PHYTOCHEMICAL IDENTIFICATION AND ANTIOXIDANT ACTIVITY OF PASSIFLORA FOETIDA FRUITS AND LEAVES EXTRACTS: A COMPARATIVE STUDY

Objective: The objective of this study was to compare the phytochemical composition and antioxidant activity of Passiflora foetida fruits and leaves extract. Methods: The parameters observed in this study were phytochemical compounds including alkaloid, flavonoid, phenolic, sterol, triterpenoid, saponin, tannin, and cardiac glycoside, total phenolic content Folin Ciocalteu method is based on reduction of Folin Ciocalteu reagent in alkaline medium; the metal complex produced measured at λ max : 760 nm; total flavonoids content with AlCl 3 Colorimetric method based on complex formation of AlCl 3 and flavonoid content in alkaline medium, the AlCl 3 - flavonoid complex produced measured at λ max : 510 nm; free radical DPPH scavenging activity; and ferric reducing power based on reduction of Fe 3+ ion into Fe 2+ ion that reacted with FeCl 3 to form a ferric-ferrous complex that measured at λ max : 700 nm. Results: Passiflora alkaloids, phenolics, flavonoids, and total phenol GAE/g sample dry base, total flavonoid GAE/g sample dry base ferric reducing power GAE/g fruits extract had phytochemical compounds such as alkaloid, phenolic, flavonoids, cardiac glycosides, total phenol was 6.53±1.02 mg GAE/g sample dry base , total flavonoids were 1.56±0.27 mg CE/g sample dry base , DPPH free radical scavenging activity was 1.00±0.15 mg GAE/g sample dry base , and ferric reducing power was 1.12±0.17 mg GAE/g sample dry base . Conclusion: Passiflora leaves extract has higher total phenol, total flavonoid and antioxidant activity measured by DPPH scavenging activity and ferric reducing power value compared with Passiflora fruits extract. flavonoid, DPPH free radical scavenging ferric reducing


INTRODUCTION
Passiflora foetida, usually called rambusa is a wild plant usually found in the tropical region and found creeping on another plant. Passiflora foetida can be eaten raw as lalapan or used as medicine to cure many diseases like fever, headache and asthma [1,2]. Passiflora foetida is grouped in the Passifloraceae family and generally grows in humid places like river and swamp [1].
Passiflora foetida can be used as a traditional medicine because it contains phytochemical compounds. The phytochemical compound in Passiflora foetida is an alkaloid, phenolic, glycoside, flavonoid and cyanogenic compound that can be used as an antioxidant [1]. Passiflora foetida has many biological activities such as antiinflammation, antitumor, anticancer, antimicrobe and many pharmacological activities. [3]. Therefore it is necessary to conduct further research on Passiflora foetida leaves and fruits as a source of antioxidants. This research was conducted to compare the phytochemical compound and antioxidant activity of Passiflora foetida leaves and fruits extracts.

Plant material
Leaves and fruits of Passiflora foetida were collected from the Mangrove forest region, Wonorejo, Surabaya. Leaves and fruits of Passiflora foetida used for this study has a different classification for Passiflora foetida leaves were green color, has a length of±9 cm and width±10 cm, intact, and not perforated while the classification for Passiflora foetida fruits was green color, has a diameter of±1.5 cm, intact, and flat skin surface. The plant was authenticated in the Herbarium of Biology and Food Industry Microbiology Laboratory at the Department of Food Technology, Agricultural Technology Faculty, the Widya Mandala Catholic University of Surabaya with voucher specimen no FTP-UKWMS-0002 for future reference.

Passiflora foetida leaves extraction
Passiflora foetida leaves that have been collected were dried at ambient temperature, ground and sieved with 28 mesh size. The dried flour of Passiflora foetida leaves was measured moisture content. For the extraction process, 2 g of Passiflora foetida leaves dried flour was packed in a tea bag and extracted with 100 ml of hot water (95 °C). Parameters were analyzed, including phytochemical content, total phenol, total flavonoid, DPPH free radical scavenging activity, and ferric reducing power.

