PHARMACOGNOSTICAL AND EXPERIMENTAL EVALUATION OF …

www.wjpps.com Vol 8, Issue 9, 2019.

754

Priya. World Journal of Pharmacy and Pharmaceutical Sciences

PHARMACOGNOSTICAL AND EXPERIMENTAL EVALUATION OF

MALLIKA (JASMINUM SAMBAC (LINN.) AIT.) WITH SPECIAL

EMPHASIS ON BREAST CANCER CELL LINE

1*Dr. Priya Pramod Wadate

1Assistant Professor, Department of Dravyaguna Vigyana,

Bhartiya Sanskriti Darshan Trusts College of Ayurved, Wagholi, Pune, Maharashtra, India.

ABSTRACT

In the plant kingdom, there are number of plants that yield medicines

useful to mankind. Jasminum sambac (Linn.) Ait. Family – Oleaceae,

called Mallika in Sanskrit is well known glabrous twining shrub widely

grown in gardens throughout the India. Breast cancer is a type of

cancer originating from breast tissue, most commonly from the inner

lining of milk ducts or the lobules that supply the ducts with

milk.According to V.G.Desai and Kirtikar & Basu Mallika acts on the

female reproductive organs (uterus and breast). The flowers of Mallika

act as „Lactifuge‟, at the puerperal state, in the case of breast abscess.

J. sambac flowers and leaves of Mallika are largely used in folk

medicine to prevent and treat breast cancer. The current in vitro

anticancer study was conducted with an aim to check its anticancer

effect on MCF-7, MDA-MB-231 breast cancer cell lines, at concentrations 10-640μg/ml. The

MTT assay protocol was followed to observe the activity of the study drug. The study drug

was tested in 96 well plates and methanolic and aqueous extract of leaf and flower of mallika

were used in different dose levels. As per MTT assay protocol, methanolic extract had shown

significant activity on MCF-7 breast cancer cell line and had shown negligible activity on

MDA-MB231.

KEY WORDS: Jasminum sambac linn. Ait, breast cancer, MCF-7, MDA-MB-231 and MTT

assay, in vitro study.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.421

Volume 8, Issue 9, 754-769 Research Article ISSN 2278 – 4357

*Corresponding Author

Dr. Priya Pramod Wadate

Assistant Professor,

Department of Dravyaguna

Vigyana, Bhartiya Sanskriti

Darshan Trusts College of

Ayurved , Wagholi, Pune,

Maharashtra, India.

Article Received on

2 July 2019

Revised on 22 July 2019

Accepted on 12 August 2019

DOI: 10.20959/wjpps20199-14612


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1.0 INTRODUCTION

Todays lifestyle are so hectic & living in such a fast growing and newly emerging world of

modern science, people are suffering from many lifestyle diseases like Alzheimer‟s disease,

Arterisclerosis, Cancer. Cancer refers to a large number of conditions that is characterized by

abnormal cell division.The cells of cancer infiltrate and destroys surrounding healthy tissue.

They have the ability to metastasize and spread throughout the body.

Cancer occurs in all ages of human being. But certain cancers are life threatening for men and

women and for so many women; there is no more deadly disease than breast cancer. Breast

cancer is a type of cancer originating from breast tissue, most commonly from the inner

lining of milk ducts or the lobules that supply the ducts with milk.

Jasminum sambac (Linn). Ait Family – Oleaceae, called Mallika in Sanskrit. It is well known

glabrous twining shrub widely grown in gardens throughout the India. As per the paper

published on antitumor activity of J. sambac flowers and leaves of Mallika are largely used in

folk medicine to prevent and treat breast cancer. Flowers are useful to women when brewed

as a tonic as it aids in preventing breast cancer and stopping uterine bleeding.

1.1 SELECTION OF DRUG

According to V.G.Desai and Kirtikar & Basu Mallika acts on the female reproductive organs

(uterus and breast). The flowers of Mallika act as „Lactifuge‟, at the puerperal state, in the

case of breast abscess.

More than 30 ayurvedic medicinal plants like Amruta, Haridra, Yashtimadhu, Kanchnar,

Guggulu etc. have proved the anticancer activity on different cancers. The species of

jasminum i.e. J. grandiflorum i.e. Jati had already studied for anticancer activity by cell line

study on MCF 7 and MDA MB231.

