INTRODUCTION:

Natural products have been used since ancient times for the treatment of many ailments^1,2. Human interest in using herbal medicines and cosmetics is increasing³, so the investigation of herbal medicines and their application in authentication becomes a very important requirement⁴.

Phytochemical investigations of plant materials are important because they are related to their biological response activities and therapeutic effects⁵. Parijoto fruit with the Latin name Medinilla speciosa Blume (Family Melastomataceae) is a typical plant that thrives in mountain slopes or in forests and is sometimes cultivated as an ornamental plant. Parijoto plants grow well on high humus and moist soil at a height of 800 to 2,300 meters above sea level. One of the areas that is a place Parijoto plants grow on the slopes of the Muria Mountains, Colo Village, Kudus District, Indonesia⁶. Melastomataceae plant has a height of 1-3 meters, has a round stem with a rough texture, serrated, old skin with a layer of cork, and brownish gray. The typical leaf type is pointed leaf tip, round leaf base, shiny upper leaf surface, leaf width 15cm, leaf length 27cm, and leaf surface color is green. The number of flower crowns owned is 5, with a crown length of 3cm and a width of 3 cm, a purplish red color, and the type of flower is perfect. Parijoto fruit is small round, reddish purple in color, the average fruit diameter is 0.7-0.8cm, and the color of the flesh is white⁷.

The methanol extract of Parijoto fruit at 500mg/KgBW was able to reduce blood sugar levels and improve sexual function in male Wistar rats with chronic DM model, the presence of secondary metabolites as antioxidants could affect the sexual function of male rats⁸. In addition, the ethanolic extract of Medinilla speciosa fruit has been reported to exhibit moderate cytotoxicity in T47D cancer cells with a value of IC₅₀ is 614.50g/ml and has the potential for chemoprevention⁹, ethyl acetate fraction of parijoto fruit (M. speciosa) has the ability to lower glucose levels with optimal reduction¹⁰, antihyperlipidemic and antiobesity¹¹. Based on the results of research conducted by⁷, Parijoto fruit contains flavonoid compounds, saponins, terpenoids, glycosides and tannins⁷, beta-carotene and antioxidants¹², and alkaloids⁶. The results of the characterization of parijoto fruit with IR showed a specific group of flavonoids flavonol group⁶.

There are so many pharmacological activities possessed by parijoto fruit. Methanol extract, insoluble and soluble fraction of n-hexane parijoto fruit were able to provide a stimulating effect on the quantity spermatozoa in SD male rats¹³, The methanol extract, soluble fraction, and n-hexane insoluble fraction at a dose of 500mg/KgBW of fruit had a significant effect on the number of Leydig cells, Sertoli cells, primary spermatocytes and seminiferous tubule diameter¹⁴. So that these plants can be developed and preserved, it is necessary to carry out continuous research, so that the types of bioactive compounds can be known¹⁵. Bioactive compounds are plant components that have medicinal value and pharmacological therapeutic effects. To obtain bioactive compounds, a series of processes are carried out called Extraction, Isolation, and Extraction Characterization¹⁶. To determine the content of active compounds from plants, it is necessary to isolate them, so that pure compounds are obtained. Furthermore, the pure compound obtained does not have meaning if the molecular structure is not known. The isolated compound contained in the methanol extract of Parijoto fruit has never been reported. The process of isolation and identification of methanol extract of parijoto fruit using Preparative Thin Layer Chromatography method. Thin Layer Chromatography is a type of liquid chromatography that can separation of chemical compounds of different structures based on the level of affinity of the compound to the stationary phase and the mobile phase¹⁷. In chromatographic techniques, the process of separating compounds depends on competition compound (solute), mobile phase and stationary phase. The stationary phase is used polar silica gel¹⁸. This method used to structurally isolate chemical compounds or chemical components from homogeneous mixtures^19,20. The obtained isolates were structurally determined using GCMS, FTIR, and NMR²¹. GCMS is used to determine the molecular weight of the parent ions, fragment ions, metastable ions and isotopes molecule along with the molecular formula²². FTIR is used to reveal the functional groups present in the molecule²². NMR spectra analyze the chemical shift values of alkyl, aromatic and different functional groups associated with the proton nucleus²². This study aims to provide information on the results of the isolation and identification of phytoconstituents from the methanol extract of parijoto fruit.

MATERIAL AND METHODS:

Materials:

The materials used in this research were parijoto fruit from Muria mountain slope, Silica gel 60 GF₂₅₄, Methanol (E Merck, Germany), n-hexane (E Merck, Germany), distilled water, ethyl acetat (E Merck, Germany), Thin-layer chromatography (TLC) plates were derivatized using a cerium sulfate reagent (Merck, Darmstadt, Germany).

