Determination of the Major Component of Water Fraction of Katuk (Sauropus androgynous (L.) Merr.) Leaves by Liquid Chromatography-Mass Spectrometry.

BACKGROUND: The katuk leaf (Sauropus androgynous (L.) Merr.) is one of the plants that are used to overcome baldness by the people of Kampung Mak Kemas, Malaysia. It is suspected that secondary metabolites contained in katuk leaves play a key role in stimulating hair growth. AIMS AND
OBJECTIVES: The aim of this study was to identify the optimum method to obtain one of the chemical compounds in the water fraction and to identify the hypothesized chemical isolates in the water fraction katuk leave's ethanol extract.
MATERIALS AND METHODS: The methods used in this study included the collection and determination of the katuk plant, the processing of the katuk, phytochemical filtrating, extracting with ethanol 96%, and fractionation using the liquid-liquid extraction method with n-hexane, ethyl acetate, and water solvents The water fraction of katuk leaves was analyzed by its components by thin-layer chromatography using the stationary phase of silica gel 60 F254, developer of n-butanol:acetic acid:water (4:1:5), and detection under ultraviolet (UV) light at a wavelength of 366 and 254nm, as well as with vanillin-sulfuric acid reagent. To isolate the compounds from water fraction of katuk leaves, it was then eluted with a vacuum column chromatography by eluent with a level polarity that would get 11 subfractions. Each subfraction was checked by two-dimensional thin-layer chromatography to see subfraction purity characterized by the appearance of a spot on the chromatogram plate. The isolate was analyzed using spot test, ultraviolet-visible spectrophotometer, infrared spectrophotometer, and liquid chromatography-mass spectrometry.
RESULTS: The isolate was an alkaloid compound with a molecular mass of 406.3131 m/z with the molecular formula C21H39N6O2 as S, S-5, 5'-amino-4,4'-dihexyl-propyldihydropyrazol-3, 3-one.
CONCLUSION: One of the chemical compounds contained in the water fraction of the ethanol extract of the katuk leaf was an alkaloid group. Copyright:

Introduction

Katuk (Indonesian) is one of the plants grown in Indonesia and is often used for fever and facilitates breastfeeding.[1] It contains tannins, saponins, flavonoids, alkaloids, proteins, calcium, phosphorus, vitamins A, B, and C so that it has the potential to be used as an alternative treatment. The katuk leaf (Sauropus androgynous (L.) Merr.) is one of the plants used to overcome baldness by the people of Kampung Mak Kemas village, Malaysia. It is suspected that secondary metabolites contained in katuk leaves play a key role in stimulating hair growth, whereas in Indonesia it can be used in jamu (Indonesian herbal medicine), which empirically is shown to restore uterus and abdomen to its normal size post giving birth. Katuk is also often believed to increase lactation. The S. androgynous is included in the Euphorbiaceae family, which has dark-colored leaves containing chlorophyll. It contains tannins, saponins, flavonoids, alkaloids, proteins, calcium, phosphorus, vitamins A, B, and C so that it has the potential to be used as an alternative treatment.[23] The katuk leaf (S. androgynous (L.) Merr.) is one of the plants used to overcome baldness by the people of Kampung Mak Kemas, Malaysia. It is suspected that secondary metabolites contained in katuk leaves play a key role in stimulating hair growth. Katuk leaves are processed to overcome the problem of baldness and fertilize hair. Katuk leaves are used by mashing with milk and are used topically.[4] On the basis of previous research, the most active fraction as a stimulator of hair growth is the water fraction.[5] This study aimed to determine the major component of water reaction from ethanol extract from katuk leaves using liquid chromatography–mass spectrometry (LC-MS). Nowadays, people are more likely to return to the herbal era, where people use effective plants as hair growers, hair fertilizers, and hair enhancers to deal with hair loss. Actually not only S. androgyny can be used for hair growth but also other plants such as aloe vera,[6] bitter melon,[7] hibiscus,[8] candlenut,[8] celery,[9] celery,[9] celery,[10] protecting aralia leaves (Polyscias scutellaria),[10] green tea leaves,[11] and fern roots (Angiopteris evecta) have been reported to be used for hair care.[12]

Materials and Methods

Materials

ME Analytical balance (Mettler-Toledo, Columbus, Ohio, USA), ultraviolet (UV) lamps 254 and 366 nm (Camag, Muttenz, Switzerland), Thin-layer chromatography (TLC) GF254 plate (Camag, Muttenz, Switzerland), Rotary evaporator (Ika RV 10 basic, Schwerte, Germany), UV- spectrophotometer (Specord 200, Analytik Jena AG, 07745 Jena, Germany), Infrared (IR) spectrophotometer (Shimadzu, PRESTIGE-21, Kyoto, Japan), and Waters LC-MS (WATERS, Xevo-Q-ToF-1, Milford, USA).

