A Simple Liquid-Liquid Fractionation (LLF) Method for Isolating Deoxyandrographolide dan Andrographolide from Herbs of Andrographis paniculata (Burm., F) Ness and Its Cytotoxic Activity on 3T3-L1 Preadipocyte Cells Metode Fraksinasi Cair-Cair (FCC) Sederhana untuk Mengisolasi Deoksiandrografolid dan Andrografolid dari Sambiloto (Andrographis paniculata (Burm., F) Ness dan Aktivitas Sitotoksik pada Sel 3T3-L1 Preadiposit

The main bitter constituents of sambiloto (Androgaphis paniculata (Burm., F) Ness) are diterpene lactones, namely andrographolide and deoxyandrographolide which have been reported to have antidiabetic, cytotoxic, antiatherosclerosis, anti-inflammatory, and antimicrobial activity. There are many studies that performed the isolation of deoxyandrographolide and andrographolide from A. paniculata herbs, but most of them included several steps that make them not efficient. This research was conducted to do an isolation of deoxyandrographolide and andrographolide through liquid-liquid fractionation (LLF) due to its simplicity, low cost, and time efficient. The extraction of deoxyandrographolide and andrographolide from the herbs was carried out using chloroform as the solvent by using Soxhlet apparatus, and LLF was performed to isolate the compounds. The identities of the compounds were confirmed by TLC scanner compared to its standard references. Hence, these present methods were successfully isolated and determined deoxyandrographolide and andrographolide of A. paniculata. The compounds were also showed relatively moderate cytotoxicity on 3T3-L1 cell lines using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, with LC50 of deoxyandrographolide and andrographolide; 29.32 μg/mL and 37.70 μg/mL, respectively.


INTRODUCTION
In recent years, treatment of diseases with traditional herbs has become an alternative. The main reasons regarded as relatively safe in long term use and it has minimal side effects. Andrographis paniculata (Burm.f.) Nees is known as a medicinal plant which can be used as an alternative treatment for various diseases. It is known as king of bitter due to the very bitter taste of all part of the plant and in Indonesia its name is sambiloto. In pharmacological research, it shows antidiabetic [1], cytotoxic effect [2], antimicrobial activity [3], anti-inflammatory [4], anti-hyperglycemia [5], antiatherosclerosis [6], antimalaria [7], hepatoprotective [8], and anticancer activity [9]; in vitro and in vivo studies.
Several methods of isolation deoxyandrographolide and andrographolide compounds from sambiloto have been carried out. Isolation of deoxyandrographolide has been evaluated by several methods, including not only the methods by Rashid et al., 2018 andHermawan et al., 1997 using Preparative Thin Layer Chromatography (PTLC), but also the method by Imanta, 2017 [12][13][14].
Isolation of deoxyandrographolide has been performed by Rashid et al., 2018 from ethanolic crude extract which then was subjected to silica gel column chromatography eluted with chloroformmethanol (99:1) [12]. More than 20 years ago Hermawan et al., 1997 isolated deoxyandrographolide using Preparative Thin Layer Chromatography (PTLC) [13]. Chromatographic methods were needed not only for isolation of deoxyandrographolide but also for separation of andrographolide and deoxyandrographolide. Isolation of andrographolide has been reported from cold maceration of leaf powder in a 1:1 mixture of dichloromethane and methanol followed by recrystallization [15]. The second major diterpene lactone of A. paniculata is 14-deoxyandrographolide (Fig. 1). It has immunomodulatory and antiatherosclerotic effects [16,17]. Therefore, the chosen isolation method must be selective to provide the expected isolation results precisely and quickly. This research focused on the isolation of deoxyandrographolide and andrographolide from the herbs of A. paniculata without chromatographic method. So, this research became a new thing to separate andrographolide and deoxyandrographolide.
On the other hand, the use of A. paniculata as remedy for curing diabetes is usually in relatively long period of time, so a question of safety arrises. Beside the chronic toxicity test, the approach of safety concern can be performed by observation of cytotoxicity. Based on this approach, this research focused also on the effect of deoxyandrographolide and andrographolide on viability of pre-adipocyte cells.

Liquid-Liquid Fractionation (LLF) Method for Isolation of 14-deoxyandrographolide and andrographolide
Briefly, the chloromic extract, the non-hexanic fraction, the isolated andrographolide and deoxyandrographolide were applied on silica gel F254 HPTLC plate with n-hexane: ethyl acetate (1:4) v/v as mobile phase and detected under ultraviolet lamp 254 nm. The authentication of isolated andrographolide and deoxyandrographolide was checked by comparison with their standard references on HPTLC system. Therefore, the pattern spectrums of each isolates were compared to each standard reference.