Instruments:

The instruments used in this research were gram and milligram electric scales (OSUKA), a set of tools for extraction and fractionation, rotary evaporator (Heidolph), Gas chromatography and Mass spectroscopy GC-MS GCMSQP2010 SE (Shimadzu), Perkin Elmer Spectrum One Fourier transform-IR spectrophotometer (Pharmacy UGM, Indonesia), and JEOL ECP 500 nuclear magnetic resonance (NMR) spectrometer (500 MHz for 1H- and 125 MHz for13C-) (Airlangga University, Indonesia).

Parijoto Fruit Methanol Extract Production:

Parijoto fruit methanol extract production was carried out using maceration method where 8kg of purple medinilla fruit was taken and had wet sorted to separate the parijoto fruit from impurities or foreign substances, then washed using running water until clean, drained, and aerated air. The dry parijoto fruit was blended. Extraction was carried out by the maceration method using methanol solvent with a ratio (1:10) and put in a container for 1 day (1x24 hours). The formed maserate was evaporated using a rotary evaporator with a temperature of approximately 45⁰C and the produced filtrate was evaporated using a waterbath at 45°C until a thick extract was obtained¹⁴.

Parijoto Fruit Methanol Extract Fractionation:

The obtained extract was partitioned using methanol and n-hexane solvents. As much 10g of the extract was dissolved in 100mL of methanol until the extract can be poured into the separatory funnel. Then, 100mL n-hexane was added into the separatory funnel and then shaken for 5 minutes while occasionally opening the valve in the separatory funnel to release the gas formed. The formed solution was left for several minutes until the methanol and n-hexane layers were separated. The top layer is non polar or n-hexane while the bottom layer is methanol layer. The layer was separated by opening the separatory funnel valve to remove the methanol layer, the top layer that was left in the separatory funnel was added with 100mL n-hexane solution. The partition was performed in the same manner until the n-hexane solvent was clear¹³.

Parijoto Fruit Methanol Extract Isolation:

The eluted fractions were subjected for fraction of n-hexane and fraction methanol parijoto fruit through preparative thin layer chromatography (PTLC) method, and the mobile phase used was n-hexane: ethyl acetate with a ratio of 5:1. Glass plates (20 x 20cm) thickly coated (0.4-0.5nm) with silica gel 'G' (45gm/80ml water) and the coated plates were activated at 100°C for 30 minutes and cooled at room temperature³. For this, the chromatographic eluent was applied to plates and plates which were dried in air and visualized using cerium sulfate spray reagent and heated. Each point that coincides with the serium sulfate spot is marked. The spots were not visible and were collected separately and eluted with n hexane:ethyl acetate (5:1). The elutant was crystallized and subjected to GCMS, IR, and NMR studies.

Gas chromatography-mass spectrometry (GC-MS):

GC/MS analyses were performed on Shimadzu – GC/MS-QP2010 Plus device equipped with RTX-5 column®, Restek Corporation (30.0m × 0.32mm; film thickness 0.50µm) fused silica capillary column (5% diphenyl polysiloxane, 95% dimethyl polysiloxane). Carrier gas was He, and gas flow rate 1.64ml/min. Mass spectra were obtained by electron impact (EI) ionization at 70 eV with an emission current of 400mA. The scan time was 1 s and the scan range was m/z 29–600. The ion source temperature was maintained at 280ºC. The identity confirmed by fragmentation pattern and by Nist and Wiley mass spectral libraries. The temperature program was as follows of the column was 100°C; 25°C/min. until 200°C and 8°C/min. until 300˚C, holding for 5.5 min²³.

Fourier Transform Infrared Spectroscopy (FT-IR):

This is the most widely used tools for the detection of functional groups in pure compounds and mixtures and for compounds comparison. An infrared spectrum represents a fingerprint of a sample with absorption peak, which correspond to the frequencies of vibrations between the bonds of the atoms making up the materials. Because each different material is a unique combination of atoms no two compounds produce the exact same infrared spectrum. Therefore, infrared spectroscopy can result in a positive identification (qualitative analysis) of every different kind of material. In addition, the size of the peaks in the spectrum is a direct indication of the amount of material present. With modern software algorithms, infrared is an excellent tool for quantitative analysis²⁴.

NMR spectroscopy:

¹H, ¹³C and Attached Proton Test (APT) NMR experiments were performed on 500 MHz Varian Unity Inova FTNMR instrument in DMSO-d₆. The ¹H chemical shift values were reported on the δ scale in ppm, relative to TMS (δ= 0.00ppm) and in the¹³C chemical shift values were reported on the δ scale in ppm, relative to DMSOd₆ (δ= 39.5 ppm)²⁵. A signal was detected only when the nuclei in the sample resonate with the energy source. The energy was transferred from the radio frequency source to the detector coil. The output signal from the detector was fed to a cathode ray oscillograph after amplification. The signal from the detector was recorded as a peak on the chart paper for proton and carbon spectra²⁶.

