Isolation and characterization of chemical constituents, cytotoxicity, antibacterial and antioxidant activity of the isolates, crude extract from Hornstedtia scyphifera var leaf

Hornstedtia scyphifera var belongs to ginger family, they are most of time used as spices for cooking, preservatives in food industry and colouring agents. It provides health-promoting effects to cutile certain diseases and ailment such as stomach-ache, Catarrh, asthma, diarrhoea, cold and cough, digestive disorder and rheumatism. In this study, we isolated phytochemicals and investigated the potentials of the leaf crude extract of hexane, dichloromethane, chloroform, ethyl acetate and methanol, cytotoxicity, antioxidant and antibacterial activity. The experiment was performed and two phytochemicals (quercetin and Dodecanoic acid) were isolated and characterised. Significant result was obtained from the isolated compounds and solvent crude extract with hexane and methanol have higher cytotoxicity of 35.462 and 34.059, chloroform and methanol with higher antioxidant of 34.46±0.32 and 35.33±0.210. There was an observed significant activity at 500 μg/mL in all the extracts against all the selected pathogen with zone of growth inhibition ranging from 07.70 + 27.30 mm to 27.30 ± 0.10 mm in all the solvent extract. The result indicated that with the isolation of quercetin and dodecanoic acid from Hornstedtia scyphifera var Leaf extract is a potential plant medicine which can be harnessed as an agent for antioxidant and potential antibacterial. The compound was isolated for the first time from Hornstedtia scyphifera var.


Introduction
Numerous efforts by researchers have been directed towards the provision of empirical proof to back the use of tropical plants in traditional medicinal practice. Focus on medicinal plant research has increased worldwide and evidence abounds in the immense potentials of medicinal plants used in various traditional systems (Dahiru et al., 2010).
Various medicinal plants have been studied using different scientific approaches and results from these studies have revealed the potentials of medicinal plants in pharmacology (Fatahi et al., 2003). These medicinal plants such as Hornstedtia scyphifera var are of great importance to the health of the individuals and communities to larger extend, and nutritional benefits are derived from these plants since they are commonly used as vegetables.
Hornstedtia scyphifera var belongs to ginger family, they are most of time used as spices for cooking, preservatives in food industry and colouring agents. However, this species Hornstedtia scyphifera var can provide health-promoting effects to cutile certain diseases and ailment such as stomach-ache, Catarrh, asthma, diarrhoea, cold and cough, digestive disorder and rheumatism (Jani et al., 2016).
There are more than ten species, including H. scyphifera, H. ophiuschus and H. phaeochoana found in Sarawak Malaysia (Hashim et al., 2014). The leaf of Hornstedtia scyphifera var leaf was consumed by the Javanese for flavouring, as well as to protect crops from insects by burning them. They are used as an external application to get relief from fever (Holtum, 1950).
The aim of this work is to isolate the phytochemicals and evaluate the potential of the leaves crude extract of its cytotoxicity, antimicrobial and antioxidant.
To the best of our knowledge, there has not been report on any isolated pure compound and bioactivities of this specie Hornstedtia scyphifera var. Here, we are reporting the isolated compound, cytotoxicity, antimicrobial and antioxidant properties of the leaves extracts of Malaysian Hornstedtia scyphifera var.

Sample Collection
The plant material was collected in Singh, Sarawak, Malaysia They were authenticated by a Botanist in the Universiti Malaysia Sarawak. The identified and certified plant materials were given a voucher number as IJU/2016/15010238 (Hornstedtia scyphifera var.) and The fresh leaves from the plant was carefully plucked and washed under running tap water. It was then air dried to be free of water and then spread in the laboratory and allow to dry at room temperature until they were fully dried.