Cytotoxicity Test of Deoxyandrographolide and Andrographolide on 3T3-L1 Pre-adipocyte Cells
Deoxyandrographolide and andrographolide were tested on 3T3-L1 pre-adipocyte cells CO2. Cells were then observed microscopically. Thereafter, cell became 80% confluence, the medium was changed to high glucose DMEM free serum, and it was incubated until 4 hours, 37 o C, 5% CO2.
After incubated for 4 hours, the high glucose DMEM free serum was discarded and then it was

Isolation of Deoxyandrographolide and Andrographolide
Extraction of A. paniculata herbs using Soxhlet apparatus gave a result of 8.26 g crude extract with dark green color, whereas the Liquid-Liquid Fractionation (LLF) method was successfully isolated the deoxyandrographolide and andrographolide from the extracts. Firstly the fractionation with n-hexane eliminated perfectly chlorophyl and other lypophilic substances without interfere deoxyandrographolide and andrographolide such as shown by HPTLC data (Fig. 2). Further, as we know that in comparison to andrographolide, deoxyandrographolide lack of oxygene moiety at 14 position in the structure (Fig. 1). That will increase lyphophilicity of the structure and we found that the mixture of n-hexane: chloroform (3:2) dissolved deoxyandrographolide but not andrographolide.
The last mentioned compound was dissolved in chloroform. Both deoxyandrographolide and andrographolide were then recrystallized and the crystals had white color. Thus, LLF was chosen for this experiment due to its simplicity, low cost, and time efficient. A simple LLF method has been developed for the isolation of deoxyandrographolide and andrographolide from chloroformic extract of A. paniculata. As the isolate was formed, the weight of deoxyandrographolide and andrographolide were measured and showed yields of 0.248 g and 0.502 g respectively, or 3.0% and In this study, deoxyandrographolide and andrographolide appeared on silica gel F254 as quenching spots under UV254 at Rf value 0.66 and 0.38, respectively (Fig.2) These spots are not only similar to those of standard references, the pattern of spectrums are also similar to those of standard references according to the data evaluated by TLC scanner (Fig.3).

Cytotoxicity Test Result of Deoxyandrographolide and Andrographolide on 3T3-L1 Pre-adipocyte Cells
Further study was designed to examine the cytotoxic effect of deoxyandrographolide and andrographolide on 3T3-L1 pre-adipocyte cells. The equation of standard curve cells was y=2063x-545.69. This equation was important because it was to convert the number of cells. Fig. 4 showed exemples of microscopic view of cells of the control group treated with DMSO and the group treated with deoxyandrographolide 50 µg/mL. Almost all cells of the control group without any treatment except the media were 3126 cells, whereas after treatment with 5.0, 10.0 and 12.5 µg/mL of deoxyandrographolide the viability of cells were lower than the control, and then the viability decrease significantly at the concentration of 25 µg/mL and at concentration more than 50 µg/mL all cells were death. It means that 12.5 µg/mL were limit of concentration which did not influence significant viability of cells (Fig. 5). Thus, the cell viability was dramatically increased (almost 100%) upon 2,5 ug/ml deoxyandrographolide treatment (Fig. 5), it was because in that dose could be happen proliferation cells. The viability of cells without any treatment except the media were 2720 cells, whereas after treatment with 3.125, 6.25, and 12.5 µg/mL of andrographolide the viability of cells were still upper than the control. At the concentration of 25 µg/mL the viability of cells were lower than the control, and than the viability decrease significantly at the concentration of 50 µg/mL and at concentration more than 75 µg/mL all cells were death, so the concentration of 25 µg/mL was limit of concentration which did not influence significant viability of cells (Fig, 6). It meaned that for further experiment the use of 12.5 µg/mL deoxyandrographolide would be safe, whereas the safe concentration of andrographolide were 25 µg/mL. The LC50 value was calculated from the correlation between percentage of death cells with the concentration using probit analysis according to Biostat Pro 5.9.8 software. The LC50 value of deoxyandrographolide and andrographolide was counted to see the effect of each isolates which was represent the 50% the death of cells. These were 29.32 µg/mL and 37.70 µg/mL, respectively. The statistical data of ANOVA showed that both isolates were significant compared to DMSO as control group (p<0.05). That was important to give not only the information about cytotoxic activity but also the cell viability for the next in vitro experiment.

CONCLUSION
By this study, we have evaluated the lilquid-liquid fractionation method for isolating deoxyandrographolide and andrographolide from A. paniculata herbs. The method was found to be suitable for analysis because its simplicity, low cost, and time efficient. The cytotoxicity of deoxyandrographolide and andrographolide on 3T3-L1 pre-adipocyte cells was also evaluated and the LC50 were 29.32 and 37.70 µg/mL, respectively.