Sushrutacharya stated that “Rakta” is one of the “Dushya” for Vidradhi (Abscess) and

Granthi (Cyst) formation. In the Susrut samhita Mallika is described as , ”Pittanashini” and in

Raj Nighantu it described as “Vranaropani” , ”Pittanashini” , “Pittaprakophrut”. Due to

“Ashraya-ashrayi Bhav” of Pitta and Rakta Mallika also acts as “Raktdushtihar”. So it can be

useful in the Arbuda and Granthi chikitsa.

Jasminum sambac is studied for antitumor activity by animal study and cervical cancer cell

line. Other species of Jasminum like jasminum grandiflorum have proved their anticancer


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activity by experimental study but still the work on anticancer activity of Mallika on Breast

cancer cell line is not found. So the further attempt has been made to scientifically evaluate

the anticancer activity on breast cancer cell line.

1.2 NEED OF STUDY

Modern science is yet to discover effective remedies for deadly diseases. These diseases are

very complicated in nature with complexity at every level–Anatomy, Physiology,

Biochemistry, Molecular biology, and Gene expression. So treating such a disease is a big

challenge, Worldwide, breast cancer comprises 22.9% of all cancers (excluding non-

melanoma skin cancers) in women. In 2008, breast cancer caused 4,58,503 deaths worldwide

(13.7% of cancer deaths in women).

Additionally toxicity of the drug, major surgeries , radiation damage , chemotherapy hazards

make the situation worse. Survival rates in the Western world are high; for example, more

than 8 out of 10 women (84%) in England diagnosed with breast cancer survive for at least 5

years. In developing countries, however, survival rates are much poorer.

The excruciating experience of dying cancer petient can be ameliorated by making use of

Ayurveda. Ayurveda can be helpful in the management of cancer in many ways such as

prophylactic, palliative, curative, supportive.

1.3 AIM & OBJECTIVES

AIM - To assess the anticancer activity of mallika with special emphasis on breast cancer cell

line by using MTT assay.

OBJECTIVES

To asses the identity of Mallika through pharmacognostical studies.

To extract the phyto-constituents of flowers and leaves of Mallika.

To evaluate anti cancerous activity by Cell based study by performing various assay.


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1.4 DRUG REVIEW

1.4.1 References of Mallika in Bruhtrayee

Table 1: References of Mallika from Bruhatrayee.

Names of sthan Charak samhita Sushrut samhita Ashtang hrudaya

Sutra - 1 2

Nidan - - -

Viman - - -

Sharir - - -

Indrya - - -

Chikitsa - - 1

Kalp - - -

Sidhi - - -

Uttartantra - - -

Utarsthana - - -

Total ref - 1 3

1.4.2 SYNONYMS

Synonyms are the word with same or similar meanings. In Ayurvedic text it holds its own

importance as it describes the morphological and pharmacological aspect of the drug.The

synonym of Mallika explained in ancient text are given below.

Table 2: Synonyms of Mallika in Nighantu.

Synonyms DN RN MN KN SHN AN BPN NA API

Asphota + +

Ashtapadi + +

Gandhanamka +

Gouri +

Girija +

Gavakshi + +

Chandrika +

Jati +

Damayanti +

Narishtha +

Pushpati +

Pramodani + + +

Priya + +

Bhadravalli + +

Bhoopadi + + + +

Madaniya + +

Madayanti + + + + +

Mallika + + + + + + + +

Malati +

Muktabandhana + +

Modini + +

Trunashunya + +


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Yuthika +

Varshika + +

Vanchandrika +

Vichakila +

Sheetbhiru + + + + + +

Soumya + +

1.4.3 GUNA – KARMA

Rasa, guna, veerya, vipak, and prabhava which are simplest parameters to ascertain the

actions of the drugs, are considered as the base of the pharmacology in Ayurveda. The

pharmacological properties of Mallika as mentioned by different acharyas, are presented in

following.

Table 3: Guna Karma of Mallika.

Guna SS DN RN MN KN BPN SHN SHAN NA API

RAS Katu + + + + + + +

Tikat + + + + + + +

Swadu + +

GUNA Laghu + + + +

VEERYA Ushna + + + +

Sheet +

VIPAKA Madhur +

KARMA

VATAHAR + + + +

PITTAHAR + + + + +

KAPHAHAR +

RAKTAJEETA +

VAJIKARAN + +

VRANAHAR + + +

CHAKSHUSHYA + + +

HRUDYA +

TWAKDOSHAHAR + + + +

URDHVAJATRUGAT

VYADHI + +

VISHAHAR + + + + +

KRIMIGHNA +

SHUKRAL +

ARUCHI + + +


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2.0 MATERIAL AND METHODS

2.1 MATERIAL

Drug:-

Drug name – Mallika.

