Antimicrobial and Antioxidative Activities of Bioactive Constituents from Hydnophytum formicarum Jack

Hydnophytum formicarum Jack. (Rubiaceae) is a medicinal plant whose tubers possesses cardiovascular, anti-inflammatory and antiparasitic effects and have been used for the treatment of hepatitis, rheumatism and diarrhea. Herein we report the isolation of its active constituents and the testing of their antimicrobial activity against 27 strains of microorganisms using an agar dilution method and of their antioxidative activity using the DPPH and SOD assays. The results show that the crude hexane, dichloromethane, ethyl acetate and methanol extracts exert such activities. Particularly, the crude ethyl acetate extract exhibits antigrowth activity against many Gram-positive and Gram-negative bacteria with MIC 256 μg/mL. Shewanella putrefaciens ATCC 8671 is completely inhibited at a lower MIC (128 μg/mL). Interestingly, Corynebacterium diphtheriae NCTC 10356 is inhibited by all the tested extracts. Significantly, the ethyl acetate extract is also the most potent antioxidant, showing 83.31% radical scavenging activity with IC50 8.40 μg/mL in the DPPH assay. The other extracts display weak to moderate antioxidative activities, ranging from 28.60-56.80% radical scavenging. The SOD assay shows that methanol extract exhibits the highest activity (74.19% inhibition of superoxide radical). The dichloromethane and ethyl acetate extracts display comparable SOD activity. The promising bioactivities of the crude ethyl acetate extract guided the first isolation of bioactive flavonoid and phenolic compounds: isoliquiritigenin (2), protocatechualdehyde (3), butin (4) and butein (5) from this species. Their structures have been fully established by 1D and 2D NMR. In addition, stigmasterol was isolated from the crude hexane and dichloromethane extracts. The antimicrobial and cytotoxic activities of compounds 3-5 were evaluated. The tested compounds were inactive against HuCCA-1 and KB cell lines, showing ED50> 10 μg/mL. Protocatechualdehyde (3) completely inhibits the growth of Plesiomonas shigelloides with MIC ≤60 μg/mL. As a result, we propose that Hydnophytum formicarum Jack. can serve as a new source enriched with potent antioxidative and antimicrobial agents.


Introduction
Hydnophytum formicarum Jack. or ant plant, a medicinal plant of the Rubiaceae family [1,2], is known in Thai as Hua roi roo [3] as well as by other local names. It is commonly found in the East and South of Thailand, South-East Asia, Papua New Guinea and Pacific Islands [2]. Its tuber possesses cardiovascular, antiinflammatory and antiparasitic effects, and is used for the relief of skin rashes and as a neurotonic [4], and as a treatment of headache [5], hepatitis, rheumatism and diarrhea [6,7].
Hydnophytum formicarum Jack. has several branches with opposite fleshy, oval leaves and small sessile, axillary white flowers and red berries [1]. The typical size varies from 10-12 cm to 30-50 cm [8]. The tubers' cavities have one or more entrance holes communicating with the outside, through which it absorbs both water and substances to obtain essential nutrients from faecal material and debris brought in by ants. In return the plants provide food for the ants in a form of nectar or other proteinrich sources and for this reason Hydnophytum formicarum Jack. is well known as the ant plant.
The plant has a long tradition of use in Thai traditional medicine as a mixture with other plant extracts for the treatment of diabetes. However, no chemical isolation and characterization of the components of this medicinal plant have been reported. The xanthine oxidase inhibitory activity of the Hydnophytum formicarum Jack. was previously reported [6]. Additionally, its antiproliferative activity against human HT-1080 fibrosarcoma cells was described [7]. The many interesting reports concerning the medicinal value of Hydnophytum formicarum Jack. as a folk remedy and its diverse reported biological activitives prompted us to investigate the bioactive ingredients of the tubers of Hydnophytum formicarum Jack to search for new sources of medicinal agents. Herein, we report the isolation, structure elucidation and biological evaluation of the antimicrobial and antioxidative activities , including cytotoxic effects, of the identifed components.

Isolation and structure elucidation
The crude hexane extract of dried Hydnophytum formicarum Jack. tubers was repeatedly chromatographed on a silica gel column using gradient elution with hexane-acetone mixtures to give a green gum containing a white solid. Recrystallization provided stigmasterol (1) of m.p. 152 -153°C [9]. The structure was confirmed by comparison of its 1 H-and 13 C-NMR spectral data with that of an authentic sample. Similarly, the crude dichloromethane extract was isolated and purified as described to give the stigmasterol (1). The crude ethyl acetate extract was separated by repeated silica gel column and preparative TLC using gradient elution to obtain four compounds: isoliquiritigenin (2), protocatechualdehyde (3), butin (4) and butein (5) (Figure 1), all of which have been isolated from Hydnophytum formicarum Jack. for the first time. The structures of 2-5 were elucidated using UV, IR, and comparison of their 1 H-and 13 C-NMR with the literature data [10][11][12][13][14][15][16][17][18]. In this study, 2D NMR spectral data was also obtained.