Sample Preparation
Dried plant materials (leaves) were ground into fine powder form using laboratory pestle and mortar and electric grinder. The finely ground powdered samples (mesh 30) were packed into clean, dry sample containers and were labelled appropriately and kept for further use. Extraction was carried out by the conventional solvent extraction method described by Fasihuddin et al. (2010) and . This was achieved by soaking the ground plant material in solvents in the order of increasing polarity as shown in Figure 2. A total of 2 kg of the dried and ground powdered sample was extracted using cold soaking method with hexane. The sample were soaked in the hexane with the ratio of 1:3 (sample: hexane) in a 5 liters Erlenmeyer flasks at room temperature for 7 days. The resulting hexane solution was then filtered using Whatman filter paper No 4 and the residue was then re-extracted with fresh hexane for another 72 hrs and filtered. Both extracts were combined and evaporated to dryness with a rotary evaporator (Heidolph Laborota 4000 efficient) under reduced pressure below 50 o C to obtain the hexane crude extract. The residue was reextracted using similar procedure with dichloromethane, chloroform, followed by ethyl acetate and methanol to obtain respective crude extracts. The dry weight and percentage yield of each crude extract were determined (simple percentage).

Column Chromatography
The basic principle of column chromatography is to separate a mixture of metabolites based on their molecular weight and polarity. A glass column of size 40/34 (large) was used for chromatography, and the sorbent used was silica gel 60 (Merck 70-230 Mesh ASTM 0.063 0.200 mm). Silica gel slurry was prepared by dissolving silica gel (150 g) with suitable solvent, usually hexane. The column was prepared by pouring a slurry mixture of silica gel and solvent, into a glass column and allow it to settle down (Firdous et al., 2013). The packed column was left overnight before 4-10 g of sample was introduced onto the top of the packed column via wet-packing method or dry-packing method. The column was eluted with suitable solvent systems with increasing polarity (Fasihuddin et al., 2010). The column's valve was then opened and about 10-30 mL fraction of the solvent coming out from the column was collected in test tubes (Patra et al., 2012). The procedure was repeated by using different solvent systems, based on increasing polarity (Table 1). Samples from the column fractions were examined by using TLC plates in few suitable solvent systems to obtain the retention factor (Rf) of any components that appeared as spots. Fractions with similar Rf values were combined (Patra et al., 2012). Fractions which contain more than one component were further isolated and purified by using smaller glass column of sizes 24/29 (medium) or 14/23 (small) with suitable solvent systems.
Fraction with single component (one spot) that appeared in TLC plate was treated as possible pure secondary metabolite. The combined fractions which contained the same single component was then allowed to air-dried or evaporated to dryness to obtain a pure secondary metabolite.

Thin Layer Chromatography (TLC)
The eluents collected from column chromatography, were subjected to thin layer chromatography (TLC) analysis. TLC was carried out using the method described by Isaac et al. (2019). A glass capillary tube was used to apply samples on the TLC plates (size 6.6 x 20 cm, 5 x 20 cm) repeatedly with a spot of about 0.3 mm in diameter. The TLC plate was then placed in a rectangular glass developing chamber with its lower marked edge (1 cm from the base) dipped into a developing solvent below the mark where the samples were spotted. The plates were allowed to develop to the level of upper mark (4 cm from the base) and then removed and dried. The TLC plates were then viewed directly for colored compounds, it was also viewed under UV box for UV fluorescent compound and stained with vanillin for compound that are neither visible nor UV fluorescence. Fractions containing similar characteristics were combined and dried.

Gas Chromatography -Mass spectrometry (GC-MS)
Gas chromatography (GC) analysis of fractions that were obtained from TLC as single spot was performed using a Shimadzu GC-Mass Spectrometry model QP2010 plus, equipped with a BPX-5 column (5% phenyl polysylphenlenesiloxane) of 30 m in length, film thickness of 0.25 µm and internal diameter of 0.25 mm. The operating method was based on the method described by Umaru et al. (2019). Ionization energy of 70 eV was used in the electron ionization energy system of the GC-MS for detection and carrier gas, helium (99.999%) at a constant flow rate of 1 mL per min was used. Exactly 1 µL of purified sample was injected into the GC-MS using a syringe and sample was analysed using split mode with ration of 25:1. Injection temperature was set at 260 o C and the oven temperature was programmed from 60 o C with an increase of 10 o C per min, isothermal for 5 min, to 280 o C, ending with 10 min isothermal at 280 o C at 70 eV. Mass spectra was taken at a scan interval of 0.5 sec and fragments from 45 to 450 Da. By matching its average peak area to the total areas, the relative percentage quantity of each component was acquired. Compound identification was obtained by matching the retention times of the compounds and the mass spectral obtained from the library data of the corresponding compound.