Botanical name - Jasminum sambac Linn. (Ait.)

Family – Oleaceae.

Useful part - flower& leaves.

Source - Anna Hajare Garden,Wadgaon Sheri pune.

Collection - Leaves collected in Varsha rutu flowers collected from plant in Grishmarutu.

Authentication - BOTANICAL SURVEY OF INDIA, PUNE.

2.2 METHODS (STEPS IN EXPERIMENTAL STUDY)

1. Collection & Authentication.

2. Drying.

3. Preparation of Herbal extract.

4. Extraction.

5. Centrifugation.

6. Filtration.

7. Preparation of concentration of extract.

8. Preparation of cell & MTT assay.

2.2.1 DRUG COLLECTION

Flower - May 2015 (Grishma rutu).

Leaf - September 2015 (Varsha rutu).

2.2.2 DRYING

Weight of wet Mallika Leaf - 3kg.

Weight of dry Mallika Leaf - 350gms.

Weight of wet Mallika Flower - 2.5kg.

Weight of dry Mallika Flower- 230gms.

STORAGE - are done as per GMP.

1. Dried samples (leaves and flowers) are stored and handled in a manner which prevents

their mixing up, contamination.

2. Containers of materials are closed, and bagged or boxed.


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3. Containers of materials are labelled with respect to identity.

4. Materials are sampled and tested or examined in conformance.

2.2.1 AUTHENTICATION

Drug has been identified and authentified at BOTANICAL SURVEY OF INDIA, PUNE.

No.BSI/WRC/Tech./2014/.

2.2.3 PREPARATION OF HERBAL EXTRACT

A) Methanolic extract

1. The plant material was ground into coarse powder and 10gm of powder was placed inside

a thimble made from thick filter paper.

2. The thimble was placed into the main chamber of the Soxhlet, extractor.The Soxhlet

extractor was placed into a round bottom flask containing the extraction solvent.

3. The solvent was heated so that the vapours travel up a distillation arm, and flood into the

chamber where the thimble was placed.

4. The chamber containing the thimble fills with warm solvent and depending upon the

solubility different bioactive compounds dissolve in the solvent.

5. When the Soxhlet chamber was almost full, the chamber was automatically emptied by a

siphon side arm, with the solvent running back down to the distillation flask.

6. This cycle was repeated 10-15 times.

7. In this manner extract of leaf and flower were prepared.

B) Aqueous extract

1. The plant material was ground into coarse powder.

2. 30gm powder was placed in the glass beaker.

3. 480 ml water was added in the beaker which was sixteen times than the powder.

4. The solution was heated on the low flame up to the one eighth solution was remaining.

5. The decoction was filtered by muslin cloth.

6. Decoction was centrifuged at 12000 rpm for 15 min.

7. Supernatant solution was kept for overnight drying at 60°C.

8. In this manner aqueous extract of leaf and flower were prepared.


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2.2.4 PREPARATION OF CONCENTRATIONS OF THE EXTRACTS

1. Weighing of herbal extract.

2. Drying it in drier for about 30 to 40 min at 60 °C.

3. Concentration=Final wt-Initial wt.

4. The concentration of methnolic exract of leaf is 4.13gm, flower is 3.2694gm.

5. The concentration of aqueous exract leaf is 3.33 gm, flower is 2.3640gm.

6. Using above value various concentrations of the extracts were prepared (10µg/ml,

20µg/ml up to 640µg/ml) in 10% DMEM.

2.2.5 PREPARATION OF CELL

Maintenance of cell lines: The obtained cell lines were maintained using sterile DMEM

(Gibco) supplemented with 10% FBS (Sigma) at 5% CO2 and 37°C in an incubator

(Thermoforma) in a T-50 flask (Falcon).

Passaging of cells: Cultured cells with adherent property form a monolayer during their

growth. These cells grow laterally to occupy the entire matrix of the culture flask, at this

stage the cells are said to be confluent and for further maintenance passaging was done.

Freezing of cells: Cells which have attained confluency are freezed to preserve them for

further use.

Thawing of cells: Thawing was performed to revive the frozen cell lines. The cryovial

containing cells was retained in warm water prior to transfer.

2.2.6 CYTOTOXICITY DETERMINATION USING MTT ASSAY

Principle

MTT (3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazoliumbromide) assay is performed to

determine the cytotoxic effect of the extract on cell lines. This assay can be used to quantify

the living cells based on the presence of active enzyme systems which is a prerequisite for

live cells.

