INTRODUCTION:

Estrogen receptors (ER) expression is the main indicator of potential responses to hormonal therapy, and approximately 80% of breast cancers are estrogen receptor (ER)-positive [1]. ER represents the primary transcription factor driving oncogenesis in hormone receptor-positive of breast cancer [2]. The biological effects of estrogens are mainly mediated by ER alpha which functions as a ligand-inducible transcription factor.

Hormone receptor was hormone therapy should be considered complementary to surgery in the majority of patients, inducing tumor remission and providing consistent clinical benefit. The two major strategies are directed at blocking ER of cancer cells or against the key enzyme in the aromatase inhibitors (AI) of estrogens.

ER activity is expressed especially in the ovary (premenopausal women), testis, placenta, brain, bone and adipose tissue (postmenopausal women)[3]. AI block aromatase enzyme activity, safely reducing circulating estrogen levels only in postmenopausal patients. aromatase [cytochrome P450 19 (CYP19)], which catalyzes the conversion of androgens, such as testosterone and androstenedione, to estrogens, such as 17-β-estradiol and estrone [4]. Herbal from Kaempferia pandurata contain some flavonoids, have been shown to be potent aromatase inhibitors by in vitro assay [5].

The rhizome of this plant is one of the components of herbal medicine jamu in Indonesia [6]. This plant has been identified to contain various essential oils (EOs) and also several flavonoid compounds that have demonstrated many biological activities. Extract of kaemferia that showed broad range of biological activities, such as strong antibacterial activity, anti-inflammatory, and anti-cancer [7-9].

Confirmation of chemical active from Kaempferia pandurata have done by in silico simulation with ER targeted. Lead discovery and identification can be performed by in-silico techniques such as molecular docking[10]. Molecular docking is an effective and fast computational technique to estimate the binding affinity of a ligand (drug candidate) in the macromolecular target site (receptor)[11]. Potent for drug discovery should be accompanied with chemical optimization and appendixes with sufficient wet laboratory in-vitro proof of the efficacy and safety of the proposed drug candidates.

Figure 1: Structure 3D estrogen receptor structure 6B0F

MATERIALS AND METHODS:

Preparation of Protein

Preparation of the estrogen receptor structure, the crystal structure was downloaded from the Protein Data Bank (PDB_ID: 6B0F) Figure 1. Hetero atoms were removed from the binding site and the chain A was selected for docking studies. Hydrogen atoms were added to the enzyme. The molecular docking method was performed using the Autodock 4.2 program to study the binding orientation of compounds into the aromatase structure. The docking experiments were performed using the binding site of estrogen receptor.

Docking Simulation:

Docking simulation of extract Kaempferia pandurata were performed. The structures of these compounds (Figure 2,3 and Table 1,2) were constructed and optimized using Marvinsketch software. Docking was carried out using Autodock 4.2 software which is based on genetic algorithm (GA). This method allows as partial flexibility of protein and full flexibility of ligand[12]. The parameters used for GA were population size (100), selection pressure (1.1), number of operations (10,000). Operator parameters default cutoff values of 3.0Å for hydrogen bonds and 6.0Å for van der Waals were employed. During docking, the default algorithm speed was selected and the ligand binding site within a 10Å. The number of poses for each inhibitor was set 100, and early termination was allowed if the top three bound conformations of a ligand were within 1.9Å RMSD.

Figure 2: Chalcone based Structure

Table 1. Secondary metabolites with Chalcone compounds of Extract Kaempferia pandurata

Compounds	R1	R2	R3	R4
1	OMe	OH	OH	H
2	OH	OH	OH	H
3	OMe	OH	OH	OH
4	OH	OMe	OH	H
5	OMe	OMe	OH	OH
6	OH	H	OH	H
7	OMe	OH	OH	H

Flavonoids are large secondary metabolites found in rhizome of K. pandurata. More than 51 flavonoid compounds from K. pandurata have been isolated and their structure was confirmed. However, only three classes of flavonoid have been reported exist in K. pandurata rhizome.