Methods

The research method used was a laboratory research method including the collection of samples in the form of katuk leaves obtained from the Manoko plantation, Lembang, West Java and determined in the Toxicology Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran No. 158/HB/10/2018.Katuk leaves were made simplicia referring to the method of making simplicia from the Indonesian Ministry of Health[13] and made a powder that was then macerated with 70% ethanol, which referred to Materia Medika Indonesia.[14] After evaporation using a rotary evaporator and water bath, a thick extract was obtained and then fractionated referred to the Rahmawati and Mustarichie method using ethyl acetate and hexane.[15] The Farnsworth method[16] was applied for determining secondary metabolites of the katuk ethanol extract. The secondary metabolites tested included alkaloids, flavonoids, tannins, saponins, quinones, triterpenoid, and steroids. The ethanol extract of katuk leaves was analyzed by its components by TLC, using the stationary phase of silica gel 60 F254, chloroform:methanol (9.5:0.5) and spotting with UV light detection at a wavelength of 366 and 254nm. The water fraction of katuk leaves was analyzed by its components by TLC, using the stationary phase of silica gel 60 F254, developer of n-butanol:acetic acid:water (4:1:5), and UV light detection at a wavelength of UV 366 and UV 254nm, as well as with vanillin-sulfuric acid reagent. To isolate the compounds from water fraction of katuk leaves, it was then eluted with a vacuum column chromatography (VCC) by eluent with a level polarity[1718] that would get 11 subfractions. Each subfraction was checked by two-dimensional TLC to see subfraction purity characterized by the appearance of a spot on the chromatogram plate. This subtraction was the selected isolate. The isolate was then identified using ultraviolet–visible (UV–Vis) spectrophotometry, IR spectrophotometry, and LC-MS.

Results

Phytochemical screening

Phytochemical screening was carried out to find out what groups of compounds were contained qualitatively in these plants. This was an initial test in isolating compounds as a guide to compounds that would be isolated by class. In this study, the part used was the leaves. Phytochemical screening results of katuk leaf extract are presented in Table 1.

Table 1

Phytochemical screening results of katuk leaf extract

Secondary metabolites	Results
Alkaloids	+
Flavonoids	+
Saponins	+
Polyphenols	+
Tannins	–
Quinone	+
Monoterpene and sesquiterpene	+
Steroids and triterpenoids	–

(+): detected

(–): not detected

The results of the chromatogram pattern of ethanol extract and leaf fraction katuk with developer 9.5:0.5 are presented in Table 2, whereas for the results of the chromatogram the fractions of water with the mobile phase 4:1:5 are presented in Table 3.

Table 2

Results of chromatogram pattern of ethanol extract and fractions of katuk leaves using chloroform:methanol (9.5:0.5)

Sample	Rf	Color
		Under UV 366 nm	Under UV 254 nm
Ethanol extract	0.17	Pink	Blue
	0.21	Pink	Blue
	0.26	Purple	Brown
	0.36	Pink	Brown
	0.41	Pink	Blue
	0.47	Purple	Blue
	0.52	Pink	Blue
	0.57	Purple	Blue
	0.93	Pink	–
	0.95	Blue	–
	0.98	Purple	–
n-Hexane fraction	0.06	Pink	Green
	0.13	Pink	Green
	0.3	Pink	Green
	0.35	Pink	Green
	0.45	Purple	Green
	0.47	Pink	Green
	0.54	Pink	Green
	0.73	Black	Green
	0.82	Pink	–
	0.89	Purple	–
	0.93	Blue	Green
	1.00	Dark red	Black
Ethyl acetate fraction	0.06	Blue	Green
	0.20	Pink	Green
	0.34	Pink	Green
	0.46	Purple	Green
	0.63	Pink	–
	0.54	Pink	Green
	0.83	Pink	–
	0.95	Pink	–
	1.00	Purple	–
Water fraction	0.13	Blue	Blue
	0.21	Pink	–
	0.41	Pink	–

Table 3

Results of chromatogram patterns of katuk leaves water fraction using n-butanol:acetic acid:water (4:1:5)

Sample	Rf	Color
		Visible light	Under UV 366 nm	Under UV 254 nm	Spotting reagent H2SO4
Water fraction	0.20	–	Pink	Brown	Brown
	0.40	–	Blue	–	–
	0.57	–	Blue	–	–
	0.71	–	Blue	–	–
	0.90	–	Pink	–	–

To purify the crystals, washing was done using methanol until the crystal was white. The results of the crystal purification results are presented in Figure 1. The results of the isolate UV spectrum are presented in Figure 2.

