Phytochemical Composition of Selaginella spp . from Java Island Indonesia

For a long time, people in West Java, Indonesia have used Selaginella as a traditional cure for several ailments including fever, minor wounds, broken bones, women’s health disorders or postnatal bleeding. However, information on the active compounds of the plant from Java Island has not been fully expounded. The objective of the research was to reveal the diversity of bioactive compounds and amentoflavone content of Selaginella from Java Island in order to optimize the use of this plant as a medicinal plant. Selaginella plants collected from 29 locations in Java were extracted and subjected to colorimetric and thin layer chromatography test to qualitatively analyze the bioactive compounds of alkaloid, phenol, and steroid. In addition, high performance liquid chromatography was performed to analyze the amentofalvone content of Selaginella extract. The research result showed that all the examined Selaginella species contained alkaloid, flavonoid, saponin, tannin, and steroid, but they did not contain hydroquinone. Seven of them contained the biflavonoid type of amentoflavone. The highest amentoflavone concentration, 6.87 ppm, was found in S. subalpina that originated from Gedung Songo, Central Java. Those results indicated that more than one Selaginella species originating from Java Island had marked potential for use as medicinal plants.


Introduction
In recent years, the need for medicine has risen in proportion to the increase of socially transmissible diseases.The use of alternative medicine from plants in traditional remedies has been used in many places around the world.Herbal products from medicinal plants are preferred because of their higher safety, efficiency, and cultural acceptability over drugs.Besides these reasons they also have less side effects and testing time.Khanna et al. [1] showed that chemical compounds of herbs is a part of the plant's physiological and ecological functions, therefore it is believed to have better compatibility with the human body.
Selaginella, a perennial herbaceous genus belonging to the Selaginellaceae (Pteridophytes) family, is distributed almost worldwide.It has simple and very small leaves, usually much less than 10 mm long without lateral veins arranged in four ranks with two lateral rows of larger leaves and two rows of smaller median leaves.All sporangia fall at the end of the branches [2].Previous studies showed that Selaginella is rich with an active compound called biflavonoid, a secondary metabolite that is formed from the dimer of flavone and flavanon structures with 5,7-4'-oxigenated pattern.For centuries, people, especially in China, have used Selaginella as a traditional medicine, for curing various diseases including hepatitits and cancer or as an antioxidant [3].Pan et al.,Gayatri et al., have evaluated the active compound and its biochemical and pharmaceutical functions in different Selaginella species.The extract of S. Doederleinii contained alkaloid, phytosterol and saponin compounds [4].The extract of S. involvens with the concentration of 2 mg/mL could inhibit lipid peroxidation at almost 50% [5].The extract of S. Bryopteris increased cell growth and protected against dead cells induced by oxidative stress [6], while the extract of S. Tamariscina contained a strong antioxidant that was able to reduce blood sugar levels and function as a lipid peroxide serum and increase insulin serum [7].
Indonesia is one of the world's countries that has mega plant diversity, including diversity of Selaginella.It has been reported by Alston [8] that there have been 23 Selaginella species identified in Java Island.For centuries people from West Java have used this genus as a remedy for curing many ailments such as fevers, wounds, broken bones, female disorders and postnatal bleeding.In spite of its benefits, Selaginella that originated from Java Island has not been expounded, scientifically examined, and exposed as a traditional medicinal plant.Therefore, scientific investigation, in particular elucidation of its active compound content is necessary in order to optimalise the use of this genus as a source of natural medicine.In the future, we hope that the active compounds of Selaginella can be extracted and massively produced.This paper revealed the content of active compounds as well as the flavonoid amentoflavone in 9 species of Selaginella originating from Java, Indonesia.

Sample collection, preparation and identification.
The sample was collected from 29 locations in Java Island and was prepared for plant extraction.Plant identification was performed by using identification keys for ferns [8][9][10] and comparing the materials to herbarium specimen collections belonging to the Research Center of Botany, LIPI, Cibinong, Bogor.
Bioactive compound analyses.The bioactive compounds were extracted and analyzed using procedures developed by Gayathri et al. [5] and de Oliveira et al. [11].All plant parts were dried and ground to powder, and then extraction was performed using ethanol and n-hexane.For the ethanol extract, dry powder of Selaginella was extracted using ethanol (5 g plant powder/100 ml ethanol) by constantly mixing the suspension for 4 hours, then it was filtered using Whatman filter paper.The filtrat was then dried using a rotary evaporator at 40 o C for 4-8 hours until all the solvent evaporated.Hexane extraction was carried out using the same procedure as ethanol extraction with 2 g plant powder per 100 ml n-hexane.
Qualitative analyses of the bioactive compounds, i.e. alkaloid, phenol, and steroid, were carried out using the colorimetric method based on the standard phytochemical analysis for plants as described by Harborne [12].The intensity of the color represents the relative content of the bioactive compounds.The bioactive compounds were then verified using thin layer chromatography (TLC) methods.In order to determine the biflavonoid content of Selaginella, a part of the plant extract was fractionated using sephadex column chromatrography with a combination of ethanol and nhexane as eluen.All fractions were then quantitatively analyzed using high performance liquid chromathography (HPLC) with amentoflavone as an internal standard.