Fourier Transform Infra-Red Spectrometry (FT-IR)
Fourier Transform Infra-Red (FT-IR) was performed using FTIR spectroscopy (Thermos Scientific, Nicolet iS10 SMART iTR) to detect the chemical bonds (functional groups) of the compounds. The operating system was based on the method described by Umaru et al., (2019). The liquid samples were introduced into the machine and scan range was set from 400 to 4000 cm -1 with a resolution of 4 cm -1 . Characteristic of the chemical bonds was read by spectrum produced through transmittance of wavelength of light. The chemical bond in a molecule were detected by interpreting the infrared transmittance spectrum and the functional groups of the compounds were identified based on the Table of Characteristic IR absorptions published in Organic Chemistry (Janice, 2008).

Nuclear Magnetic Resonance (NMR)
Nuclear Magnetic Resonance (NMR) spectrometry was performed using JEOL JNM-ECA 500 Spectrometer. The operating system was based on the method described by Efdi et al. (2010). Sample was dissolved in 0.8 mL chloroform D1 (CDCl3) or Acetone D6 and placed into NMR tube to a sample depth of 4 cm and the 1 H (500 MHz) and 13 C (125 MHz) spectra were measured. Chemical shifts were reported as δ units (ppm) with tetramethysilane (TMS) as internal standard and coupling constants (J) in Hz. Integration of the 1 H-NMR and 13 C-NMR data was performed by using DELTA version 5.0.4 software by JEOL. The identification of each 1 H-NMR and 13 C-NMR detected was based on the Table of Characteristic NMR absorptions published in Organic Chemistry (Janice, 2008) and with the guide of the possible proposed structure given by NIST library.

Melting Point
The melting point of the compounds isolated was determined using a melting point apparatus (Stuat model SMP3). Small amount of the sample was put into a small capillary tube and was inserted into the machine melting point heating bath. The heating process was monitored and the temperature at which the sample begins to melt and completely melted was recorded.

Antibacterial assay
Antibacterial activity of leaf Hornstedtia scyphifera var was determined against four pathogenic bacterial strains E. coli, K. pneumonia, S. typhii and S. aureus using disk diffusion method as reported by various authors (Boyan et al., 2005;Prashanth et al., 2006). The extract was dissolved using dimethyl sulfoxide (DMSO) and sterilized by filtration and stored at 4 o C until use. Standard antibiotics (tetracycline) was used for comparison of the zone of inhibition of the pure strains of the bacteria. The extracts were then screened for their antibacterial activity against the bacterial strains. Set of five dilutions for antibacterial activity (25, 50, 100, 250, 500 μg/mL) of the leaf extracts of Hornstedtia scyphifera var. and standard drug (tetracycline) was prepared in distilled water. Sterile plates containing Mueller-Hinton agar were seeded with indicator bacterial strains and control experiment using tetracycline as standard drug were kept for 3 hrs at 37 o C. They were then incubated for 18 to 24 hrs at 37 o C, and the zones of growth inhibition around the disks were measured in mm. The antibacterial activity of the test organisms on the plant extracts were determined by measuring the diameter of the inhibitory zones on the surface of the agar around the disk, and the values <9 mm were considered as not active against the microorganism for antibacterial activity (Prashanth et al., 2006). The experiment was carried out in triplicate and the mean values of the diameter of zones of inhibition was calculated using statistical software SPSS 22.

