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Phytochemical Screening and Antioxidant Activity of Hippobroma longiflora Extracts

This study aims to identify the secondary metabolite compounds and to test the antioxidant activity of ethanol extract, n-hexane fraction, and chloroform fraction of Hippobroma longiflora leaves. Extraction was carried out by the maceration method using ethanol. The resulting crude ethanol extract was then partitioned with n-hexane and chloroform. Each extract and fraction was then tested by phytochemical screening. Antioxidant activity testing was carried out using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. The phytochemical screening test showed that the ethanol extract contained flavonoids, saponins, triterpenoids, and alkaloids; the n-hexane fraction contained steroids, and alkaloids; and the chloroform fraction contained flavonoids, steroids, and alkaloids. Analysis of the antioxidant activity revealed the following Inhibitory Concentration (IC50 values): 9.57 ppm of ethanol extract, 99.59 ppm of n-hexane fraction, 48.54 ppm of chloroform fraction, and 4.30 ppm of ascorbic acid. Based on these results, the antioxidant activity of the ethanol extract was more potential than the n-hexane and chloroform fractions, but smaller than ascorbic acid.


Introduction
The rapid development of technology and industry has changed the behavior and lifestyle of the community. Unhealthy lifestyles such as consuming fast food, lack of time to exercise, and smoking habits as well as environmental factors such as vehicle fumes and solar radiation can cause an increase in the number of free radicals in the body. This condition will trigger oxidative stress [1]. The magnitude of the negative effect of free radicals on human health causes the body to require an intake that contains an antioxidant compound that will be able to capture and neutralize free radicals. It happens because antioxidants can provide electron pairs to free radicals so that they can stop further reactions that cause oxidative stress and cell damage to be avoided [2]- [4].
There are various kinds of food ingredients that have natural antioxidant properties such as apples, tomatoes, and green tea. Besides, there are also synthetic antioxidants that are produced through several chemical reactions, such as Tert-Butyl Hydroxy Quinone (THBQ), Butyl HydroxyAnisol (BHA), and Butyl Hydroxy Toluene (BHT). However, these synthetic antioxidants have side effects on the body and harmful to human health so that their use must be monitored [5]. The leaves of H. longiflora contain alkaloids, phenolics, flavonoids, triterpenoids, steroids, coumarins, saponins, and tannins [6]- [7]. The activity of alkaloids and flavonoids has been reported to have many pharmacological effects including anti-inflammatory, antioxidant, anticancer, antidiabetic, antibacterial, antimalarial, antitumor, antimicrobial, antifungal, anti-insecticide, and antiseptic [8]- [9]. Flavonoids are a group of polyphenolic compounds that act as antioxidants, namely to capture free radicals [10]. The presence of polar and non-polar compounds in H. longiflora is the reason to research the activity fractionation of leaves of H. longiflora by using three types of solvents, namely ethanol, n-hexane, and chloroform. This study aim to identify the secondary metabolite compounds and test the antioxidant activity of ethanol extract, n-hexane fraction, and chloroform fraction of H. longiflora leaves.

Materials
The chemicals used for the extraction are ethanol, n-hexane, and chloroform. Dragendorff reagent, Liebermann-Burchard reagent, Mayer reagent, Bouchardat reagent, Wagner reagent, sulphuric acid, sodium hydroxide, magnesium ribbon, hydrochloric acid, and ferric chloride for the phytochemical screening test. Methanol, DPPH, and ascorbic acid for antioxidant activity test. The tools which used in this study included a rotary vacuum evaporator, UV-VIS spectrophotometer, and other glass tools.

Plant sample
Fresh leaves of H. longiflora were collected from Singaraja Region, Bali, Indonesia. The botanical identification and authentication were confirmed at the Indonesian Institute of Sciences (LIPI) UPT Bali Botanical Garden Eka Karya Bali.

Preparation of samples
H. longiflora leaves are taken from the plant and then washed in running water. Leaves that have been washed are cut into small pieces, then air-dried without being exposed to sunlight at room temperature for 7 days. The dried leaves are then ground to a powder.

Extraction procedure
The crude powder that has been weighed (100 gm) is macerated with ethanol. Every 24 hours the extract is filtered and replaced with a solvent. This extraction was repeated until the last extract contained no metabolites. The obtained ethanol filtrate is evaporated to produce a crude ethanol extract. Furthermore, the crude extract (10 gm) is dissolved in 100 mL of water. This water extract was partitioned with n-hexane, then separated and the n-hexane layer was evaporated, to obtain the nhexane fraction (2,7 gm). The residue (water layer) is partitioned with chloroform and separated. The chloroform layer was evaporated to obtain the chloroform fraction (1,1 gm).

