Dendrophthoe Petandra L. Miq.s Progesterone-Like Effect

Dendrophthoe Petandra L. Miq.s Progesterone-Like Effect ABSTRACT The in-vivo study on healthy adult female rat were known that extract methanol of Dendrophthoe Petandra L. Miq by intramuscular injection (w/v) at dose 100 mg/kg body weight for four days (s.d.d.) can be produce progesterone hormone two times more than usual levels. That phenomenon apparently indicates that plant as an Indonesia namely Benalu duku were probably consist of steroid with carbon bonding substances at number 1st to 21st namely pregnane derivate. These research aims to determined specificity active substances 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 analysis grade by shaker maceration method at 200C during the 72 hours. Semi solid crude extract were drying by vacuum dried apparatus and UV exposure at 15 minutes and analysis by FT-IR and LC ESI MS. Research result obtained that active substances with main chemical structure of pregnane deriva te were progesterone at about 30%, medroxy progesterone acetate at about 66%, megestrol acetate at about 3% and 1% dydrogesterone. Key words: 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 several essential active substances and useful for treated proliferation cell (NURAINI et al., 2000, ROOSTANTIA et al., 2000 and RATNA et al., 2007). Some researcher reported that plants have a beneficially active substance like alkaloid, flavonoid, polyphenol, terpenoid and free steroid (ARIFA et al., 2009). New research report explained that crude methanolic extract of Benalu duku leaves can be increasing progesterone hormone two times more than usual levels, but giving FSH levels on female adult rat (BAMBANG, LAZUARDI, 2014). The last research report indicate that plants have a several substances with characterize as a progesterone like effect. Characterization progesterone like effect was known have a specific chemical main structure namely pregnane derivate with consist of carbon at 1st to 21st at their double bonded chain structure 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 structure of pregnane derivate were known as an anabolic hormone and usefully for treatment prostate cancer in adult man (RANDOX LABORATORY LTD, 2013). Pregnane derivate were known have some specific anabolic steroid structure i.e cortisone, hydrocortisone, progesterone, medroxy progesterone acetate, megestrol acetate, 17- ÃŽ ±- hydroxyl progesterone acetate, dydrogesterone. Other herbal medicine with anabolic steroid action was found at Mexico namely Codices plant. That plant was similar with Benalu duku as a parasites plant growing at subtropics seasons (LERNER, ARNWINE, 2002). With analog to Codices plant in Mexico, might be benalu duku leaves after extracted by methanol were containing specific anabolic steroid. By background research at above, will be determined progesterone like effect substance like progesterone, medroxy progesterone acetate, megestrol acetate, dydrogesterone from base on overview of identic molecule ion of LC-ESI MS and function compound of FT-IR. Materials and methods Benalu duku leaves were obtained from Muara Enim region of west Sumatera-Indonesia and identification as a Dendrophthoe Petandra L. Miq at Botany Research Institute at Tangerang – Jakarta Indonesia as described by BAMBANG, LAZUARDI (2014). Reference substance of progesterone was obtained from Sigma-aldrich product 46665 batch SEBA XV. Reference substance of medroxy progesterone acetate was obtained from Harsen pharmaceutical Industry Jakarta – Indonesia at pharmaceutics grade. Megestrol acetate reference material was obtained from Sigma Aldrich at product 46420 batch SZB9173XV. Dydrogesterone reference material was used from European Directorate for quality medicines and health care, France by catalog code Y0001004. A 400 gram of pulverize benalu duku leaves were extracted on 2 L of methanol pro analysis on to the 1 L separated flask during the 72 hours (movement shaker maceration method at 200C). The semi-solid of crude extract were dried on vacum drying and exposure to uv light (15 minutes) for kept contaminate from the mold and some bacteria. Some amount of semi-solid crude extract benalu duku leaves was determined specific molecule structure compound by FT –IR PerkinElmer Frontier 89485 and LC-ESI MS Accela TSQ Quantum Acess Thermo. The semi-solid crude extract methanol of benalu duku leaves was determined to molecule function of progesterone like effect substances at 400 cm-1 to 4000 cm-1 . Performance control of LC-ESI MS were adjusted as follows; column hypersil Gold 0.2  µm length 10 cm gradient method with mobile phase 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 100 µl.second-1, velocity injector 8  µl.second-1, height of suction apparatus at about 2 ml capacity autosample vial at 1.2 ml. Temperature column was adjusted at 220C at maximum pressure pump 1250 PSI and stabile in 10 BAR (THERMO ELECTRON CORPORATION, 2007). Results Analysis Spectrum infra-red 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 appeared s orbital of stretching aromatic compounds. 