Brine shrimp (Artemia salina) Lethality Test
The LC50 of the plant extracts was determined using brine shrimp lethality test. The test was conducted using larvae of Artemia salina based on method developed by McLaughlin et al. (1991). One spatula full of brine eggs was placed into a 250 mL beaker containing 150 mL of sea water placed under light environment. A source of O2 supply was connected to the beaker using water pump at reduced pressure and allowed for 72 hrs to hatch. The brine shrimp (nauplii) were then used for the test. Exactly 4 mg of each extract was dissolved in 200 µL of DMSO (RCI Labscan limited) and a lower series of chosen concentration was prepared by serial dilution with DMSO. The assay system was prepared with 5 mL of filtered seawater containing chosen concentration of extract and 1% yeast extract (for feeding) in a pre-marked 6well microplate and 10 brine shrimps were carefully taken with micropipette and introduced into each microplate. This was done in triplicates making a total of 30 brine shrimps per concentration. Filtered seawater was added to DMSO and 10 brine shrimps in triplicates and this was used as the control groups. If the brine shrimp in these microplates shows a rapid mortality rate, then the test is considered invalid as the nauplii might have died due to some reasons other than the cytotoxicity of the extracts. The setup was allowed to remain for 24 hrs under constant illumination of fluorescent and number of survived nauplii were counted with a hand lens. Based on the data obtained, the average death of the brine shrimp at different concentrations of the extract and the LC50 of the extract was calculated using probit regression by statistical software SPSS 22 and the result was expressed as Mean + SD at the 95% level of confidence (p < 0.05).

DPPH (2,2-diphenyl-1-picryl-hydrazyl) Free Radical Scavenging Assay (Antioxidant)
The free radical scavenging assay of compound 2,2-diphenyl-1-pycryl-hydrazyl (DPPH) was used to evaluate the antioxidant properties of the crude extract. The measurement was based on the method described by Wang et al. (2008). The sample was prepared by diluting 6 mg of crude extract into 6 mL of methanol, producing a concentration of 1000 µg/mL. The stock solution was sonicated to ensure the homogeneity of the sample. Three other concentrations were prepared at 10, 50 and 100 µg/mL, diluted from the 1000 µg/mL stock solution. Sample of 5000 µg/mL was prepared separately by diluting 25 mg of crude extract into 5 mL of methanol.
Approximately 3 mL of 0.1 mM solution of 2,2-diphenyl-1-pycrylhydrazyl (DPPH) in methanol was each added into five series of prepared concentrations (10, 50, 100, 1000 and 5000 µg/mL) of sample solutions (1 mL). Analysis was done in triplicate. The solution was mixed vigorously and left to stand at room temperature for 30 minutes in the dark after which its absorbance was measured spectrophotometrically at 517 nm using Jasco ultra violet spectrophotometer model V-630. Methanol was used as blank (only methanol) and negative control (1 mL methanol mixed with 3 mL DPPH), while ascorbic acid (vitamin C) as the standard. The concentration of the sample required to inhibit 50% of the DPPH free radical was calculated as IC50 and the value was determined using Log dose inhibition curve which performed by using PRISM version 3.02 software, based on the calculated values of the DPPH scavenging activity (%) of the sample (Tailor & Goyal, 2014).

Purification of Compounds 1, and 2 from methanol Hornstedtia scyphifera var Leaf extract
Compounds 1 and 2 were isolated from methanol crude extract of Hornstedtia scyphifera var Leaf extract. About 20 g of the crude extract was loaded using dry pack method into a column packed with silica gel in hexane (100%). The crude extract was then eluted from the column with solvent system in the sequence as indicated in Table 1.