Figure 1

Crystal F.B isolates

Figure 2

The ultraviolet spectrum of isolates in methanol

Determination of functional groups in isolates was carried out using IR spectrophotometry. IR spectrophotometry results can be seen in Figure 3. To determine the molecular weight of the isolates, they were identified using LC-MS. On the basis of the results of LC-MS analysis, there were several peaks produced. This showed that the isolate was not really pure or there were still many impurities.[26] The results of the chromatogram LC-MS are shown in Figures 4 and 5.

Figure 3

Isolates IR spectra

Figure 4

LC isolate result

Figure 5

MS of isolate

Discussion

Extraction

From the extraction process of katuk leaves, the yield was 11.56%. The yield obtained had met the requirements according to BPOM RI (Indonesian Drug and Food Control Agency 2004), which stated that the yield of katuk leaf extract was not less than 9.2%.

The results of phytochemical screening carried out by Susanti et al.[19] showed that 90% ethanol extract of katuk leaves contained compounds of alkaloid, triterpenoid, saponin, tannin, polyphenols, glycosides and flavonoids. In addition, Selvi and Baskar[20] reported that katuk leaf simplicia contains alkaloids, flavonoids, tannins, saponins, polyphenols, and terpenoids. Differences in the content of secondary metabolites could also be influenced by differences in the origin of the plant area, harvest time, and extraction method used.

Thin-layer chromatography of extract and water fraction

The extracts and fractions obtained were then carried out by initial identification, TLC. The first thing to do was to optimize TLC to get the appropriate mobile phase in separating the compounds in the sample. After testing several solvent mixtures, the corresponding mobile phase was obtained, namely 9.5:0.5 and 4:1:5 as the lower phase. By using the silica gel 60 F254 stationary phase and the mobile phase of 9.5:0.5, each ethanol extract, n-hexane fraction, and water fraction produced several spots seen in UV light at a wavelength of 254 and 366nm. With the help of 10% vanillin in H2SO4 spots in methanol, the intensity of the spots was seen more clearly. For water fraction, TLC was also used by the mobile phase of 4:1:5; the lower phase produces several spots that appear on UV light at a wavelength of 254 and 366nm and visible light with 10% vanillin in H2SO4 spotting. This was because spots in methanol were the appearance of universal spots that most organic compounds would form color complexes with 10% vanillin in H2SO4. The results of the chromatogram pattern of ethanol extract and leaf fraction katuk with developer 9.5:0.5 can be seen in Table 2, whereas for the results of the chromatogram the fractions of water with the mobile phase 4:1:5 can be seen in Table 3.

Vacuum column chromatography

The water fraction was then separated using column chromatography.[21] The stationary phase used was cellulose microcrystalline for column chromatography and the eluent used was methanol:water (7:3). The mobile phase was chosen based on the optimization that had been done using paper chromatography with the stationary phase of Whatman No.1 paper. In this study, 60 subfractions were obtained, and then monitoring with TLC was conducted. Monitoring was carried out using the stationary phase of silica gel 60 F254 and the mobile phase of ethyl acetate:methanol (3:7). On the basis of the TLC pattern obtained, the subfractions were grouped according to the same spotting pattern, so that three subfraction groups were produced, namely F.A (1–4), F.B (5–20), and F.C (21–60). From the TLC description obtained, F.B showed better separation so that the sub-F.B fraction was then evaporated using a rotary evaporator so that a thick and crystalline subfraction was formed. To purify the crystals, washing was done using methanol until the crystal was white. The results of the crystal purification results can be seen in Figure 1.

Isolate identification

The resulting isolates were then identified using three instruments, namely UV spectroscopy, IR spectroscopy, and MS other than melting point apparatus. Previously, a compound test of isolate compounds was carried out using a specific spotting appearance. The appearance of the spots used was KOH, Dragendorff, Lieberman-Burchard, and anisaldehyde. The isolates showed positive results by producing orange spots meaning that isolates were alkaloid groups.[22]

The purity test was carried out using the melting point apparatus. The purity test was carried out up to 400°C. The melting temperature of a solid was the temperature at which solids converge and melt perfectly. A substance would be pure if the melting temperature obtained is the same as found in the literature. On the basis of the literature, the compound C21H39N6O2 will decompose at 304°C.[23] It was found that the melting point was 305°C.

