MATERIALS AND METHODS

Plant materials: The fresh bud of R. deceptor and root and stem of T. papillosum was collected from study site in Biology Research and Educational Forest, Andalas University, Padang, during research on October, 2014. All the plants from this study was taxonomically identified in Herbarium Universitas Andalas (ANDA), Padang.

Extraction of plant materials: Samples in powder form has taken each ±20 g to measure water levels. Then the rest of samples going to extract with maceration method with ethanol for approximately 24 h at room temperature. The filtrate is obtained to 100 ml with a rotary evaporator at temperature 40-50°C and 50 rpm.

Phytochemical testing: Extract of materials has been used on qualitative phytochemical test in g performed to detect the presence of alkaloids, phenolics, flavonoids, saponins, terpenoids and steroids. The phytochemical testing were screened using standard procedures, as described by Harborne (1987). Total phenolic content was determined as Gallic Acid Equivalent (GAE) according to Pourmorad et al. (2006).

Brine Shrimp Lethality Test (BSLT) bioassay (Meyer et al., 1982): Brine shrimp lethality test is a convenient bioassay for plants constituent (Meyer et al., 1982). Artemia salina was used to monitor the screening. The eggs of brine shrimp (Artemia salina Leach.) were collected from a shop in faculty of marine and fisheries, Bogor Agricultural University and hatched in a tank with seawater and constant oxygen supply for approximately 24-48 h, until the nauplii matured.

The extract dissolved by DMSO and then test solution prepared at a concentration of 1000, 500, 100 and 30 μg mL^–1. The BSLT done by inserting 20 A. salina shrimp larvae in each test solution by repeating three times at each concentration. Observations were made after ±24 h to see the number of people, who lived and died. The data was obtained and analyzed by probit analysis method to determine the LC₅₀ with 95% confidence interval. The LC₅₀ is the concentration of the extract that is able to kill 50% of the population tested shrimp larvae. The lower the LC₅₀ value means higher toxicity of the extracts were tested.

DPPH (1,1-diphenyl-2-picrylhydrazyl) antioxidant activity test (Sari et al., 2011): The extract was dissolved in ethanol, according to the desired concentration. The solution was incubated at 37°C for 30 min and then the absorbance was measured using UV-VIS spectrophotometry at a wavelength of 517 nm. The antioxidant activity of each sample was determined by calculating the percent inhibition, which is calculated by the following equation:

where, A is the absorbance of negative control (DPPH+ethanol) and B is the absorbance of test extracts (DPPH+ethanol+test extract). Value of sample concentration and percent inhibition plotted, respectively on the x-axis and y in the linear regression equation. Linear regression equation obtained in the form of the equation y = a+bx used to find the value of IC₅₀ (inhibitor concentration of 50%) of each sample with a stated value of y is 50 and the value of x that will be obtained as IC₅₀. The IC₅₀ declare the concentration of the sample solution is needed to reduce DPPH free radicals by 50%.

Data analysis: The results of in vitro study are expressed as Mean±Standard Deviation (SD) obtained from triplicate experiments. The data analyzed with Student’s t-test and a ‘p’ value less than 0.05 was considered as significant difference in analysis.

RESULTS AND DISCUSSION

Extraction of buds of R. deceptor (RHZ) and part of the root (AKR) and stem (BT1) from T. papillosum with ethanol solvent showed varies yield value with different physical form (Table 1). The highest yield resulting from the extraction of R. deceptor buds, which is almost 3 times the yield of extract its host. Differences in levels and physical form extracts showed that the content of extractive substances differ between different parts of the network though, the tree is extracted with the same solvent. This is evidenced by research (Alimpic et al., 2014) that the ethanol extraction Salvia amplexicaulis flowers produce a higher yield than the stalks. Ethanol considered as, a good solvent, because it has been extensively used to extract compounds from various plants and plant-based foods especially to extract antioxidant compounds.

The results of the qualitative phytochemical testing showed that all the samples contain alkaloid, phenolic and flavonoid. Terpenoid detected in bud extract and root extract and Saponin detected in root and stem extract. Steroid detected only in stem extract (Table 2).

Secondary metabolites that detected in all the extract should tell about the bioprospecting potential of R. deceptor and its host T. papillosum. Alkaloids are known as source to play a defensive role in the plant against herbivores and pathogens. Due to their potent biological activity, many of the approximately 12,000 known alkaloids have been exploited as pharmaceuticals, stimulants, narcotics and poisons (Wink, 1998). Sofiyanti et al. (2008) reported that Rafflesia hasseltii and its host T. leucostaphylum contain nicotine and caffeine as members of alkaloids group.