Purification and Structural Elucidation of Compound 1
Purification Compound 1 was isolated from combined fraction HSMtol 9 of 220.9 mg dark brown. TLC analysis of the combined fraction HSMetol 9 was carried out in different solvent system. It was observed under UV light and recorded as shown in Table 3. Light colored spots seen under UV light with the same Rf values collected from fractions HSMtol 9-1 to HSMtol 9-15 were targeted and were combined, and it was labelled as HSMtol 9-A. Separation using small column of combined fractions HSMtol 9-A was performed and combined fraction HSMtol 9-A1 was obtained, it was then separated using small column and combined fraction HSMtol 9-A2 was obtained. Preparative TLC analysis of the combined faction HSMtol 9-A2 was performed in the solvent systems hexane: chloroform (8:2) which gave a good separation from the other spots present.
The targeted spot was labelled as HSMtol 9-A3. TLC analysis of HSMtol 9-A3 was performed in different solvent systems and the result obtained under UV light and vanillin staining showed 2 spots as shown in Table 4. HSMtol 9-A3 was further purified in a smaller column using the solvent system hexane: chloroform (8:2) which gives a better separation as examined on the TLC profile. Fractions with similar Rf values of the targeted spots were combined and labelled as HSMtol 9-A4. TLC analysis of HSMtol 9-A4 was performed in different solvent systems and the spot obtained with its Rf value was recorded as shown in Table 5. Table 5 Rf values of combined fraction HSMtol 9-A4 in different solvent system under UV light

Solvent system (v/v) Number of spots Rf values
Hexane: chloroform (8:2) 1 0.48 Figure 3 Shows the TLC profile for the combined fraction HSMtol 9-A4 in hexane: ethyl acetate (8:2) as a single spot which suggest that it is a pure compound.
GC analysis of the fraction of HSMtol 9-A4 was then carried out, and the result from the gas chromatogram ( Figure 4) showed a single peak at a retention time of 24.48 min. This confirmed that HSMtol 9-A4 is a pure compound and it was renamed as Compound 1.

Figure 4
Gas chromatogram of Compound 1.

Structural Elucidation
Compound 1 was isolated from the methanol leaf crude extract of Hornstedtia scyphifera, with its physical appearance as a yellow crystal and a melting point at 322-324 o C with molecular formula C15H10O7. The mass spectrum of Compound 1 in Figure 5 shows a similarity index of 98.9% with the mass spectrum of the suggested structure of Compound 1 by the NIST library in Figure 6. On the mass spectrum of Compound 1 one of its molecular ion peak was observed at m/z 302 which was found to correspond to the molecular ion peak and molecular ion weight of the suggested structure of Compound 1 by the NIST library which has a chemical formula of C15H10O7. Figure 4 also shows base peak for Compound 1 at m/z 75 which was observed in the mass spectrum of the suggested structure for Compound 1.     Figure 9 shows the result of the 13 C-NMR spectrum of Compound 1. From the result every carbon NMR signal that was observed was assigned to the proposed chemical structure of Compound 1 which is based on the table of 13 C-NMR characteristics absorption reported in Organic Chemistry by Janice (2008).

Purification and Structural Elucidation of Compound 2
Purification Compound 2 was isolated from the combined fraction HSMtol 7 of 180.6 mg dark brown (Methanol crude leaf extract of Hornstedtia scyphifera) Table 2. TLC analysis of the combined fraction HSMtol7 was performed in different solvent systems and the result as observed under UV light was recorded as shown in Table 8. Fractions containing a light yellowish spot were targeted and combined, it was labelled as HSMtol7-A. Combined fraction HSMtol 7-A. was then further purified two successive times in a smaller column using the solvent system hexane: ethyl acetate (7:3), and each fraction collected (HSMtol 7-A1 and HSMtol 7-A2) were observed under UV light and those containing the light yellowish spot were combined and labelled as HSMtol 7-A3. The Combined fraction HSMtol 7-A3 was then tested using TLC and observed under UV light. The result is shown in Table 9. Combined fraction HSMtol7-A3 was further purified using small column and fractions containing the targeted spots from HSMtol 7-A3 were then combined and labelled as HSMtol7-A4. Combined fraction HSMtol 7-A4 was further purified using the solvent system hexane: ethyl acetate (7:3), which gives a better separation. TLC of the fractions collected was performed and examined under UV light. Fractions containing the target spots were combined and labelled as HSMtol7-A5. TLC of the combined fraction HSMtol7-A5 was performed in different solvent system and the result was again examined under UV light as well as vanillin staining. It showed a single sport as shown in Table 10.

