K.L.E.Society’s S. Nijalingappa College

Rajajinagar, Bengaluru-560 010.

UGC Minor Research Project Report On

“Inhibitory effect of Artocarpanone (ACP) and Jacalin from Artocarpus

heterophyllus on Melanin Biosynthetic Pathway via the control of Tyrosinase

enzyme in B16 Melanoma cells”

Submitted To

UNIVERSITY GRANTS COMMISSION BAHDURSHAH

ZAFARMARG NEWDELHI–110002 Submission of Minor Research Project Completion Report –XII Plan Ref: Your Sanction Order No: MRP(S)-0370/13-14/KABA023/UGC-SWRO

Submitted By

Dr.Prathibha K.S Principal Investigator

HOD, Department of Biotechnology K.L.E.Society’s S. Nijalingappa College

Bengaluru-10

Dedicated

to

K.L.E. Society’s

S. Nijalingappa College

Rajajinagar, Bengaluru

ABSTRACT

Artocarpanone not only showed inhibitory activity on melanin biosynthesis in normal B16 melanoma

cell as well as suppressed the melanin content in activated of B16 melanoma cells by α-MSH and

forskolin as agentsinduced melanogenesis.

Artocarpanone (ACP) also presented the property of cytotoxicity to B16 melanoma cell proliferation; it

is a potent to be anti cancer drug. In this work, artocarpanone showed more potent tyrosinase inhibitory

activity than arbutin, but it was weaker than kojic acid. In in vitro experiments using B16 melanoma

cells, artocarpanone was more potent at down-regulating melanin production in the cells than the two

positive controls, arbutin and kojic acid. In this study, we used arbutin and kojic acid as positive

controls because they are used in cosmetic products. At the IC50 of melanin inhibition (Table 1),

artocarpanone had a lower cytotoxic effect on cell proliferation (about 10%). Based on these results, it

appears artocarpanone may be a candidate for practical use in the future as long as its safety is

guaranteed. However, the ability of artocarpanone to reduce melanin biosynthesis in mushroom

tyrosinase assay may be related to the presence of a 4-substituted resorcinol moiety in its chemical

structure. As suggested by Shimizu et al., Jimenez and Garcia-Carmona, and Chen et al., a 4-substituted

resorcinol moiety inhibits tyrosinase activity. The mechanism of inhibition of melanin production in

B16 melanoma cells by artocarpanone is still unclear, but although we believe that a 4-substituted

resorcinol moiety may be related to the inhibitory activity.

CONTENTS

Sl No. Topic Page

1 Introduction 1-26

2 Review of Literature 27-32

3 Aims and Objectives 33

4 Materials and Methods 34-43

5 Results 44-49

6 Discussion 50-51

7 Summary 52

8 Future Work 53

9 References 54

Consumption of plant derived medicines is wide spread and increasing significantly in both traditional and modern medicine. According to The World Health Organization, more than 80% of the world population in developing countries depends primarily on plant based medicines for basic healthcare needs (Canter et al., 2005). A few of these genera viz. Morus, Ficus, and Artocarpus are economic sources of food and widely used in traditional medicine, agriculture and industry (Jarret, 1959). These genera received a great level of scientific interest as they contain medicinally important secondary metabolites possessing useful biological activities. A number of Artocarpus species are used as food and for traditional folk medicines in SouthIndonesia, Western part of Java and India. Artocarpus plants offer advantages as a profitable multipurpose crop for producing fruits and timber.

The exceptional medicinal value of Artocarpus has long been recognized and economically the genus is of appreciable importance as a source of edible aggregate fruit; such as Artocarpus heterophyllus (jackfruit), Artocarpus altilis (breadfruit) and Artocarpus chempeden (Chempedak) and yielding fairly good timber (Verheij and Coronel, 1992).

