each
microphyll and sporophyll. Unusually for the lycopods, each microphyll contains a branching
vascular trace. Roots borne on wiry rhizophores. Life cycle includes various stages having
micro sporangia, megasporangia etc. Microspores are small, numerous, megaspores large, 4
per megasporangium. The gametophyte develops inside the megaspore.

Chemical composition –

1. Amentoflavone (C30H18O10)

2.Amentoflavone (C30H18O10) is a common biflavonoid chemically named as 8-[5-(5,7-

dihydroxy4-oxo-4H-chromen-2-yl)-2-hydroxyphenyl]-5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-
chromen-4-one

3.Bilobetin

4.sequoiaflavone

5.Heveaflavone

6.(2S)-2,3-dihydroamentoflavone

7.(200S)-200,300-dihydroamentoflavone
The herb contains a variety of secondary metabolites and bioactive compounds such as
alkaloids, phenol (flavonoids, tannins, saponins), and terpenoids (triterpene, steroid).A series
of eleven biflavonoids containing amentoflavone and hinokiflavone2,3-Dihydrohinokiflavone
Flavonoid.Biflavonoid that has been identified from Selaginella, among others amentoflavone,
2', 8’’-biapigenin, delicaflavone, ginkgetin,heveaflavone, hinokiflavone, isocryptomerin,
kayaflavone, ochnaflavone, podocarpusflavone A, robustaflavone, sumaflavone, and
taiwaniaflavone.

Pharmacological Activity --

Reported pharmacological effects are anti-bacterial and anti-protozoal activities, growth promote
on , anti-stress cell death , relief from heat stroke and the burning sensation during urination,
memory enhancement, anti-hyperglycemic activity , relief from stomach-ache and anti-
depression activity .these compounds act as antioxidants,anti-inflammatory,anti-cancer,anti-
allergic,anti-microbial,anti-fungal,antibacterial, antiviral, protective against UV irradiation,
vasorelaxant, heart boosters, anti-hypertensive,anti-clotting,and affect the metabolism enzymes.

figure 1: selaginella bryoperis figure 2: selaginella bropteris leaves

Figure 3:amentoflavone Figure 4:Bilobetin

Figure 3:sequoiaflavone

Figure 4: Heveaflavone
Chapter 3 Aim

Chapter3
Aim
The aim of our study is to determine the cytotoxic effects of some plant extract using brine
shrimp assay.The medicinal plants selected is Selaginella bryopteris (whole plant).Also
identification of chemical constituentsis to be done using tlc and hptlc method.
Chapter 4 Material And Method

Chapter4
Material And Method
4.1 material
1.Measuring cylinder (100mL)
2.Measuring cylinder (50 mL)
3.30 test tubes
4.Test tube Stand
5.Air pump
6.1 mL pipette
7.Analytical balance
8.Test sample of methanol extract of Selaginella bryopteris plant
9.Spatula
10.Brine shrimp eggs (a few gram)
11.Sodium chloride(Central Drug House (P) Ltd.)
12.Sodium sulphate(Avra Syntheis Ptd. Ltd.)
13.Potassium chloride(Central Drug House (P) Ltd)
14.sodium hdrogen carbonate(SDFCL)
14.Potassium bromide
15.Boric acid(SDFCL)
16.Beaker (1000 mL)
17.Tlc plate
18.solvent system chloroform-acetone-formic acid (75:16.5:8.5)
19.Tlc chamber
20.forceps
21.UV chamber