Table 10
Rf values of combined fraction HSMtol 7-A5 in different solvent system under UV light.

Solvent system (v/v) Number of spots Rf values
Hexane: Ethyl acetate (7:3) 1 0.62 Figure 10 shows the TLC profile for the combined fraction HSMtol7-A5 in hexane: ethyl acetate (7:3) as a single spot which suggest that it is a pure compound  Figure 10 show that combined fraction HSMtol7-A5 is a pure compound. GC analysis of the combined fraction HSMtol7-A5 was then carried out, and the result from the gas chromatogram ( Figure 11) showed a single peak at a retention time of 19.020 min. This confirmed that HSMtol 7-A5 is a pure compound and it was renamed as Compound 2. It is a white compound and 15 mg was obtained.

Structural Elucidation
Compound 2 was isolated from the methanol crude leaves extract of Hornstedtia scyphifera, its physical appearance as a light compound with a melting point of 44.6 o C. The mass spectrum of Compound 2 ( Figure 12) show a similarity index of 96.5% with the mass spectrum of the compound suggested by the NIST library in Figure 13. The mass spectrum of Compound 2 showed an ion base peak which was observed at m/z 73 and a molecular ion peak of m/z 73 was also observed in the mass spectrum of the suggested structure of Compound 2. The mass spectrum of Compound 2 has one of its molecular ion peak observed at m/z 200, this corresponded to the same molecular ion peak and molecular ion weight of the suggested structure of Compound 2 by NIST library with a chemical formula of C12H24O2.   Figure 15 and Figure 16 ( 1 H-NMR), Figure 17 and Figure 18 ( 13 C-NMR). Based on the table of 1 H-NMR characteristics absorption and 1 H-NMR peaks splitting pattern as reported in Organic Chemistry by Silverstein (2005), the proton signals were all integrated and were assigned to every proton NMR of Compound 2 as the proposed chemical structure.
The 1 H-NMR spectrum of Compound 2 exhibited 13 proton resonates. A singlet proton signal was observed at δ 9.47 (1H, s) indicating the presence of an OH group (hydroxyl) of the structure. A doublet proton signal was observed at δ 2.28, δ 1.63, δ 1.33, δ 1.32, δ 1.31, δ 1.31, δ 1.31, δ 1.31, δ 1.30 and δ 1.37, respectively. Indicating the presence of a methylene group of the long chain of Compound 2 and was assigned to H-2, H-3, H-4, H-5, H-6, H-7, H-8, H-9, H-10, and H-11. A multiplet proton signal was observed at δ 0.99 (3H, m) which correspond with a methyl group and was assigned to H-12. From the result of the 13C-NMR spectrum of Compound 2 every carbon NMR signal that was observed was assign to the proposed chemical structure of Compound 2 which is based on the table of 13C-NMR characteristics absorption reported in Organic Chemistry Silverstein (2005).
Chemical shift of every proton and carbon NMR for Compound 2 is shown in Table 12 and Table 13 and comparison was made with NMR data of similar compound reported by Yamashita et al., (2015) and Zhu (2014).     From the data obtained, the GC spectrum of Compound 2 gave a similarity index of 96.5% with the mass spectrum of the proposed structure by the NIST library, which matched the characteristic of dodecanoic acid (2) with chemical formula C12H24O2. The melting point of Compound 2 is 44.6 o C. The proton and carbon NMR data of Compound 2 were mostly identical to match the NMR signal of dodecanoic acid (2) as reported by Zhu (2014).