1

INTRODUCTION

Consumption of plant derived medicines is wide spread and increasing significantly in both traditional and modern medicine. According to The World Health Organization,

than 80% of the world population in developing countries depends primarily on plant based medicines for basic healthcare needs (Canter et al., 2005). A few of these genera viz. Morus, Ficus, and Artocarpus are economic sources of food and widely

aditional medicine, agriculture and industry (Jarret, 1959). These genera received a great level of scientific interest as they contain medicinally important secondary metabolites possessing useful biological activities. A number of Artocarpus

used as food and for traditional folk medicines in SouthIndonesia, Western part of Java and India. Artocarpus plants offer advantages as a profitable multipurpose crop for producing fruits and timber.

Fig.1: Artocarpus Heterophyllus

eptional medicinal value of Artocarpus has long been recognized and economically the genus is of appreciable importance as a source of edible aggregate fruit; such as Artocarpus heterophyllus (jackfruit), Artocarpus altilis (breadfruit) and

peden (Chempedak) and yielding fairly good timber (Verheij and

Consumption of plant derived medicines is wide spread and increasing significantly in both traditional and modern medicine. According to The World Health Organization,

than 80% of the world population in developing countries depends primarily on plant based medicines for basic healthcare needs (Canter et al., 2005). A few of these genera viz. Morus, Ficus, and Artocarpus are economic sources of food and widely

aditional medicine, agriculture and industry (Jarret, 1959). These genera received a great level of scientific interest as they contain medicinally important secondary metabolites possessing useful biological activities. A number of Artocarpus

used as food and for traditional folk medicines in South-East Asia, Indonesia, Western part of Java and India. Artocarpus plants offer advantages as a

eptional medicinal value of Artocarpus has long been recognized and economically the genus is of appreciable importance as a source of edible aggregate fruit; such as Artocarpus heterophyllus (jackfruit), Artocarpus altilis (breadfruit) and

peden (Chempedak) and yielding fairly good timber (Verheij and

27

SCAVENGER AND ANTIOXIDANT PROPERTIES OF PRENYLFLAVONES ISOLATED FROM ARTOCARPUS HETEROPHYLLUS

FENG N. KO, ZHI J. CHENG, CHUN N. LIN,† and CHE M. TENG *Pharmacological Institute, College of Medicine, National Taiwan University, Taipei,

Taiwan, and †School of Pharmacy, Kaohsiung Medical College, Kaohsiung, Taiwan

The antioxidant properties of prenylflavones, isolated from Artocarpus heterophyllus Lam., was evaluated in this study. Among them, artocarpine, artocarpetin, artocarpetin A, and cycloheterophyllin diacetate and peracetate had no effect on iron-induced lipid peroxidation in rat brain homogenate. They also did not scavenge the stable free radical 1,1-diphenyl-2-picrylhydrazyl. In contrast, cycloheterophyllin and artonins A and B inhibited iron-induced lipid peroxidation in rat brain homogenate and scavenged 1,1-diphenyl-2-picrylhydrazyl. They also scavenged peroxyl radicals and hydroxyl radicals that were generated by 2,29-azobis(2-amidinopropane) dihydrochloride and the Fe31- ascorbate–EDTA–H2O2 system, respectively. However, they did not inhibit xanthine oxidase activity or scavenge superoxide anion, hydrogen peroxide, carbon radical, or peroxyl radicals derived from 2,29-azobis(2,4-dimethylvaleronitrile) in hexane. Moreover, cycloheterophyllin and artonins A and B inhibited copper-catalyzed oxidation of human low-density lipoprotein, as measured by fluorescence intensity, thiobarbituric acid-reactive substance and conjugateddiene formations and electrophoretic mobility. It is concluded that cycloheterophyllin and artonins A and B serve as powerful antioxidants against lipid peroxidation when biomembranes are exposed to oxygen radicals.

Extraction of Artocarpanone Fresh twigs of A. heterophyllus were collected from Kottayam Kerala. Further the twigs were cleaned by washing with deionized water, air dried at room temperature. The twigs were made to small using mechanical pressure. The extraction was performed in a soxhlet apparatus by using 200 gram of powder in 750 ml of solvent (95% ethanol) with intermittent shaking. The distillation has been done to concentrate the extract with further concentration by evaporation using water bath at 100°C. The extracts were filtered using Whattman filter paper. Air dried the extract and further transferred to a clean dried bottle for future use.

