Dendrophthoe Petandra L. Miq.’s Progesterone-Like Effect

ABSTRACT

The in-vivo analysis on healthy adult feminine rat were noted that extract methanol of Dendrophthoe Petandra L. Miq by intramuscular injection (w/v) at dose 100 mg/kg bodyweight for four times (s.d.d.) can be produce progesterone hormone 2 times more than usual amounts. That phenomenon apparently indicates that plant as an Indonesia namely Benalu duku were in all probability contain steroid with carbon bonding chemicals at number 1^st to 21^st specifically pregnane derivate. These exploration aims to identified specificity active chemicals of methanol crude extract Benalu duku leaves with pharmacodynamics effect like progesterone hormone. Pulverized Benalu duku leaves at 400g was extracted with 2 L methanol pro evaluation grade by shaker maceration technique at 20⁰C through the 72 hours. Semi sound crude extract had been drying by vacuum dried apparatus and UV publicity at quarter-hour and examination by FT-IR and LC ESI MS. Research effect obtained that active substances with main chemical framework of pregnane derivate were progesterone at about 30%, medroxy progesterone acetate at about 66%, megestrol acetate at about 3% and 1% dydrogesterone.

Key phrases: Benalu duku, pregnane derivate, anabolic steroid, leaves extract FT-IR, Methanolic leaves extract LC-ESI MS.

Introduction

Dendrophtoe Petandra L Miq (Benalu duku) as a parasites plant growing on Lancium demesticum were known have a more than a few essential active chemicals and useful for cured proliferation cell (NURAINI et al., 2000, ROOSTANTIA et al., 2000 and RATNA et al., 2007). Some researcher reported that crops have a beneficially lively material like alkaloid, flavonoid, polyphenol, terpenoid and free of charge steroid (ARIFA et al., 2009). New research survey described that crude methanolic extract of Benalu duku leaves can be increasing progesterone hormone two times a lot more than usual levels, but giving FSH amounts on female adult rat (BAMBANG, LAZUARDI, 2014). The last research report indicate that crops have a several substances with characterize as a progesterone like effect.

Characterization progesterone like impact was known have a particular chemical main structure namely pregnane derivate with consist of carbon at 1^st to 21^st at their dual bonded chain composition or (8S,9S,10S,13R,14S,17S)-17-ethyl- 10,13-dimethyl-2,3,4,5,6,7,8,9,11,12, 14,15,16,17-tetradecahydro-1H- cyclopenta [a] phenanthrene. Identically of their main framework of pregnane derivate had been called an anabolic hormone and usefully for treatment prostate cancers in adult person (RANDOX LABORATORY LTD, 2013). Pregnane derivate were known have some specific anabolic steroid composition my spouse and i.e cortisone, hydrocortisone, progesterone, medroxy progesterone acetate, megestrol acetate, 17- α- hydroxyl progesterone acetate, dydrogesterone.

Other herbal treatments with anabolic steroid actions was found at Mexico specifically Codices plant. That plant was comparable with Benalu duku as a parasites plant developing at subtropics seasons (LERNER, ARNWINE, 2002). With analog to Codices plant in Mexico, could possibly be benalu duku leaves after extracted by methanol were containing specific anabolic steroid. By backdrop research at over, will be determined progesterone like effect element like progesterone, medroxy progesterone acetate, megestrol acetate, dydrogesterone from base on summary of identic molecule ion of LC-ESI MS and function substance of FT-IR.

Materials and methods

Benalu duku leaves had been received from Muara Enim location of west Sumatera-Indonesia and identification as a Dendrophthoe Petandra L. Miq at Botany Exploration Institute at Tangerang – Jakarta Indonesia as defined by BAMBANG, LAZUARDI (2014). Reference chemical of progesterone was attained from Sigma-aldrich product 46665 batch SEBA XV. Reference element of medroxy progesterone acetate was acquired from Harsen pharmaceutical Sector Jakarta – Indonesia at pharmaceutics quality. Megestrol acetate reference material was obtained from Sigma Aldrich at merchandise 46420 batch SZB9173XV. Dydrogesterone reference materials was applied from European Directorate for quality medicines and health care, France by catalog code Y0001004.

A 400 gram of pulverize benalu duku leaves had been extracted on 2 L of methanol pro analysis to the 1 L separated flask during the 72 hours (activity shaker maceration approach at 20⁰C). The semi-sturdy of crude extract were dried on vacum drying and contact with uv light (quarter-hour) for retained contaminate from the mold plus some bacteria. Some volume of semi-sound crude extract benalu duku leaves was established specific molecule structure substance by FT –IR PerkinElmer Frontier 89485 and LC-ESI MS Accela TSQ Quantum Acess Thermo. The semi-stable crude extract methanol of benalu duku leaves was motivated to molecule function of progesterone like result chemicals at 400 cm^-1 to 4000 cm^-1 .

Performance control of LC-ESI MS were adjusted the following; column hypersil Gold 0.2 µm duration 10 cm gradient approach with mobile stage at bottle A 0.1% in Aqua bidest and bottle B 0,1% formic acid in acetonitrile pro HPLC. The gradient was perform at 35% to70% of B in 20 minute at 254 nm UV detector. Automatic sucking and injection capacity was adjusted at 10 µl, flushing capacity 400 µl with velocity at 100µl.second^-1, velocity injector 8 µl.second^-1, height of suction apparatus at about 2 ml ability autosample vial at 1.2 ml. Temperatures column was adjusted at 22⁰C at maximum pressure pump 1250 PSI and stabile in 10 BAR (THERMO ELECTRON CORPORATION, 2007).