4.2 Methods
4.2.1 Collection And Authetication Of Plants
All the plant material were commercially purchased from Dehradun (India) and was
authenticated from Department Of Natural Product ,NIPER(National Institute of Pharmaceutical
Education and Research) SAS nagar Punjab (India)
4.2.2Preparation Of Plant Extract
The plant materials were powdered and passed through seive (BSS ~10) Powdered
material (30 gm) were subjected to ultrasonication for 1 hr . With Methanol(200 mL)The extracts
prepared were filtered and concentrated to dryness using rotary evaporator.
4.2.3Hatching The Brine Shrimp
Half capsule of Brine shrimp eggs were added to artificial sea water (500 mL)attracted to an
areator to provide oxygen for these eggs for hatching.Artificial sea water contains Sodium
chloride(2.39%),Sodium sulphate(0.4%),Potassium chloride(0.067%),sodium hdrogen
carbonate(0.02%),Potassium bromide(0.098%),Boric acid (0.0026%). After 24 hours incubation
at room temperature (25-29°C), nauplii (larvae) were collected.
4.2.4Bioassay
Clean test tubes were taken and labeled. Plant extract of 10 mg was weighed by an analytical
balance. Then stock solution was prepared by dissolving 10 mg of Selaginella bryopteris (whole
plant)i 1 ml of water,10 mg of Berberis aristata (fruit) in 1 mL of water,3 mg of Acacia catchu(
heartwood) in 1 mL of water ,2 mg of Ficus glomerata (fruit) in 1 mL of water ,9 mg of Thea
sinensis (leaf) in 1 mL of water. Concentrations of 1 mg/ mL, 100 μg/ mL, 10 μg/ mL and 1 μg/
mL were prepared by serial dilution from the stock solution. Then 1 mL of prepared solution was
taken into the respect test tubes containing 10 nauplii and 1 mL of seawater. The number of dead
nauplii was counted after 24 hours.
4.2.5 Hptlc method

Chapter 5 Result

Chapter5
Result
concentration 1 2 3 average

10mg/ml 100% 100% 100% 100%

1 mg/ml 50% 50% 30% 43%

100ug/ml 90% 20% 30% 46%

10ug/ml 60% 60% 0% 40%

1 ug/ml 60% 30% 20% 36%

Table no 1 showing mortality rate of brine shrimp against different concentration

of whole Plant extract of Selaginella bryopteris
Chapter 6 Discussion

Chapter6
Discussion
.Selaginella bryopteris contains a variety of secondary metabolites such as alkaloids, phenol
(flavonoids, tannins, saponins), and terpenoids (triterpene, steroid)The main secondary
metabolite of this plant is bioflavonoid, whose type varies depending on the species.
Bioflavonoid that has been identified from Selaginella, among others amentoflavone, 2',
8‟‟biapigenin, delicaflavone, ginkgetin, heveaflavone, hinokiflavone,
isocryptomerin,kayaflavone, ochnaflavone, podocarpusflavone A, robustaflavone,
sumaflavone,and taiwaniaflavone.Cytotoxic properties of selaginella may be due to the presence
bioflavonoid as reported in the paper.[9].

Chapter 7 Conclusion

Chapter7
Conclusion
1.Cytotoxicty study was performed using brine shrimp assay and results obtained in
dilutions of 10mg/ml,1mg/ml,100ug/ml;,10ug/ml,1 ug/ml was 100%,43%,46%,40%,36%
respectively.
2.
References:
1.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614850/
2.https://www.medicinenet.com/script/main/art.asp?articlekey=19883
3.https://repository.up.ac.za/bitstream/handle/2263/24934/06chapter6.pdf?sequence=7
4.https://www.thermofisher.com/in/en/home/life-science/cell-analysis/cell-viability-and-
regulation/cytotoxicity.html
5.https://www.sciencedirect.com/topics/neuroscience/cytotoxicity
6.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3035885/
7.https://www.omicsonline.org/open-access/some-phytochemicals-found-in-medicinal-plants-
used-in-cancer--a-review-2161-0444-1000491-99121.html
8.Jitender M., Chetan CS., Manu S. Human epithelial carcinoma cytotoxicity and inhibition
of DMBA/TPA induced squamous cell carcinoma in Balb/c mice by Acacia catechu willd
heartwood. J of Pharm. and Pharmacology, 2011; 63: 1470-82
9.https://www.researchgate.net/publication/277750708_An_Overview_on_Properties_Therapeuti
c_Efficacy_of_the_Indian_Magical_Herb-_SANJEEVANI