The MTT dye is reduced to form purple formazan crystals in living cells by various enzymes

of the class oxidoreductase. The formazan crystals are dissolved in solvents like SDS, DMF

etc.

In this experiment the presence of a mitochondrial inner membrane bound enzyme, succinate

dehydrogenase was detected using MTT dye. The presence of this enzyme ensured the

viability of the cells.


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Procedure

Day 1: Seeding of cells in 96 well plate.

Day 2: Dosing of cells in 96 well plate.

Day 3: The media containing the dose was discarded and 100 µl of the MTT solution was

added to every well.

2.3 OBSERVATIONS

2.3.1 Pharmacognostical Study

Standardisation and phyto-chemical analysis of leaf and flower powder is done at IDRAL,

Pune. Report No. 272,273.

Table 4: Organoleptic study.

Sr. No. Characters Leaves Stem Root

1 Sound No significant No significant No significant

2 Touch Smooth Rough Hairy

3 Color Green Greenish brown Light brown

4 Taste Bitter Bitter Bitter

5 Odour Characteristic Characteristic Characteristic

2.3.2 Microscopical study

Leaf- petiole - shows single layered upper and lower epidermis consisting of tubular cells,

covered with striated cuticle; trichomes of two types, non-glandular, uniseriate, 1-5

celled, warty, and with pointed apical cell.

Midrib - cut at basal region shows both upper and lower single layered epidermis,

externally covered with cuticle.

Lamina- shows a dorsi ventral structure, epidermis single layered, externally covered with

cuticle.

Microscpical study of powder

Leaf Powder - Dark green; Palsied tissue in surface view, Stomata with two guard cells,

parenchymatous epidermal hairs multi cellular, spongy cells with chlorophyll, conducting

strands with spiral thickening, cells with brown coloured substance, crystals of calcium.

Flower Powder - Brown coloured; polygonal parenchymatous cells in surface view, few

cells with brown coloured pigment, small pollen grains are seen, no vascular tissue.


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Table 5: Phyto-Chemical Analysis.

Test leaf Flower

pH 4.61% 4.84%

Moisture 7.88% 8.73%

Total Ash 14.12% 10.29%

Acid soluble Ash 13.80% 10.29%

Water soluble Ash 2.34% 6.64%

2.3.3 CALCUTATIONS

(Absorbance of test sample/ Absorbance of control) × 100=% Viability

Table No. 6: Methanolic leaf extract- MDA MB231 (Breast Cancer Cell).

Concentrations 1 2 3 Average %Viability

Control 0.163 0.165 0.159 0.162333 100

10 ug 0.149 0.123 0.139 0.137 84.45929

20 ug 0.159 0.167 0.15 0.158667 97.69925

40 ug 0.239 0.236 0.251 0.242 149.1726

80 ug 0.279 0.291 0.312 0.294 181.2519

160 ug 0.312 0.315 0.326 0.317667 195.7839

320 ug 0.42 0.423 0.427 0.423333 260.8619

640 ug 0.353 0.385 0.361 0.366333 225.6473

Table No. 7: Aqueous leaf extract- MDA MB231 (Breast Cancer Cell).


Control 0.128 0.14 0.137 0.135 100

10 ug 0.118 0.1 0.124 0.114 84.70901

20 ug 0.126 0.123 0.137 0.128667 95.43155

40 ug 0.162 0.175 0.175 0.170667 126.4332

80 ug 0.21 0.225 0.245 0.226667 167.8696

160 ug 0.252 0.278 0.289 0.273 202.1318

320 ug 0.292 0.318 0.371 0.327 242.0236

640 ug 0.272 0.279 0.334 0.295 218.5271

Table No. 8: Methanolic flower extract- MDA MB231 (Breast Cancer Cell).


Control 0.168 0.173 0.154 0.165 100

10 ug 0.149 0.157 0.17 0.158667 96.61051

20 ug 0.165 0.185 0.193 0.181 110.1585

40 ug 0.231 0.26 0.258 0.249667 151.7738

80 ug 0.251 0.288 0.254 0.264333 160.2713

160 ug 0.314 0.323 0.321 0.319333 194.0172

320 ug 0.39 0.391 0.387 0.389333 236.4844

640 ug 0.326 0.314 0.307 0.315667 191.6337


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Table No. 9: Aqueous flower extract- MDA MB231 (Breast Cancer Cell).