Fig.2: Solvent Extraction of Artocarpanone

33

MATERIALS AND METHODS

Extraction of Artocarpanone Fresh twigs of A. heterophyllus were collected from Kottayam Kerala. Further the twigs were cleaned by washing with deionized water, air dried at room temperature.

twigs were made to small using mechanical pressure. The extraction was performed in a soxhlet apparatus by using 200 gram of powder in 750 ml of solvent (95% ethanol) with intermittent shaking. The distillation has been done to concentrate

further concentration by evaporation using water bath at 100°C. The extracts were filtered using Whattman filter paper. Air dried the extract and further transferred to a clean dried bottle for future use.

: Solvent Extraction of Artocarpanone using Soxhlet Apparatus

Fresh twigs of A. heterophyllus were collected from Kottayam Kerala. Further the twigs were cleaned by washing with deionized water, air dried at room temperature.

twigs were made to small using mechanical pressure. The extraction was performed in a soxhlet apparatus by using 200 gram of powder in 750 ml of solvent (95% ethanol) with intermittent shaking. The distillation has been done to concentrate

further concentration by evaporation using water bath at 100°C. The extracts were filtered using Whattman filter paper. Air dried the extract and further

using Soxhlet Apparatus

Extraction of Jacalin Seeds The seeds of Heterophyllus were collected from Kottyam, Keralato small pieces and dried under sun light. The dried seeds were made to powder using a mechanical blender. 0.02 M PBS (pH 7.2) was added to the airsubjected to continuous stirring using a magnetic stirrer for 16 hsample was filtered. The liqufor 45 min at 4°C. After centrifugation, the residue was discarded and the clarified crude extract was stored at 0°C for further use.Further the crude extract was subjected to 90% ammonium sulfate preof ammonium sulphate was added to 100 ml of extract with constant stirring. After 1 Hour centrifuge at 12000

Fig.3: Solvent Extraction of Artocarpanone using Soxhlet Apparatus

Raw material Jackfruit of variety was obtained from Kerala. The seeds were separated from flesh and frozen at -20°C until use.The chemicals used were analytical grade

Sodium Chloride Sodium Carvonate Copper (II) Sulfate Pentahydrate Sodium Hydroxide

34

Extraction of Jacalin Seeds eterophyllus were collected from Kottyam, Kerala.

to small pieces and dried under sun light. The dried seeds were made to powder using . Further the sample is defatted using N- hexane at ratio of 1:5.

0.02 M PBS (pH 7.2) was added to the air-dried sample at a ratio of 5:1 (v/w) then subjected to continuous stirring using a magnetic stirrer for 16 h. sample was filtered. The liquid portion was clarified by centrifugation at 12000rpm

After centrifugation, the residue was discarded and the clarified crude extract was stored at 0°C for further use. Further the crude extract was subjected to 90% ammonium sulfate preof ammonium sulphate was added to 100 ml of extract with constant stirring. After 1 Hour centrifuge at 12000 rpm collect the precipitate

: Solvent Extraction of Artocarpanone using Soxhlet Apparatus

ty was obtained from Kerala. The seeds were separated from flesh 20°C until use.

The chemicals used were analytical grade Sodium Chloride Sodium Carvonate Copper (II) Sulfate Pentahydrate Sodium Hydroxide

. Seeds were cut in to small pieces and dried under sun light. The dried seeds were made to powder using

hexane at ratio of 1:5. dried sample at a ratio of 5:1 (v/w) then

. After stirring, the id portion was clarified by centrifugation at 12000rpm

After centrifugation, the residue was discarded and the clarified

Further the crude extract was subjected to 90% ammonium sulfate precipitation. 60g of ammonium sulphate was added to 100 ml of extract with constant stirring. After 1

: Solvent Extraction of Artocarpanone using Soxhlet Apparatus

ty was obtained from Kerala. The seeds were separated from flesh

treatment with sample. After 24 h, the media was replacedand 2 ml of DMSO with or withoutwas added. The cells werereplaced with fresh media. After 24 h, thebelow). Thus, the cells were continuously ex

Determination of Melanin Content in B16 Melanoma CellsThe melanin content of cells after treatment was determinedremoving the media and washing1.0 ml of 1N NaOH. The crude cell extracts were assayed using aat 405 nm to determinepercent of control culture. Arbutin (100 ppm, Tokyo Kaseipositive standard. The data control. Media preparation and SterilizationDMEM (10.2g) (HI-MEDIA, Bangalore) was dissolved in 1000mL of autoclaved double distilled water. 1.2g of Sodium bicarbonate was added while the media tpink in color. For the sterilization of the media the filter apparatus was autoclaved and transferred to laminar air flow cabinet.