Figure 3: Flavanones based structure

Table 2. Secondary metabolites with flavanones compounds of Extract Kaempferia pandurate

Compounds	R1	R2	R3	R4	R5
8	OMe	OH	H	H	H
9	OH	OH	H	H	H
10	OMe	OMe	H	H	H
11	OH	OMe	H	H	H
12	OMe	OH	H	H	OH
13	OH	OMe	H	H	OH
14	OMe	OH	H	H	OH
15	OMe	OMe	H	H	OH
16	OMe	OMe	OMe	H	H
17	OMe	OMe	H	H	OMe
18	OMe	OH	H	H	OMe
19	OMe	OMe	H	OMe	OMe
20	OMe	OH	OMe	H	OMe
21	OMe	OH	OMe	OMe	OMe

Extraction of Kaempferia pandurate:

Extraction was done by maceration, the whole or coarsely powdered crude (100 Gram) is placed in a stoppered container with the solvent (1 L) of ethyl acetate, hexane and aqueous and allowed to stand at room temperature for a period of at least 3 days with frequent agitation until the soluble matter has dissolved. The mixture then is strained, the marc (the damp solid material) is pressed, and the combined liquids are clarified by filtration or decantation after standing. After that the extract was filtered and evaporated with evaporator.

In vitro Assay:

The cell lines were prepared and cryopreserved using reagents such as DMSO which preserve the cell during freezing. DMSO is toxic at room temperature. The freezed ampoule is brought to room temperature by slow agitation (thawing). The freezed cryovials plunged into the water bath and is rapidly thawed until it gets liquified. Solution, centrifuged with saline for 10 mins to remove the DMSO. The saline is discarded and aliquot is taken for cell counting, cell viability and for sub culturing. MTT assay is a quantitative colorimetric assay for measuring cellular growth, cell survival and cell proliferation based on the ability of living cells. The assay was carried out using (3-(4, 5-dimethyl thiazol-2yl) -2, 5-diphenyl tetrazolium bromide (MTT)[13-16]. The result of In vitro cytotoxic activity of hexane, ethyl acetate and aqueous extracts of Kaempferia pandurata Breast cancer cell lines (MCF 7 cell line) is tabulated in Table 4.

RESULT AND DISCUSSION:

Structure-based drug design begins with the identification of a molecular target such as a protein. This structure of protein used as a targeted for the drug discovery of a lead compound. The compounds are constructed for their fit in the active site of the target and functional group interactions. The selected docked conformations of chemical compounds from extract Kaempferia pandurata into the ESR binding site were shown in Figure 4. The docked conformations revealed that all ligands were located in the hydrophobic binding pocket. In this study, all docked compounds were found to have some interactions between an oxygen atom of the compounds and ESR. Moreover, these docked conformations also formed an H-bonding interaction within the active site with active finder shown that active site order (Glu323, Pro324, Ile327, Met343, Leu346, Thr347, Glu353, Leu391, Phe404 and Met421) Figure 4. The binding region ESR with site order 309….546 are ligand binding domain of Estrogen receptor, which are activated by the hormone 17beta-estradiol estrogen. Order site (345,350,352,386,393,520,523) are ligand binding site with chemical binding. Order site of ESR (357, 361, 374. 375, 379, 537, 541. 542) as coactivator recognition site of polypeptide binding[17].

Figure 4: Active site (red cycle) of Estrogen Receptor with PDB code 6B0F

The docking results shown as Table 3, compounds Helichrysetin has the lowest ΔG and Ki values, even lower compared to gossypol, which means that this molecule is most active when bound to estrogen receptors a. The hydrogen bonds that occur between the Helichrysetin and the receptor are similar to the hydrogen bonds that are present between gossypol and its receptor, called Thr347, Phe404. This hydrogen bonding as active site of ESR.