Results and Discussion
The qualitative phytochemical compositions of Selaginella is shown in Table 1.All tested Selaginella species contained alkaloid, flavonoid, saponin, and steroid compounds.Tannins were only found in some species.This research showed that Selaginella was not only rich in flavonoid compounds, but also contained all the most important bioactive compounds of plants, such as alkaloid, tannin, flavonoid, saponin, and phenolic compounds [13].All compounds have a good effect on human health when they are used properly.Saponins form strong insoluble complexes with cholesterol therefore, it is believed to be useful in the human diet for controlling cholesterol.Beside that it has antifungal and antibacterial properties that are important as components of cosmetic products [14].
Apart from the antioxidant activity, flavonoid also possesses anti-inflammatory, anti-allergic, hepatoprotective, anti-thrombic, antiviral, and anticarcinogenic activities.Alkaloids have diuretic, antipasmodic, anti-inflammatory, and analgesic effects [15].Different classes of steroids have different functions.Anabolic steroids increase muscle mass.Anti-inflammatory steroids can reduce swelling, pain, and other manifestations of inflammation [14].Tannins content:-= absent, + = low, ++ = medium, +++= high may be used medicinally as antidiarheal, haemostatic, and antihemorhoidal compounds.Tannins can also be effective in protecting the kidneys [16].Tannins have also shown to have potential antibacterial and antiparasitic effects [17][18][19].It is believed that tannins isolated from the stem bark of Myracrodruon urundeuva may have neuroprotective functions capable of reversing hydroxydopamine-induced toxicity.The plant is also potential as a therapeutic agent, which may be beneficial to patients with neurological disease [20].Sung et al. [21] showed that tannins extracted from green tea waste product could be a natural source for antioxidants as well as antibiotics.All those properties possessed by the bioactive compounds of plants may explain the use of Selaginella species as a medicinal plant in West Java communities.
Table 1 also showed that all the tested Selaginella extracts did not contain hydroquinone.The absence of this compound makes them safe to be consumed because it is extremely dangerous and may lead to the development of cancer.Hydroquinone is an aromatic organic compound that is a type of phenol, having the chemical formula C 6 H 4 (OH) 2 .This compound has been used for decades as a skin lightening agent.However, the use of hydroquinone in cosmetics has been banned since January 2001 because of the mid-term effects like leukomelanoderma en confetti and exogenous ochronosis [22].
The verification of phytochemical components using TLC analysis showed that the type of eluen affected the result.The TLC analysis for alkaloids showed that the eluen n-hexane was able to extract alkaloid from all tested Selaginella species, but analysis using ethanol eluen was only able to extract alkaloid from two species (Table 2).Those results indicated that the eluen nhexane was better than that of ethanol for extracting Selaginella alkaloids.Flavonoid was found in all tested Selaginella species, and the highest level of flavonoid was in S. remotifolia (Table 1).The TLC analysis showed that ethanol eluen was better for extracting flavonoid • +: present -: absent than that of n-hexane (Table 3).Tannins were found in 5 species, namely; S. ornata, S. remotifolia, S. aristata, S. Involvens and S. Intermedia (Table 1).Meanwhile saponins were identified in all the tested species, and the highest saponin content was found in S. willdenovii.Steroids were also found in all species (Table 1 and 4).
In general, the TLC analysis showed that the n-hexane eluen was better than that of the ethanol eluen.The identification of active compounds of Selaginella extraction indicated that Selaginella has good potential as a source of active compounds (secondary metabolites), especially flavonoid, which has great potential as a source of natural medicine.
The active compounds identified from 9 Selaginella species were described in Table 5. Extract contents were varied among species ranging from 6.01 to 14.22% of plant dry weight.The content of active compound was highest in S. willdenovii, while the lowest content of active compound was found in S. intermedia.Both these species originated from Cangkuang, Sukabumi (West Java).Flavonoid amentoflavone was identified in seven Selaginella species, but it was not detected in two other species.The concentration of amentoflavon varied among seven species ranging from 1.44 to 6.87 ppm with the highest content found in S. subalpina that originated from Gedung Songo (Central Java).Even though amentoflavone was not detected in S. plana, and S. ornata, it does not mean that the species have no flavonoid because we know from Table 1 that all species have flavonoid, indicating that those two species have different types of flavonoid.There are several types of flavonoids known in Selaginella, such as gingketin and robusflavon [23].It is also possible that one Selaginella species has more than one phenolic compound.For example, the extract of S. Doederleinii has 11 phenolic compounds, which are 5 lignans: (-)lirioresitol A, lirioresitol B, (+)-wikstromol, (-)nortracheloside and matairesinol; 2 phenylpropanon: 3hydroxy-1(3-methoxy-4-hydroxy-phenyl)-propan-1-one and 3-hydroxy-1-(3,5-dimethoxy-4-hydroxyphenil)propan-1-one; and 4 biflavonoids: amentoflavone, 7,7"di-0-methylamento-flavone, 7, 4', 7", 4"'-tetra-0methylamentoflavone and heveaflavone [24].The results of this research suggested that more than one species of Selaginella originating from Java Island have great potential for medicinal plants.

Conclusions
All the examined Selaginella species contained alkaloid, flavonoid, saponin, and steroid compounds, but only some Selagnella species contained tannins.Hydroquinone was not found in all the examined Selaginella species.The content of each compound varied among species.The extract concentration ranged from 6.01-14.22% of the dry matter with the highest concentration found in S. willdenovii that originated from Cangkuang, Sukabumi (West Java).
Seven species have flavonoid amentoflavone with various concentrations ranging from 1.44 to 6,87 ppm.The highest concentration of amentoflavone was found in S. subalpina originating from Gedung Songo (Central Java).More than one Selaginella species has the potential as medicinal plants.