Passiflora foetida fruits extraction
Passiflora foetida fruits that have been collected were weighted 250 g and crushed with the addition of aqua dest (fruit: aquadest= 1:3). The mixture of crushed fruit was macerated with a magnetic stirrer at ambient temperature for 3 h. The mixture was filtered and the filtrate was dried with a freeze dryer for±72 h. Dried powder of Passiflora foetida fruits extracts weighed 1 g and dissolved in 50 ml water. Parameters were analyzed, including phytochemical content, total phenol, total flavonoid, ferric reducing power, and DPPH free radical scavenging activity.

Moisture content
Moisture content of Passiflora foetida dried leaves flour and fruit are determined with the thermogravimetric method [4]. One gram of samples is measured with the oven at 105 °C. The difference weight between before and after heating was the moisture content of the sample.

Yield analysis
Yield analysis of Passiflora foetida fruits was measured with the comparison between the weight of dried fruit extract and initial fresh fruit weight (% w/w dry base). The yield of Passiflora foetida fruits was used to determine the concentration of the antioxidant compound in fresh fruits.

Phytochemical identification
Phytochemical identification was done to determine phytochemical content in samples such as alkaloid, flavonoid, phenolic, sterol, triterpenoid, saponin, tannin, and cardiac glycoside in Passiflora foetida leaves and fruits extracts [5].

Total phenol analysis
Total phenol analysis was determined by spectrometry method [6]. 100 µl sample was added with 1 ml Folin Ciocalteu 10% and 2 ml Sodium Carbonate 7.5%. The mixture was added with water in a 10 ml volumetric flask and shook. The solution was incubated at ambient temperature for 30 min and the absorbance of the sample was measured at λ 760 nm. The total phenolic content of the sample was stated by gallic acid equivalence (GAE)/g sample dry base.

Total flavonoid analysis
Total flavonoid analysis was determined by the AlCl3 colorimetry method [7]. 200 µl sample was added with 0.3 ml NaNO2 5% (b/v), 0.3 ml AlCl3 10% (b/v), and 2 ml NaOH 1 M in 10 ml volumetric flask. The mixture shook and diluted with water until volume 10 ml. The absorbance of the sample was measured at λ 510 nm. Total flavonoid content of the sample was stated by catechin equivalence (CE)/g sample dry base

DPPH radical scavenging activity
Passiflora foetida leaves and fruits extract antioxidant activity was measured by spectrophotometer [8]. 3 ml of DPPH solution (4 mg/100 ml methanol) was added to 100 µl sample in a test tube and diluted until 5 ml. The mixture was incubated in ambient temperature for 30 min. The absorbance of the sample was measured at λ 517 nm. DPPH radical scavenging activity was stated as % inhibition using the equation: % inhibition: Abst=0= Control absorbance Abst=30= Sample absorbance

Ferric reducing power
Ferric reducing power of Passiflora foetida leaves and fruits extracts were measured by spectrophotometer [9]. 200 µl sample mixed 2.5 ml phosphate buffer (pH 6.6) and 2.5 ml potassium ferricyanide 1% and incubated at 50 °C for 20 min. 2.5 ml chlorogenic acid 10% was added to the solution and the mixture centrifuged at 3000 rpm for 10 min. 2.5 ml of supernatant was added with 2.5 ml aquabidest and 0.5 ml ferric chloride 0.1% and incubated for 10 min. The absorbance of the sample was measured at λ 700 nm. High absorbance indicates an increased ferric reducing power. The ferric reducing power of the sample was stated as a gallic acid equivalence (GAE)/g sample dry base.