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 area (k orbital) and inside of area (l orbital). To compare similarities of infra-red spectrum between reference material vs., crude extract methanol of Benalu duku leaves at high intensity (%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 intensity (%T) on the finger print area 750 cm-1 to 1000 cm-1 were identic infra-red spectrum between reference materials vs., crude extract methanol benalu duku leaves. Table 1 at bellow w as 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 Stage Quadruple Mass Spectrometers were 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. Table 2 at bellow explained that retention time and ion molecule of ESI (m/z) as dependent variable of extract methanol Benalu duku leaves was similar to retention time and ion molecules of ESI references material. Table 1. Infra-red spectrum (cm-1) and intensity (%T) crude extract benalu duku and reference materials of progesterone like effect substances Extract benalu duku Progesterone Med. Prog. acet Megestrol acetate Dydrogesterone Identified function group, cm-1  ± %T cm-1 %T cm-1 %T cm-1 %T cm-1 %T 3500-3400 15-11 3436.69 72 3434,34 0,11 3436,39 10,16 3435,72-3374,34 14,17-14,58 0-H 2927,33 12,87 2969,05-2925 10,25-8,22 Absent 2946,49-2927,66 12,04-12,17 2989,16-2930,19 5,05-13,35 Unsymmetrical vibration, stretching C-H 1632,88 17,15 1699,13-1616,17 49-8,83 1638,95 1,05 1664,2-1629,09 6,49-10,9 1659,51-1620,82 1,24-2,94 Stretching aromatic ring (1,s) 1458,07 18,49 1438,76 15,45 1401,19 3,29 1458,7 14,88 1452,06 14,48 Stretching aromatic ring (s) 1272,22 19,21 1279,25-1268,89 17,85-18,67 Absent 1269,83 10,1 1277,7 12,4 Flexible C-H on orbital (l) 1203,09 19,36 1204,91 14,97 Absent 1206,31 14,18 1193,1 7,97 Alkene, R-OH, stretching vibration C-H, aromatic 1169,44 19,73 1179,06-1162,48 17,64-16,36 Absent 1166,41 16,27 1174,79-1162,58 17,08-14,51 -CH—CH-(trans),R-CH2 1062,81 19,17 1116,32 19,96 1112,60 3,28 1083,18-1058,62 16,29-15,81 1064,16 21,79 Cà ¢Ã¢â‚¬ ¢Ã‚ CH2, mono substitute alkenes 826,55 23,84 871,18 17,15 Absent 877,33-796,78 14,9-21,26 831,21-793,86 24,45-23,51 Tri substitute alkenes, meta-di subtitute Benzene (aromatic) 721,48 23,67 778,14-687,31 21,77-21,17 Absent 755,83-713,19 20,62 727,77 23,21 cis-di substitute alkenes, mono substitute benzene, meta-disub. benzene (Aromatic) 639,21 23,8 687,31 21,17 644,79 2,57 634,94 20,65 630,99 18,55 cis-di substituted alkenes (vinyl, C-H) Table. 2. Result analysis retention time and molecules ion of ESI between crude extract methanol benalu duku leaves and reference material as medroxy progesterone acetate, progesterone, megestrol acetate and dydrogesterone Analytes Weight ( µg/ml) Area Retention time (minutes) Electron Spray Ionization (ESI) (m/z) Crude extract benalu duku leaves 0,0646 227044 4.40 387.000 Medroxy progesterone acetate 0,15 99585 4.40 387.000 Crude extract benalu duku leavesFigure 1 to Figure 4 at below obtained that LC ESI-MS of extract methanol benalu duku leaves were containing identic active substances of medroxy progesterone acetate, progesterone, megestrol acetate and dydrogesterone. Discussion Analysis by retention time and ESI (m/z) of LC ESI-MS were apparently just two of progesterone like effect substances on crude extract methanol benalu duku leaves that very identic substances to reference material namely medroxy progesterone acetate and progesterone. Other progesterone like effect substances as a megestrol acetate and dydrogesterone on crude extract methanol benalu duku leaves were mostly identic to reference material of megestrol acetate and dydrogesterone by different shift at 0.01 and 0.6 retention time of reference materials. Difference in retention time shift is closely linked nature of polarity samples bound to the column Hypersil Gold of LC. Thus the most likely molecular structure of megestrol acetate and dydrogesterone on methanol extract of benalu duku no resemblance to 100% identically as reference materials of megestrol acetate and dydrogesterone. Analysis to compare between the values of molecule ion fragments (m/z) and ESI (m/z) reference materials of f ourth progesterone like effect substances versus identically substances of progesterone like effect on crude extract methanol benalu duku leaves were apparently have a differences values (p