Table 1:	Extracts yield and extracts physical form of R. deceptor (RHZ), root of T. papillosum (AKR) and stem of T. papillosum (BT1)

Table 2:	Result of qualitative phytochemical testing and total phenolics content of R. deceptor (RHZ), root of T. papillosum (AKR) and stem of T. papillosum (BT1)
+: Detected, -: Undetected, TCP: Total phenolics content

Table 3:	Mortality and LC50 values of ethanolic extract from bud of R. deceptor (RHZ), root of T. papillosum (AKR) and stem of T. papillosum (BT1)
*Mean±SD

It is possible that R. deceptor and its host contain some valuable members of alkaloids but we can’t prove it from this research. Rhizanthes deceptor and its host also contain phenolics and flavonoids in their ethanolic extract. Phenolics and flavonoids known as, source of natural antioxidant and also are related with antioxidant activity of some plants (Demiray et al., 2009; Hossain et al., 2011; Patel et al., 2012). Although, in some cases the relation is low (Omonhinmin et al., 2015). The high values of total phenolic content from R. deceptor and its host indicated the probability of high antioxidant ability (Table 2).

The results of toxicity tests on all samples with BSLT method showed that all the samples test edare toxic. An extract said to be toxic, if it is capable of killing Artemia salina more than 50% for 24 h (Meyer et al., 1982). The highest LC₅₀ value is stem extract ethanol of T. papillosum with 277.65 μg mL^–1 followed by bud extract of R. deceptor and root extract of T. papillosum with 283.21 and 719.95 μg mL^–1 (Table 3). The BSLT toxicity test method is one of the most convenient bioassay to see the potential for a kind of compound. Meyer et al. (1982) states that, if the value LC₅₀ extract below 1000 μg mL^–1, there is a chance of compound or active extract to fa sample with the potential bioactive, while Carballo et al. (2002) states that if the LC₅₀ value of a small natural extract slower than 1000 μg mL^–1, then its potential as, an anti-cancer. Meyer et al. (1982) also stated that the most active compounds in plants are toxic at certain doses, so that, the toxic values obtained are evidence that there is the content of bioactive compounds in the extracts tested. The toxicity results of the test could be potential anti-cancer oranti-pest (Ghisalberti, 1993). The existence of a diverse group of chemical compounds in the extract were tested in the study confirm the possibility of potential antioxidants and other potentials of the samples tested.

The results of radical scavenging activity test by DPPH showed that all tested extracts have high antioxidant activity. Table 4 shows the percentage of DPPH inhibition from each extract and regression equation of the relationship interpolation. The results of the regression equation, this variety will produce for different IC₅₀ values for each sample extract. The IC₅₀ values were obtained from the regression equation in Table 4 shows that all the extract has the ability of radical scavenging activity.

Table 4:	IC50 values from bud of R. deceptor (RHZ), root of T. papillosum (AKR) and stem of T. papillosum (BT1)
*Mean±SD

The highest IC₅₀ value is the extract of bud of R. deceptor with IC₅₀ value of 31.97 μg mL^–1, as well as extracts of root sand stems T. papillosum with IC₅₀ value of 21.71 and 35.23 μg mL^–1, respectively. The antioxidant activity which is owned by third extract can be quite high, if it refers to Blois (1958) in Hanani et al. (2005) which states IC₅₀ of 200 μg mL^–1 Lasa strong antioxidant activity limits.

Several species of Rafflesia which has been studied previously also represents a good potential as antioxidant. Zulkffle et al. (2014) stated that the Rafflesia cantleyi and host T. tuberculatum absorption of free radical activity is quite high with IC₅₀ value of 14.58 and 19.12 μg mL^–1, as well as research and Puttipan and Okonogi (2014) on the Rafflesia kerri, which concludes the high antioxidant potential in the Rafflesia. Hossain et al. (2011) stated that the flavonoids and terpenoids are potent antioxidants and the close relationship between the total phenolic content of the extracts with antioxidant activity. From the results obtained in the study and compared with several previous studies of antioxidant activity, it can be said that R. deceptor also has potential as a natural source of antioxidants.

CONCLUSION

The study concluded that the qualitative phytochemical testing showed that all the extracts containing alkaloid, phenolic and flavonoid. The toxicity test of BSLT showed the ethanol extract of R. deceptor bud sand the roots and stems of T. papillosum are toxic with LC₅₀ values for 283.21, 719.95 and 277.65 μg mL^–1, respectively. The DPPH radical scavenging test showed ethanol extract R. deceptor bud and the roots and stems of T. papillosum have radical scavenging activity which capable of capturing DPPH with IC₅₀ values are 31.97, 21.71 and 35.23 μg mL^–1, respectively. All the test results showed chemical compounds contained in all the extracts tested samples are supposed to influence the antioxidant activity and toxicity of the extracts. Overall results of the tests that have been done indicate that R. deceptor and T. papillosum containing compound has potential as an antioxidant and other potential possibilities indicated by its toxicity. This study certainly not been able to reveal the overall potential intended. Further research needs to bed one to isolate the compounds related to the anti-cancer potential and antioxidant that contained in R. deceptor and T. papillosum.