RESULTS AND DISCUSSION:

The methanol extract of parijoto fruit was diffracted using n-hexane, so that the n-hexane fraction and methanol fraction were obtained. Each fraction was then continued with the isolation process. The process of isolation and identification of methanol extract of parijoto fruit using Preparative Thin Layer Chromatography method resulted in three chemical compounds (1–3) being obtained. One compound from the n-hexane fraction is stigmasterol (1), and two compounds from the methanol fraction are stigmasterol and 1-hexanol (2 and 3). Structure these compounds were determined based on the Gas chromatography-mass spectrometry (GC-MS), Fourier Transform Infrared Spectroscopy (FT-IR) and NMR spectra. These values are also compared with those reported in previous studies.

Figure 4. ¹H-NMR spectral data of Stigmasterol (1)

Table 1. NMR data of Stigmasterol

S. No.	Type	Compound (1)		Stigmasterol²⁸
S. No.	Type	δ_C(ppm)	δ_H, m (J; ΣH)	δ_C(ppm)	δ_H, m (J; ΣH)
1	CH₂	37.3	1.84, m (2H)	37.6
2	CH₂	31.7	1.51, m (2H)	32.1
3	CH	71.9	3.51, m (1H)	72.1	3.51, tdd (4.5, 4.2, 3.8; 1H)
4	CH₂	42.4	2.24, dd (10.5, 2.1; 2H)	42.4
5	C	140.8	-	141.1
6	CH	121.8	5.34, d (5.04; 1H)	121.8	5.31, t (6.1; 1H)
7	CH₂	31.7	1.84, m (2H)	31.8
8	CH	31.9	1.93, m (1H)	31.8
9	CH	50.2	1.44, m (1H)	50.2
10	C	37.1		36.6
11	CH₂	21.2	1.47, m (2H)	21.5
12	CH₂	39.8	2.00, dd (8.6, 3.6; 2H)	39.9
13	C	42.4	-	42.4
14	CH	56.8	1.10, m (1H)	56.8
15	CH₂	24.4	1.55, m (2H)	24.4
16	CH₂	29.0	1.25, m (2H)	29.3
17	CH	55.2	1.07, m (1H)	56.2
18	CH₃	12.3	0.67, s (3H)	12.2	0.71, s (3H)
19	CH₃	18.9	0.99, s (3H)	18.9	1.03, s (3H)
20	CH	40.6	1.99, m (1H)	40.6
21	CH₃	19.9	0.91, d (6.6; 3H)	21.7	0.91, d (6.2; 3H)
22	CH	138.4	5.00, dd (15.1, 8.6; 1H)	138.7	4.98, m (1H)
23	CH	129.4	5.12, dd (15.1, 8.6; 1H)	129.6	5.14, m (1H)
24	CH	45.9	1.93, m (1H)	46.1
25	CH	29.2	1.65, m (1H)	29.6
26	CH₃	19.5	0.81, d (6.8; 3H)	20.2	0.82, d (6.6; 3H)
27	CH₃	19.1	0.79, d (6.8; 3H)	19.8	0.80, d (6.6; 3H)
28	CH₂	25.5	1.25, m (2H)	25.4
29	CH₃	12.1	0.84, t (3.7; 3H)	12.1	2.83, t (7.1; 3H)

Table 2. NMR data of β-sitosterol

No.	Type	Compound (2)		β-sitosterol²⁸
No.	Type	δ_C(ppm)	δ_H, m (J; ΣH)	δ_C(ppm)
1	CH₂	37.3	1.84, m (2H)	37.3
2	CH₂	32.0	1.52, m (2H)	31.9
3	CH	71.9	3.51, m (1H)	71.9
4	CH₂	42.4	2.24, dd (10.5, 2.1; 2H)	42.3
5	C	140.8	-	140.8
6	CH	121.8	5.34, d (5.04; 1H)	121.8
7	CH₂	31.7	1.84, m (2H)	31.7
8	CH	31.9	1.93, m (1H)	32.0
9	CH	50.2	1.44, m (1H)	50.2
10	C	36.7	-	36.6
11	CH₂	21.2	1.47, m (2H)	21.2
12	CH₂	39.9	2.00, dd (8.6, 3.6; 2H)	39.8
13	C	42.4	-	42.4
14	CH	56.8	1.10, m (1H)	56.8
15	CH₂	24.4	1.55, m (2H)	24.4
16	CH₂	28.3	1.25, m (2H)	28.3
17	CH	56.1	1.07, m (1H)	56.1
18	CH₃	12.1	0.67, s (3H)	11.9
19	CH₃	19.5	0.99, s (3H)	19.5
20	CH	36.2	1.99, m (1H)	36.2
21	CH₃	18.9	0.91, d (6.6; 3H)	18.9
22	CH₂	34.0	1.20 (m, 2H)	34.0
23	CH₂	26.1	1.65 (m, 2H)	26.2
24	CH	45.9	1.93, m (1H)	46.0
25	CH	29.2	1.65, m (1H)	29.2
26	CH₃	19.9	0.81, d (6.8; 3H)	19.9
27	CH₃	19.1	0.79, d (6.8; 3H)	19.1
28	CH₂	23.1	1.25, m (2H)	23.1
29	CH₃	12.3	0.84, t (3.7; 3H)	12.1