Control 0.131 0.152 0.139 0.140667 100

10 ug 0.119 0.111 0.117 0.115667 82.67956

20 ug 0.139 0.131 0.151 0.140333 100.3081

40 ug 0.189 0.216 0.222 0.209 148.6974

80 ug 0.263 0.259 0.29 0.270667 193.2637

160 ug 0.312 0.304 0.304 0.306667 218.9577

320 ug 0.372 0.386 0.422 0.393333 280.5047

640 ug 0.313 0.349 0.432 0.364667 259.776

Table No. 10: Methanolic leaf extract- MCF7 (Breast Cancer Cell).


Control 0.246 0.228 0.226 0.233333 100

10 ug 0.265 0.267 0.278 0.27 115.9459

20 ug 0.31 0.293 0.309 0.304 130.4169

40 ug 0.259 0.241 0.243 0.247667 106.1695

80 ug 0.246 0.273 0.234 0.251 107.7589

160 ug 0.23 0.197 0.217 0.214667 91.97238

320 ug 0.236 0.211 0.189 0.212 90.70238

640 ug 0.158 0.161 0.12 0.146333 62.64634

Table No. 11: Aqueous leaf extract- MCF7 (Breast Cancer Cell).


Control 0.273 0.242 0.274 0.263 100

10 ug 0.27 0.27 0.26 0.266667 101.7873

20 ug 0.373 0.361 0.322 0.352 134.4406

40 ug 0.32 0.264 0.276 0.286667 109.0123

80 ug 0.36 0.388 0.385 0.377667 144.2366

160 ug 0.36 0.345 0.32 0.341667 130.4061

320 ug 0.444 0.487 0.402 0.444333 170.1975

640 ug 0.342 0.345 0.375 0.354 134.8993

Table No. 12: Methanolic flower extract- MCF7 (Breast Cancer Cell).


Control 0.268 0.263 0.275 0.268667 100

10 ug 0.317 0.332 0.334 0.327667 121.9913

20 ug 0.269 0.354 0.313 0.312 116.264

40 ug 0.284 0.303 0.292 0.293 109.1204

80 ug 0.315 0.24 0.245 0.266667 99.29433

160 ug 0.262 0.322 0.303 0.295667 110.1255

320 ug 0.274 0.267 0.208 0.249667 93.13203

640 ug 0.177 0.184 0.163 0.174667 65.09316


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Table No. 13: Aqueous flower extract- MCF7 (Breast Cancer Cell).


Control 0.268 0.267 0.21 0.248333 100

10 ug 0.255 0.257 0.236 0.249333 101.2616

20 ug 0.328 0.356 0.329 0.337667 137.4627

40 ug 0.326 0.28 0.271 0.292333 118.5194

80 ug 0.351 0.307 0.304 0.320667 130.2378

160 ug 0.37 0.397 0.349 0.372 150.9798

320 ug 0.487 0.482 0.385 0.451333 181.858

640 ug 0.383 0.377 0.312 0.357333 144.2268

Figure No. 1: MTT on MDA MB231 (Breast cancer cell).

Figure No. 2: MTT on MCF7 (Breast cancer cell).

3.0 RESULT AND DISCUSSION

3.1 RESULT

MTT assay was performed to define the optimal concentration at which extract was toxic

to cells.

Aqueous extracts of both leaf and flower were found to be non-toxic upto 640µg/ml in

both breast cancer cell lines.


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Methanolic extract of leaf and flower were found to be toxic on MCF-7 at the higher two

concentrations (320, 640µg/ml) and non-toxic to MDA MB231 cell line as evident from

graph.

3.2 DISCUSSION

As per the references collected from ayurvedic literature, it is observed that sushrutacharya

and Ashtang Hrudaya karas mentioned mallika as name Mallika itself .All the nighantukaras

gave the synonyms of mallika and they included it in differant vargas.

In pharmacognostical and microscopical analysis there is no significant result found. In

phytochemical analysis pH of leaf and flower is nearby same but other values like moisture

total ash values are varies.

From experimental study it is observed that methanolic extracts of mallika have significant

cytotoxicity on MCF7 breast cancer cells and other extracts are not showing such a type of

activity on both MCF7& MDAMB 231 cell lines.

4.0 CONCLUSION

Mallika has the anticancer activity on MCF7 cell line. There is cytotoxic effect found of

methanolic extracts of leaf and flower of mallika in MCF-7 breast cancer cell line at 320 and

640µg/ml.

Further Scope of Study

Further studies need to be conducted to investigate the cytotoxic activity of the extract in

higher concentrations and also in various assays and in other cancer cell lines.

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