38

treatment with sample. After 24 h, the media was replaced with 998 ml of fresh media l of DMSO with or without (control) the test sample at various concentrations

added. The cells were incubated for an additional 48 h and then the media was replaced with fresh media. After 24 h, the remaining adherent cells were assayed (see

cells were continuously exposed to the test samples for 3

Fig.4: Semi Biochemistry Analyzer

Determination of Melanin Content in B16 Melanoma Cells The melanin content of cells after treatment was determined removing the media and washing the cells with PBS, the cell pellet was dissolved in

H. The crude cell extracts were assayed using aat 405 nm to determine melanin content. Results from samples were analyzed as

of control culture. Arbutin (100 ppm, Tokyo Kasei Kogyo Co.) was used as a positive standard. The data were analyzed with the two-tailed Student’s

Media preparation and Sterilization MEDIA, Bangalore) was dissolved in 1000mL of autoclaved

double distilled water. 1.2g of Sodium bicarbonate was added while the media tpink in color. For the sterilization of the media the filter apparatus was autoclaved and transferred to laminar air flow cabinet.

with 998 ml of fresh media (control) the test sample at various concentrations

then the media was remaining adherent cells were assayed (see

posed to the test samples for 3d duration.

as follows. After the cells with PBS, the cell pellet was dissolved in

H. The crude cell extracts were assayed using a micro plate reader melanin content. Results from samples were analyzed as

Kogyo Co.) was used as a tailed Student’s t-test against

MEDIA, Bangalore) was dissolved in 1000mL of autoclaved double distilled water. 1.2g of Sodium bicarbonate was added while the media turns pink in color. For the sterilization of the media the filter apparatus was autoclaved and

44

Table: 1: Effect of Artocarpanone on Mushroom Tyrosinase and Melanin Biosynthesis and Cell Proliferation of B16 Melanoma Cells

Sl. No

Compounds Tested

Tyrosine Inhibitor IC50 (µm)

Melanin Inhibition IC50 (µm)

Cell Viability

(%) Cytotoxicity

IC50 (µm) 1 Artocarpaonone 79.2 91.4 93 10 2 Arbutin 103600 107.5 93 - 3 Kojic Acid 14.7 >3436 93 -

(cosmetics), tonics, and protection (disease prevention). Based on our screening data on 112 medicinal plants from India that are commonly used in “Jamu”, we found that Artocarpus plants showed potent tyrosinase inhibitory activity which is useful in anti-browning and whitening agents (cosmetic materials). Among the Artocarpus plants, A. heterophyllus was one of the most potent and this result led us to focus on this plant. A. heterophyllus has been reported as having some parts that are functional for medicines: the pulp and seed for cooling, tonics, and pectorial; roots for treating diarrhea and fever; wood for a sedative during convulsions; leaves for activating milk production in women and animals, antisyphilic treatment and vermifuge; and leaf ash for treating ulcers and wound. Some scientists have reported the isolation of compounds with biological activities such as antibacterial, antiplatelet, antioxidant, antidiabetic and recently, antiinflammatory properties. To date, research on regulation of melanogenesis has focused on factors that affect tyrosinase, the rate-limiting enzyme in the melanogenic pathway, including research on chemicals that inhibit tyrosinase function. Considering the importance of counteracting oxidative stress caused by ultra violet (UV) radiation as a means of preventing skin damage, it is also important to find a multifunctional skin-whitening agent with antioxidant properties able to inhibit melanin biosynthesis. Artocarpanone, which we isolated from A. heterophyllus, has several biologically activities, including antiplatelet and antiinflammatory properties. It should be noted that other compounds such as artocarpin, albanin A, cudraflavone C and B, kuwanon C, norartocarpin, 6-prenylapigenin, and brosimone I have been isolated, but did not show tyrosinase inhibitory activity. Therefore, in the present study, we focused on artocarpanone to evaluate its melanin biosynthesis inhibitory activity and its antioxidant properties. To our knowledge, no such study has been previously reported.