2
Dodecanoic acid (1) is a compound containing a carboxylic group attached to the terminal end of carbon one in the structure. The acid was report to have showed virucidal effects on enveloped RNA and DNA viruses. It was also observed to have inactivate bacteria, yeast, fungi, and enveloped viruses. It is also an active anti-microbial, that is alpha-and beta-MG. Also, it is mentioned that the anti-microbial effects of the dodecanoic acid are additive and total concentration is critical for inactivating viruses (Yamashita et al., 2015). Dodecanoic acid has greater anti-viral properties and many of the pathogenic organisms reported to be inactivated by these antimicrobial compound. It is known to be responsible for opportunistic infections in HIV-positive individuals. It can kill harmful pathogens like bacteria, viruses and fungi. This important compound can be beneficial to infants in reducing the cancer risk and future heart disease (Niknamian and Niknamian, 2015).
In a study reported by Rayan and McDonnell (2014), that dietary supplementation of dodecanoic acid in maternal mice enhances resistance to giardia duodenalis infection in suckling Neonatal pups. Yamashita reported the identification of self-growth-inhibiting compounds of dodecanoic acid from Helicobacter pylori (Yamashita et al., 2015).

Cytotoxicity of Hornstedtia scyphifera var leaf extract using Brine Shrimp (Artemia salina)
Result in Table 13 shows the leaf crude extract of hexane, dichloromethane. Chloroform, ethyl acetate and methanol fraction. Highest brine shrimp lethality was observed in methanol crude and hexane crude extract with LC50 value of 34.059 µg/mL, 35.462, respectively. Ethyl acetate crude extract exhibited the lowest activity with LC50 value 62.220 µg/mL when compared to the test control thymol of 1.16 µg/mL. There was an observed concentration dependent increment in mortality rate of the brine shrimp. The isolated compound as shown in table 14, dodecanoic acid exhibited higher toxicity of LC50 of 46.23 µg/mL and Quercetin of LC50 56.66 µg/mL.
In toxicity evolution of plant extracts by brine shrimp lethality bioassay, LC50 values lower than 1000 µg/mL are considered active (Umaru et al., 2018). This has been well utilized for screening and fractionation of physiologically active plant extracts and has also been demonstrated to correlates reasonably well with cytotoxic and other biological properties . Brine shrimp bioassay has been established as safe, practical and economical method for determination of bioactivities of synthetic compounds (Almeida et al., 2002). It has established a significant correlation with in-vitro growth inhibition of human solid tumor cell where it shows the value of this bioassay as a pre-screening tool for antitumor drug research . Which support the results obtained from crude and isolated compound of the leaf extract of Hornstedtia scyphifera var as an agent to be considered. The result is Mean + SD. N = 30 There was an observed concentration dependent increment in mortality rate of the brine shrimp.

Antioxidant Activity of Hornstedtia scyphifera var leaf crude extract
Many medicinal plants as well as the pure bioactive isolates have demonstrated tremendous beneficial therapeutic potentials, and many herbs were reported to contain antioxidant properties, and most of these activities are largely attributed to the phytochemicals (Aqil et al., 2006). Antioxidants are substances that possess free radical chain reaction breaking properties , antioxidant activity is a fundamental important property for life (Velioglu et al., 1998) and has been shown to reduce oxidative stress-induced tissue injury (Pourmorad et al., 2006).
Free radical scavenging activities of leaf Hornstedtia scyphifera from different solvent systems of varying polarity as while as the isolated compounds were evaluated using DPPH a stable free radical method that is sensitive in determining the antioxidant activity of plant extracts and vegetables. It has been reported that natural antioxidant presents in plants scavenge harmful free radicals from human body and exert their mode of action by suppressing the formation of reactive oxygen species either by inhibition of enzyme or by chelating trace elements (Asok Kumar et al., 2009).
The antioxidant effect of Hornstedtia scyphifera is shown in Table 15 and Figure 19 for crude extract and Table 16 and Figure 20 for isolated compounds. The results of the study showed that methanol and chloroform fractions of the leaf extract exhibit strong antioxidant activity with IC50 values of 34.43 + 0.110 μg/mL and 34.46±0.32 μg/mL, respectively. The strong antioxidant activity exhibited by the methanol extract in this study is congruent to the work of Mariya and Reena (2007) in which they reported that methanolic extract was found to be most effective antioxidant property. It was observed also that hexane and dichloromethane fractions exhibited weak antioxidant activity with IC50 values 63.4±0.11 and 51.91±0.921 when compared to the standard ascorbic acid of 17.27±10.16μg/mL. Table 16 and Figure 19 shows the dodecanoic acid to have higher antioxidant potential of 30.63 and lower was observed with dodecanoic acid of 39.47 when compared to the control ascorbic acid of Figure 20.