The structure of β-sitosterol is further determined using APT and ¹H NMR spectral data. The APT experiment shows that this compound contains 29 carbons consisting of six methyls, nine methylenes, 11 methines, and three quarternary carbons (Figure 7). A methine carbon at δ_C 71.9 (C-3) indicates a carbon attached to an oxygen atom (oxymethine) which reveals the existence of a hydroxyl group. The presence of one pair of highly deshielded carbons at δ_C 140.8 (C-5) dan 121.8 (C-6) suggests that there is a double bond in the molecule. The DBE value of five suggests that this compound is a tetracyclic compound containing one double bonds. Table 2 lists the carbons and protons of β-sitosterol.

The 1H NMR experiment of β-sitosterol (Figure 8) displays information of the presence of two singlet methyl signals at δ_H 0.67 (H-18) and 0.99 (H-19) indicating that they are bound directly to the ring system. Three doublet methyls at δ_H 0.91 (H-21), 0.81 (H-26) and 0.79 (H-27), and one triplet methyl at δ_H 0.84 (H-29) are located at the side chain outside the rings of the main skeleton. A proton at δ_H 3.52 (H-3) is attached to the oxygen-binding carbon (C-3). The occurrence of three highly-deshielded protons at δ_H 5.34 (H-6) indicates the presence of one pair of double bonds. The typical signal for the olefinic H-6 of the steroidal skeleton was evident from a proton at δ_H 5.34 integrating for one-proton. This result is in line with the results of the study^30,31.

1-Hexanol was obtained as colourless needle crystal. The mass spectral data indicated the molecular formula of this compound is C₆H₁₄ONa with m/z of [M+Na] 125 (Figure 9).

Figure 10. The FT-IR spectral data of 1-Hexanol (3)

The FT-IR spectral data (Figure 10) shows the stretching vibration band of O-H at 3336 cm^-1, C-O bend at 1062 cm^-1. The peaks of stretching Csp3-H appear at 2921 and 2851 cm^-1, while the bending vibrations of Csp3-H of the methyl groups give absorption at 1465 (asymmetrical) and 1372 cm^-1 (symmetrical).

Figure 11. APT NMR spectral data of 1-Hexanol (3)

The structure of 1-hexanol is further determined using APT and ¹H NMR spectral data. The APT experiment shows that this compound contains 6 carbons consisting of one methyl dan five methylenes (Figure 11). A methylene carbon at δ_C 63.2 (C-1) indicates a carbon attached to an oxygen atom (oxymethine) which reveals the existence of a hydroxyl group, another four methylenes ei. δ_C 22.8 (C-2), 25.8 (C-3), 32.0 (C-4), 32.9 (C-5), while a methyl δ_C14.2 (C-6). The DBE value of zero suggests that this compound is an aliphatic compound without double bond.

Figure 12. ¹H NMR spectral data of 1-Hexanol (3)

The ¹H NMR experiment of 1-hexanol (Figure 12) displays information of the presence of one triplet methyl signals at δ_H 0.87 (t, J = 8.0 Hz, 3H). four multiplet methylenes at δ_H 1.33-1.56 (H-2,5), and one triplet methylene at δ_H 3.63 (t, J = 8.0 Hz, 2H) is bind with -OH group.

Table 3. NMR data of 1-Hexanol

No	Compound 3		1-Hexanol^{32, 33}
No	δ_C(ppm)	δ_H, m (J; ΣH)	¹³C (ppm)
1	63.2	3.63 (t, J = 8.0 Hz, 2H)	14.07
2	22.8	1.56 (m, 2H)	22.75
3	25.8	1.33 (m, 6H)	25.59
4	32.0		31.80
5	32.9		32.79
6	14.2	0.87 (t, J = 8.0 Hz, 3H)	62.80

CONCLUSION:

This study succeeded in isolating three compounds from the methanol extract of parijoto fruit

ACKNOWLEDGEMENT:

Thanks to the Ministry of Education and Culture of the Republic of Indonesia for the grant “Research on Decentralization, National Competitive and Assignment Programs “Penelitian Disertasi Doktor (PDD) Tahun 2021”.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 12.09.2021 Modified on 25.12.2021

Research J. Pharm. and Tech 2022; 15(10):4395-4404.

DOI: 10.52711/0974-360X.2022.00737