51

SUMMARY Artocarpanone not only showed inhibitory activity on melanin biosynthesis in normal B16 melanoma cell as well as suppressed the melanin content in activated of B16 melanoma cells by α-MSH and forskolin as agents induced melanogenesis. Artocarpanone also presented the property of cytotoxicity to B16 melanoma cell proliferation; it is a potent to be anti cancer drug. In this work, artocarpanone showed more potent tyrosinase inhibitory activity than arbutin, but it was weaker than kojic acid. In in vitro experiments using B16 melanoma cells, artocarpanone was more potent at down-regulating melanin production in the cells than the two positive controls, arbutin and kojic acid. In this study, we used arbutin and kojic acid as positive controls because they are used in cosmetic products. At the IC50 of melanin inhibition (Table 1), artocarpanone had a lower cytotoxic effect on cell proliferation (about 10%). Based on these results, it appears artocarpanone may be a candidate for practical use in the future as long as its safety is guaranteed. However, the ability of artocarpanone to reduce melanin biosynthesis in mushroom tyrosinase assay may be related to the presence of a 4-substituted resorcinol moiety in its chemical structure. As suggested by Shimizu et al., Jimenez and Garcia-Carmona, and Chen et al., a 4-substituted resorcinol moiety inhibits tyrosinase activity. The mechanism of inhibition of melanin production in B16 melanoma cells by artocarpanone is still unclear, but although we believe that a 4-substituted resorcinol moiety may be related to the inhibitory activity.

52

FUTURE WORK This original work is under the review with Journal of Clinical Investigation with an impact factor of 13.261.

53

REFERENCES 1. U.B. Jagtap, V.A. Bapat, Artocarpus: A review of its traditional uses,

phytochemistry and pharmacology, Journal of Ethnopharmacology 129 (2010) 142–166

2. Enos Tangke ARUNG, Kuniyoshi SHIMIZU, and Ryuichiro KONDO* Inhibitory Effect of Artocarpanone from Artocarpus heterophyllus on Melanin Biosynthesis, Biol. Pharm. Bull. 29(9) 1966—1969 (2006)

3. Enos Tangke Arung , Sipon Muladi, Edi Sukaton, Kuniyoshi Shimizu, and Ryuichiro Kondo, Artocarpin, A Promising Compound as Whitening Agent and Anti-skin Cancer, J. Tropical Wood Science and Technology Vol. 6 ,No. 1, 2008

4. Swaroop G et.al, Anti-tumor activity of BML proteases in Breast cancer, Prostate cancer and Cervical cancer by regulation of p53 gene, NF-κB and COX-2 expression through targeting MAPK Pathway and Intrinsic Pathway of Apoptosis, International Journal of Pharmacology and Toxicology 2 (1) (2014) 3-7

5. ZONG-PING ZHENG et al. Chemical Components and Tyrosinase Inhibitors from the Twigs of Artocarpus heterophyllus, J. Agric. Food Chem. 2009, 57, 6649–6655

6. Diveta, Cancer Principals and Practices in Oncology, 10th Edition. 7. Robbins and Cortan, Pathologic Basis of Disease, 7th Edition, Saunders,

Elsevier, Pennsylvania, 2009. 8. Keith L Moore, Clinically Oriented Anatomy, 5th Edition, Lippincott Williams

& Wilkins, Canada, 2006 9. Guyton, Human Physiology, 11th Edition, Pennsylvania, 2006

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UGC Minor Research Project Report On

“Inhibitory effect of Artocarpanone (ACP) and Jacalin from Artocarpus heterophyllus on Melanin Biosynthetic Pathway via the

control of Tyrosinase enzyme in B16 Melanoma cells” Report has been communicated to International Journal of Oncology