RESULTS AND DISCUSSION
Passiflora foetida dried leaves flour and fruits had moisture around 13.51±0.34% and 85.36±0.36%, respectively. The yield obtained from fruits extraction with aqua dest was 8.81±1.36%. Passiflora foetida leaves and fruits extracts contained phytochemical compounds that were shown in table 1. Data-informed that both leaves and fruits extract contained alkaloid, flavonoid, phenolic compound and cardiac glycoside. The difference was saponin content in leaves extract that wasn't detected in fruit extract.
Tannin, terpenoid, and sterol weren't detected in both extracts. Terpenoid and sterol was nonpolar compound [10] and the solvent used to extract was aqua dest which is a polar solvent. Tannin is a water-soluble active compound that can be found in the plant. In this study, tannin wasn't detected in both extracts. A different result was obtained from [11] who found tannin in both extract and [12] found tannin in Passiflora leaves. The difference result was caused by a different place of plant growth that can influence the nutritional value and phytochemical content of plants [13].
Total phenol, Flavonoid, DPPH scavenging activity, and ferric reducing power shown in fig. 1, 2, 3 and 4, respectively. Total phenol and flavonoid of Passiflora leaves extract (22.92±0.18 mg GAE/g Sample dry base and 7.01±0.10 mg CE/g Sample dry base respectively) was higher compared with Passiflora fruits extract (6.53±1.02 mg GAE/g Sample dry base and 1.56±0.27 mg CE/g Sample dry base respectively). Consequently leaves extract of Passiflora leaves extract scavenging activity and reducing power was higher (2.76±0.01 and 3.20±0.04 mg GAE/g Sample dry base respectively) than Passiflora fruits extract (1.00±0.15 and 1.12±0.17 mg GAE/g Sample dry base respectively).  Total phenolic content of determined by some factors like enzyme activity of Phenylalanine Ammonia-Lyase (PAL) and Chalcone Synthase (CHS) in plants [14] and the number of free hydroxyl group in the sample [15]. The formation of flavonoids is influenced by the action of CHS enzymes that form chalcone compounds which are subsequently isomerized by CHI enzyme (chalcone isomerase) into another flavonol compounds [16].
Total phenol and flavonoid from Passiflora leaves and fruits extracts were different because a different part of the plant has different functions and nutrition content. Leaves have a function for photosynthesis and place to storage nutrition [5] meanwhile fruit has the function to protect the seeds by surrounding it with flesh containing mineral, simple organic compound, and substrate and facilitate its dispersal [17].
Data in fig. 1 and 2 showed that flavonoid content in both extracts was too low compared to total phenolic content. 80% of the phenolic compound in the plant was flavonoid [3]. High results in total phenol assay correlated with the Folin-Ciocalteu method. Folin-Ciocalteu method wasn't a specific method to measure total phenol [18]. The presence of another reducing agent like aromatic amine, reducing sugar and ascorbic acid in a sample can increase the result of total phenol.
Antioxidant activity related to total phenol and flavonoid in Passiflora leaves and fruits extracts that can be measured as free radical scavenging activity and ferric reducing power. The measurement was based on the donation of a hydrogen atom or electron from the antioxidant compound to free radical. Total phenol and flavonoid were correlated with DPPH scavenging activity and ferric reducing power [3,19,20,21]. DPPH assay was based on the decolorization of DPPH free radical from purple into yellowish color in the presence of antioxidant compounds [22,23]. DPPH scavenging activity stated as inhibition rate that calculated from the difference of control and sample absorbance divided with control absorbance. Ferric reducing power was measured as a secondary antioxidant activity [24]. Ferric reducing power was measured based on the ability of an antioxidant compound to reduced ferric (III) iron ion to ferrous (II) iron ion that can be seen from the change of color from yellow to green-Prussian blue color [25]. Data showed that Passiflora leaves extract has higher DPPH free radical scavenging activity and ferric reducing power compared with fruit extract. This caused by a higher concentration of phenolic and flavonoid content in leaves extract. The higher concentration of the phenolic compound in the sample, the scavenging activity and ferric reducing power value will be increased [20].

CONCLUSION
The result obtained in this study showed that Passiflora leaves extract has higher total phenol, total flavonoid and antioxidant activity measured by DPPH scavenging activity and ferric reducing power value compared with Passiflora fruits extract.