Antibacterial Activity of Leaves extract of Hornstedtia scyphifera
Over the years, there has been a tremendous increase on the report of antimicrobial properties of medicinal plants by researchers worldwide and this has contributed enormously to the understanding and discovery of natural agents that could be effective in combating microorganism in human health delivery. Table 17 and Table 18 show the mean values of the zone of growth inhibition of leaf extract and isolated compound of Hornstedtia scyphifera against bacterial strains in mm compared to tetracycline.

Figure 19
Radical scavenging activities of leaves extract in different solvents at absorbance of 517 nm.

Figure 18
Radical scavenging activities of Isolated pure compounds at absorbance of 517 nm.
In this study, the leaf extracts and the isolated compounds quercetin and dodecanoic acid of Hornstedtia scyphifera showed significant bactericidal effects against the test bacterial strains; Escherichia coli (E. coli), Salmonella typhi (S. typhi), Klebsiella pneumonia (K. pneumonia) and Staphylococcus aureus (S. aureus), as shown in Table 17 and Table 18.
There was an observed significant activity of the crude extract at 500 µg/mL in all the extracts against the selected pathogen with zone of growth inhibition ranging from 07.70 + 27.30 mm to 19.30 ± 0.10 mm. The activity was observed in Salmonella typhi ranged from 7.87 + 0.06 mm in ethyl acetate extract to 17.97 + 0.06 mm in methanol extract. The activity against E. coli was observed in all the extract which ranged from 7.47 ± 0.15 mm in chloroform extract to 19.73 ± 0.06 mm in hexane extract, as compared to standard drug tetracycline with zone of growth inhibition of 19.77 ± 0.38 mm.
The activity against S. aureus was observed in all the extract range from 9.67 ± 0.06 mm methanol to 18.06 ± 0.06 mm methanol at 500 µg/mL. The activity of leaf extract of Hornstedtia scyphifera on this pathogen K. pneumonia range from 10.0 ± 0.00 mm ethyl acetate to 16.10 ± 0.10 mm ethyl acetate. However higher inhibition was observed with ethyl acetate and methanol crude on Escherichia coli with inhibition value of 20.80 ± 0.10 mm and 20.80 ± 0.10 mm, respectively. Lower inhibition was generally observed with hexane crude extract as shown in the Table 17.  However, at lower concentrations of the crude extract, activity was observed but they are not significant as compared to the standard drug as while as to the standard inhibition where inhibition < 9 is considered inactive reported by Jan Hudzicki, (2009).

Conclusion
In this study, we extracted, isolated and characterized two pure compound quercetin and dodecanoic acid from Hornstedtia scyphifera var leaf, a series of biological activity was experimented, demonstrating the necessity of the crude extract as an agent for proper function as an antioxidant and antibacterial potential. These results help to explain the high sequence of medicinal activity deposited and observed at this position in all the solvent extract and the isolated pure compounds. We subsequently found that the crude extract, because of the composition of the phytochemical deposit such as quercetin and dodecanoic acid, the plant extract and its isolate could be used as an agent in pharmaceutical industry. To the best of our knowledge this known compound was first isolated in this plant Hornstedtia scyphifera var leaf.