Preliminary phytochemicals screening
Each extract and fraction are tested by phytochemical screening. The test is performed according to various standard methods such as flavonoids, alkaloids, tannins, saponins, and steroids/triterpenoids [11]- [12]. The results are represented as + orwhich indicate the presence or absence of classes of compounds in the extract and fractions.

Antioxidant activity
Antioxidant activity testing used the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method with the ascorbic acid as a positive control. In this research, the DPPH method was described by [13], with minor modification. Five concentration series were created for each extract and fractions. Each concentration was taken 2 milliliter and added as much as 1 milliliter of DPPH (0.1 mM). The blank (DPPH in methanol) and positive controls were prepared in the same way without any extract. Then, the mixture was left to stand for 30 minutes in a dark place and the absorbance was measured at λ 517 nm using a UV-Vis spectrophotometer. The repeated of this treatment is three times. Antioxidant activity can be expressed in units of percent inhibition. This value is obtained by the following equation. After the inhibition percentage of each extract and fractions has been obtained, it is followed by a linear regression calculation (x, y) to obtain the IC 50 (inhibition concentration) value. x is the concentration (µg/mL), while y is the percentage of antioxidant activity (%). The IC 50 value is obtained by the equation y = ax + b.

Results and Discussion
The phytochemical screening test showed the ethanol extract which contained flavonoids, saponins, triterpenoids, and alkaloids; the n-hexane fraction contained steroids and alkaloids; and the chloroform fraction contained flavonoids, steroids, and alkaloids. These phytochemical compounds are known to support bioactivity, thus responsible for the antioxidant activities. Phytochemical screening results showed in Table 1. Antioxidant activity for each extract and fractions was evaluated using the DPPH method. This method was chosen because it is a simple, fast, and easy method for screening the radical scavenging activity of several compounds. In addition, this method has proven to be accurate and practical [14]. DPPH is a free radical that is stable at room temperature and is purple. If DPPH is reacted with free radical-reducing compounds (for example flavonoids), the intensity of the purple color will decrease and if the amount of free radical-reducing compounds that react is large, the DPPH can change color to yellow. This color change can be measured its absorbance with a UV-Vis spectrophotometer. DPPH provides strong absorption at a wavelength of 517 nm with a dark violet color. The free radical scavenging causes the electrons to pair then causes a decoloration proportional to the number of electrons taken [15]. In samples containing antioxidant compounds, the higher concentration means the more compounds will donate electrons or hydrogen atoms to the DPPH free radicals. This reaction also causes color fading in the DPPH. DPPH, which was originally dark purple, when reacted with large amounts of antioxidant compounds, will turn yellow. The DPPH color change is also related to the energy possessed by DPPH free radicals. In the form of radicals, DPPH tends to be unstable (reactive) and has large energy because it always reacts looking for its electron pair, but after getting an electron pair, DPPH becomes more stable (low energy).
The results of the analysis of the antioxidant activity for each H. longiflora extract and fraction and compared with ascorbic acid as a positive control are presented in Table 2. Based on this table, it can be seen that there is an increase in the percentage of inhibition along with the increase in the concentration value. This shows that there is a relationship between an increase in the concentration of the test sample and an increase in free radical reduction. This relationship is given by the linear regression equation of the sample. Based on ref [16], a compound is stated as a very strong free antiradical if the IC 50 value is <10 μg/mL, strong if the IC 50 value is between 10-50 μg/mL, moderate if the IC 50 value ranges from 50-100 μg/mL, weak when the IC 50 value ranges from 100-250 μg/mL. Moreover, it will be inactive when the IC 50 is above 250 μg/mL. Based on the results of the antioxidant activity test, it was found that the ethanol extract of H.longiflora leaves had very strong antioxidant activity, n-hexane fraction had moderate antioxidant activity, and the chloroform fraction had strong antioxidant activity. The results showed that the antioxidant activity of the ethanol extract was more potential than the n-hexane and chloroform fractions, but smaller than ascorbic acid. The flavonoids and alkaloids compounds contain in ethanol extract might be responsible for the activity. This type of compound has been reported to have antioxidant activity [17].

Conclusion
To sum up, ethanol extract, n-hexane fraction, and chloroform fraction of H. longiflora have the potential and can be developed as a source of natural antioxidants. Further research is needed for the isolation and identification of the compounds responsible for antioxidants.