ESR-2

ESR-3

Figure 5: Conformation changes the active site of 3D structure ESR-2 to ESR-3

Table 3. Docking of complex ER with compounds of extract Kaempferia pandurata

No.	Compounds	ΔG	pKi	Hbond
1	Cardomonin	-9.1008	5.672	-
2	Pinocembrine chalcone	-10.4711	7.823	Thr347, Glu353
3	Helichrysetin	-11.7522	8.334	Thr347, Glu352, Phe404
4	2,6-dihydroxy-4-methoxychalcone	-6.7889	4.251	-
5	Flavokawain C	-10.2832	7.567	Glu323, Pro324,
6	2’,4’,6’-trihydroxychalcone	-9.7232	5.671	Ile327
7	Uvangoletin	-8.5130	6.356	Ile327
8	Pinostrobin	-7.3501	4.562	-
9	Pinocembrine	-6.5418	5.752	-
10	5,7-dimethoxyflavanone	-6.7563	5.431	-
11	Alpinetin	-6.8931	4.936	-
12	Sakuranetin	-10.5349	7.380	Glu323, Phe404
13	7,4’-dihydroxy-5methoxyflavanone	-9.8648	6.065	Met343, Leu346
14	Tectochrysin	-9.2663	5.987	Glu323
15	5,7-dimethoxyflavone	-6.4512	5.096	-
16	5-hydroxy-3,7-dimethoxyflavone	-7.5887	5.167	-
17	5,7,4’-trimethoxyflavone	-7.9826	5.611	-
18	5-hydroxy-7,4’dimethoxyflavone	-7.1251	5.226	-
19	5,7,3’,4’-tetramethoxyflavone	-8.5862	6.562	Met343
20	5-hydroxy-3,7,4’trimethoxyflavone	-9.6579	6.182	Leu346
21	5-hydroxy-3,7,3’,4’tetramethoxyflavone	-8.8776	6.351	Glu323
22	Gossypol	-10.8754	7.012	Thr347, Phe404

Figure 5 shows that helichrysetin compounds changes conformation the active site of 3D structure ESR. Pinocembrine chalcone compounds from docking showed low ΔG value, but Helichrysetin compounds were more effective, because of the 3D structure this compound did not change the active site of the ESR.

Based on insilico study, were confirmed on invitro assay by MTT. The cytotoxic activity of the hexane, EtoAc and aqueous extracts of Kaempferia pandurata on MCF-7 cells from human breast cancer was investigated in vitro 3-(4) 5-Dimethyl-thiazol-zyl) -2,5 biphenyl tetrazoliumbromide (MTT). The results showed decreased cell viability and cell growth inhibition in a dose dependent manner. The IC50 value of standard cisplatin, gossypol, hexane extract was 8.42; 21.98; 26.70 μg/ml respectively. Hexane extracts of Kaempferia pandurata demonstrated anti-proliferative activities. Many studies in the past have assumed that the free hydroxyl groups of the flavonoids and other polyphenols are necessary for biological effects. The long half belief that their structural requirements for antiproliferative activity on breast cancer cells [18].

The docking results agreed well with the observed in vitro data, which showed that the Estrogen Receptor inhibitory activity of helichrysetin (-11.7522 KCal/mol) was higher than those of other compounds. Our investigations shown that Cisplatin and gossypol has good inhibitory activity on ER and this can be helpful for further investigations. The docking results data supports the inhibitory activity of pinocembrine chalcone and helichrysetin.

CONCLUSION:

In conclusion show that the flavonoid compound against the target ER breast cancer with insilico have enthalpy energy ΔG -11.75 kcal/mol, in vivo test shown that IC₅₀value of standard cisplatin, hexane extract was 8.42; 26.70 μg/ml respectively to MCF-7 cell line showed that Kaempferia pandurata Roxb extract very potential as antibreast cancer. the flavonoids compound dominates the composition of the Kaempferia pandurata Roxb. and Pinocembrine chalcone and helichrysetin potent as anticancer activities to breast cancer cell line.

ACKNOWLEDGEMENTS:

We would like to thank our team. We are grateful to PITTA 2018 from DIKTI and DRPM Universitas Indonesia for funding our research projects at present times.

CONFLICT OF INTREST:

The Authors declare no conflict of interest.

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Received on 12.02.2019 Modified on 14.03.2019

Research J. Pharm. and Tech. 2019; 12(5):2391-2395.

DOI: 10.5958/0974-360X.2019.00400.1

Concentration	Extract Hexane	Extract EoAc	Extract Aq	Cisplatin	Gossypol
3.125	95,008	113,547	86,987	73,440	83,778
6,251	67,379	85,3832	64,349	52,406	58,110
12.5	58,645	55,7932	50,980	42,245	45,454
25	47,237	43,1372	45,811	39,215	44,741
50	42,245	39,3939	39,215	32,442	46,345
100	39,572	39,5721	39,215	31,550	39,572
IC₅₀	26.70	55.70	45.26	8.42	21.98