Results

Analysis Spectrum infra-reddish colored of crude extract methanol Benalu duku leaves obtained that stretching aromatic compound specific overtone was presented at 1650 to 2000 cm^-1. Especial in 1550 cm^-1 to 1600 cm^-1 was appeared stretching aromatic compound (1,s). In 1450 cm^-1 to 1500 cm^-1 was came out s orbital of stretching aromatic substances. At finger print area wavelength number of 600 cm^-1 to 900 cm^-1 and 1000 cm^-1 to 1300 cm^-1 were indicate of flexible carbon atom and hydrogen outside of spot (k orbital) and inside of area (l orbital). To do a comparison of similarities of infra-red spectrum between reference materials vs., crude extract methanol of Benalu duku leaves at substantial strength (%T) indicated that wavelength number of 1400 cm^-1 to 1050 cm^-1 and 1600 cm^-1 to 1660 cm^-1 were identic both of them. Even at low strength (%T) on the finger print region 750 cm^-1 to 1000 cm^-1 were identic infra-reddish spectrum between reference components vs., crude extract methanol benalu duku leaves. Table 1 at bellow was presented specific function molecules of crude extract methanol benalu duku leaves after assessment by FT-IR by references of NOERDIN (1985).

Analysis LC ESI-MS by Triple Level Quadruple Mass Spectrometers had been obtained that compound of

progesterone like effect in extract methanol Benalu duku leaves were identic to reference materials of medroxy progesterone acetate, progesterone, megestrol acetate and dydrogesterone. Desk 2 at bellow explained that retention period and ion molecule of ESI (m/z) as dependent variable of extract methanol Benalu duku leaves was comparable to retention time and ion molecules of ESI references materials.

Table 1. Infra-reddish colored spectrum (cm^-1) and strength (%T) crude extract benalu duku and reference materials of progesterone like effect substances

Table. 2. Result evaluation retention time and molecules ion of ESI between crude extract methanol benalu molar mass of cuso4.5h2o duku leaves and reference material as medroxy progesterone acetate, progesterone, megestrol acetate and dydrogesterone

Discussion

Analysis by retention period and ESI (m/z) of LC ESI-MS were apparently simply just two of progesterone like result chemicals on crude extract methanol benalu duku leaves that extremely identic chemicals to reference material namely medroxy progesterone acetate and progesterone. Different progesterone like effect substances as a megestrol acetate and dydrogesterone on crude extract methanol benalu duku leaves had been mainly identic to reference materials of megestrol acetate and dydrogesterone by numerous change at 0.01 and 0.6 retention period of reference resources. Difference in retention time shift is closely linked characteristics of polarity samples bound to the column Hypersil Gold of LC. So the probably molecular framework of megestrol acetate and dydrogesterone on methanol extract of benalu duku no resemblance to 100% identically as reference elements of megestrol acetate and dydrogesterone. Examination to compare between your values of molecule ion fragments (m/z) and ESI (m/z) reference supplies of fourth progesterone like effect chemicals versus identically substances of progesterone like effect on crude extract methanol benalu duku leaves were apparently have a distinctions values (p<0.05). The molecule ion fragments ideals were apparently lowest than ESI ideals as represented at Table 3.

Table 3. Research molecule ion fragments vs., ESI between reference components and crude extract methanol benalu duku leaves.

Note: Superscript a vs., b of molecules ion fragment vs., ESI at the same Desk were significant distinct at p<0.05 by t. test method

The distinctions of the values between both of them were triggered difference technology of sensing ion molecule analytes after ionization on ionized chamber by molecule ion fragments technique versus ESI method of LC. The amount of sensitivity of the mass range of the device ESI LC-MS Accela TSQ known to the value of m / z 3000 with accompanying Quantitation-Enhanced Data-Dependent MS / MS (QED-MS / MA) with the capacity of monitoring up to make three points of data. Behavior noisy devices have also been suppressed due to the development of methods such section curved space after ionization chamber (collision unit cell) made ​​900. Study the TSQ Quantum robustness Access Ion Relative Common Deviation gets to% = 2.9% (THERMO SCIENTIFIC, 2007).

Composition of the crude extract benalu duku leaves at 0.0656 µg/ml were apparently containing progesterone like impact substances at majority component of medroxy progesterone acetate (0.062 µg/ml) from then on the second amounts as a progesterone (0.028 µg/ml) and the third level namely megestrol acetate (0.003 µg/ml) and dydrogesterone as a minority compounds at 0.0009 µg/ml. That composition of the progesterone like influence on crude extract methanol benalu duku leaves already predicted by BAMBANG, LAZUARDI (2014)., at take care of the implication of samples at adult woman rat with resulted progesterone hormone at twice level than usual level.

Conclusions

The crude extract methanol benalu duku leaves including identic chemicals as a Medroxy progesterone acetate at about 66%, progesterone at about 30%, megestrol acetate at about 3% and dydrogesterone at about 1%. The definitely identic compounds were attained at medroxy progesterone acetate and progesterone. But the significantly less than absolute identic substance were obtained at megestrol acetate and dydrogesterone. Recommended of the study result the following: i., for next study must be explored isolation and identification of the effective substance as a progesterone like jj thomson contribution to atomic theory impact to influenced the physiologic pet subject.

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