Rhizanthes deceptor is a species that found only in Sumatera, Indonesia. Crude ethanolic extract of Rhizanthes deceptor and its host, T. papillosum papillosum were examined for their phytochemical properties and antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH). The phytochemical qualitative analysis showed that all the extracts, containing alkaloid, phenolic and flavonoid. Triterpenoid detected in bud extract and root extract, saponin detected in root and stem extract and steroid only detected in stem extract. The result showed high level of total phenolic content from all the extract with their value are 431.52, 323.93 and 271.38 mg GAE/g from bud extract of R. deceptor, root and stem of T. papillosum, respectively. Presence of detected secondary metabolites thought to contribute to antioxidant activity of this extract. Antioxidant activity of the plants showed that bud extract of R. deceptor and root and stem extract of T. papillosum had fairly high DPPH antioxidant activity with IC50 are 32, 22 and 35 μg mL1, respectively. The result of antioxidant activity test indicates that R. deceptor and T. papillosum as, natural sources of antioxidant.
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Plants has been used by human for millennia, maybe as old as, humanity itself. Cures as yet undiscovered may exist in plants as yet undescribed. Currently, it is estimated over 50,000 species used worldwide for medicine purposes (Schippmann et al., 2002). Demand for traditional medicine is increasing around the world alongside with growing environmental awareness and a desire for natural healing through natural products (Hawkins, 2008).
Rhizanthes deceptor belongs to a parasitic plant, Rafflesiaceae. Unlikely the famous member of Rafflesiaceae, Rafflesia, R. deceptor found less popular. The knowledge about R. deceptor is extremely poor, even traditional knowledge. Taxonomic revision, biology and some ecological information given by Banziger and Hansen (2000). Some books considered R. deceptor as, medicinal plant, but no information about what exactly the utility of R. deceptor. Traditional people often utilize the plant as, medicinal plant but its not happen to R. deceptor. Although, this flower have found only in Sumatera, no information so far about utilization of this flower in Sumatera.
Unlike R. deceptor, Rafflesia has known in some region as, traditional medicinal plant. This traditional information used by scientist as, starting point for bioprospecting. Research about bioprospecting of Rafflesia has been done in some country. Latest research about Rafflesia kerri carried by Puttipan and Okonogi (2014) while research about Rafflesia cantleyi and its host Tetrastigma tuberculatum carried by Zulkffle et al. (2014). Both of this research found high antioxidant ability from their samples.
Host of R. deceptor is T. papillosum and T. pedunculare (Banziger and Hansen, 2000). On Tetrastigma genera, there has been some researched about chemical compound and other potential. Tetrastigma also well known as traditional medicine in some country. Krishna et al. (2013) has examined the content of the chemical compound and test the proximate leaf of T. leucostaphylum. T. hemsleyanum note useful as, an anti-inflammatory, relieving pain and improve blood circulation (Liu et al., 2002), while T. hypoglaucum useful as drugs for broken bones and relieving pain (Liu et al., 2003) and T. angustifolia leaf extract potentially anti diabetes (Junejo et al., 2014).
Currently no study about chemical compounds or bioprospecting of R. deceptor and its host. If we look at bioprospecting of Rafflesia, it is possible that R. deceptor have some potential too. This study is proposed to report a preliminary result about potential of R. deceptor and its host T. papillosum through, the phytochemical screening and antioxidant activity test.
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 mL1. 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 Students 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)|
It is possible that R. deceptor and its host contain some valuable members of alkaloids but we cant 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 mL1 followed by bud extract of R. deceptor and root extract of T. papillosum with 283.21 and 719.95 μg mL1 (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 mL1, 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 mL1, 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)|
The highest IC50 value is the extract of bud of R. deceptor with IC50 value of 31.97 μg mL1, as well as extracts of root sand stems T. papillosum with IC50 value of 21.71 and 35.23 μg mL1, 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 mL1 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 mL1, 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.
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 mL1, 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 mL1, 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.
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