Ultraviolet–visible spectroscopy

Identification of isolates using UV–Vis spectrophotometry was carried out by looking at absorption in the UV region at a wavelength of 200–400nm. Isolates would produce absorbance at certain wavelengths. The UV spectrum used the principle of antagonism of the electromagnetic group with molecular compounds associated with transition energy between electron transitions π–π* and n–π*. The resulting wavelength depends on the electron promotion ability of a compound. The easier a compound promotes its electrons, the more energy needed will be lower and produce a higher wavelength.[24] The isolate was identified by UV spectrophotometer with methanol solvent. Methanol solvents were used because they were organic solvents that dissolved both polar and nonpolar compounds. Based on the results of the examination of isolates using UV-spectrophotometry obtained a maximum wavelength at 201 nm. This could be caused by the presence of an auxochrome group. One of the auxochromes was the presence of nitrogen substituents. In conjugation of free electron pairs on nitrogen atoms, it will produce maximum absorption at wavelengths of 190–230nm.[17] The results of the isolate UV spectrum are presented in Figure 2.

Infrared spectrophotometry

Determination of functional groups in isolates was carried out using IR spectrophotometry. On the basis of the IR spectrophotometry results, isolates contained NH-stretching groups at a wave number of 3120.82 cm–1 (sharp-band intensity), CH stretching at a wave number of 3034.03 cm–1 (medium-band intensity), and OH bending at a wave number of 1404.18 cm–1 (sharp-band intensity).[25] IR spectrophotometry results are presented in Figure 3.

Liquid chromatography–mass spectrometry

To determine the molecular weight of the isolates, they were identified using LC-MS. On the basis of the LC-MS analysis results, there were several peaks produced. This showed that the isolate is not really pure or there were still many impurities.[26] Image of the results of the chromatogram LC-MS can be seen in Figures 4 and 5.

The LC-MS method used was the ESI (electrospray ionization) method which was used in the use of LC-MS because ESI has a very high sensitivity in detecting samples.[2627] In the ESI method, compound molecules were not fired using electron beams, but by providing very high electrical energy (2–6kV) in the sample so that changing samples from solution form into aerosol forms from high-charged electrospray droplets. The formed droplet would enter into the heated capillary so that an evaporation process occurred which caused a reduction in the size of the droplet and an ionic form that would be forwarded to the mass analyzer. The analyte ion that was read on the detector was an analytic molecule and the form of fractional ion because the ion that enters the mass spectrometry would remain in its full form, which did not go through the gas phase ion activation process.[28] There were two methods of charge administration, namely the positive ion and negative ion modes. In this study, the tools used to analyze isolates use positive ions, so that the process of loading the analyte occurred with the addition of protons to the analyte molecule so that the analyte ions that were read were not the original molecule but in the form (M + H)+, where M was original molecular ion form. The mass analyzer used in this tool was quadrupole-time of flight (Q-ToF). The compound observed was a compound that had a retention time of 10.08min because the compound had a high peak. Estimates of compounds shown at the base peak molecular compounds of (M + H)+ at 407.3131 m/z indicated that the original molecular weight was 406.3131 m/z, whereas the molecular weight of 429.2966 m/z occurred because of isotopes on Na atoms. Other peaks formed were other compounds contained in the isolate, so that the isolate was not really pure. The results of the MS spectra are shown in Figure 5. On the basis of the results of other instruments, such as IR and UV, it was suspected that the chemical formula of the isolate was C21H39N6O2. This was because this compound had a functional group C, H, O, and N in the IR spectrum and the functional group had a small difference in the value of mDa with the results of MS analysis, which was equal to 0.3. So far there is no publication mentioning that S. androgynous contain this compound, but in the literature, this formula was called (S, S)-5, 5′-amino-4,4′-dihexyl-propyldihydropyrazol-3, 3′-one23.

Conclusion

By using the method of extraction, isolation, and determining by spot test, IR spectrometer, and MS described in this study, it was found that the major component of water fraction of katuk (S. androgynous) was an alkaloid compound with a molecule mass of 406.3131 m/z with C21H39N6O2 formula, and so it was called (S, S)-5, 5′-amino-4,4′-dihexyl-propyldihydropyrazol-3, 3′-one.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.