Antimicrobial activity
The crude hexane, dichloromethane, ethyl acetate and methanol extracts, and the isolates 3-5 from Hydnophytum formicarum Jack. were tested for antimicrobial activity against 27 strains of microorganisms using the agar dilution method [19]. The results ( The antimicrobial activity of pure compounds 3 -5 isolated from the crude ethyl acetate extract was similarly evaluated. It was found ( Table 1) that only compound 3 displayed complete inhibiton against P. shigelloides, with MIC ≤ 60 μg/mL. In comparison ampicillin, a positive control, showed antigrowth activity against P. shigelloides at MIC 10 μg/mL. a: compound 4 was tested at 64 μg/mL, no growth inhibition.
The SOD activity at 300 μg/mL shows that the crude methanol extract exhibits the highest (74.19 %) inhibition of superoxide anion. The native SOD, a positive control (7.6 U) exhibits 75.98 % inhibition. The crude dichloromethane and ethyl acetate extracts exert moderate SOD activity (62.24 and 67.91% inhibition, respectively) as shown in Figure 3. The hexane extract exhibits weak SOD activity. a: α-Tocopherol was used as a positive control.
b: Native SOD (7.6 U) from bovine erythrocytes was used as a positive control.

Cytotoxic activity
The isolates 3, 4 and 5 were evaluated for cytotoxic activity [22] against two cell lines: HuCCA-1 and KB, using etoposide as a positive control. It was observed that the tested compounds 3 -5 (Table  3) were all inactive, showing ED 50 > 10 μg/mL. The antimicrobial and antioxidative assay results (Tables 1 and 2) have shown that all the tested extracts exert such some biological activity. Particularly, the crude ethyl acetate extract exhibited antigrowth activity against many Gram-positive and Gram-negative bacteria with MIC 256 μg/mL, while S. putrefaciens ATCC 8671 is completely inhibited at a lower MIC of 128 μg/mL. It is interesting to note that all the tested extracts can inhibit the growth of C. diphtheriae NCTC 10356. However, antigrowth activity against the yeasts tested, S. cereviseae ATCC 2601 and C. albicans ATCC 90028, was not observed.
The antioxidative activity (DPPH assay) of the tested extracts indicated moderate to high (55.70-83.31 %) radical scavenging activities, except for the hexane extract, is a weak antioxidant (28.60 % radical scavenging). Significantly, the ethyl acetate extract is the most potent in this respect, exhibiting 83.31% radical scavenging activity with IC 50 8.40 μg/mL. In the SOD assay the methanol extract exerted the highest inhibition (74.19 %) of superoxide radical. It is interesting to note that the activity increases with higher polar solvent extracts. It was reported that methanol and methanol-water extracts of Hydnophytum formicarum Jack. showed xanthine oxidase inhibitory (XO) activity (10.3 and 32.5%, respectively) at 50 μg/mL, while its water extract at 100 μg/mL exhibited < 0.1% XO activity [6]. In addition, methanol and methanol-H 2 O (1:1) extracts of Hydnophytum formicarum Jack. displayed potent antiproliferative activity against human lung A549 adenocarcinoma cell, with an ED 50 value of less than 4 μg/mL. Such activity was observed against other cell lines, such as highly metastatic human HT-1080 fibrosarcoma, human cervical HeLa and murine colon 26-L5 carcinoma cells [7]. Our findings have led to the first isolation of flavonoid and phenolic pure compounds 2-5 from the ethyl acetate extract of Hydnophytum formicarum Jack. so it is conceivable that the potent antioxidative activity (DPPH assay) of the ethyl acetate extract may result from these constituents 2-5. Their structures have been fully established by 1D and 2D NMR. Additionally, stigmasterol was also isolated from the crude hexane and dichloromethane extracts.
The isolated compounds 3-5 were tested for antimicrobial and cytotoxic activities. The tested compounds were inactive against HuCCA-1 and KB cell lines, ED 50 >10 μg/mL. Only protocatechualdehyde (3) exhibits antigrowth activity against P. shigelloides, with MIC ≤60 μg/mL. Due to the limited quantity available of the isolated compounds, compounds 4 and 5 were tested at 64 and 115 μg/mL, respectively. No antimicrobial activity was observed at these concentrations.
Diverse biological activities of isoliquiritigenin were reported. For example, it exhibited effects on cardiac [28] and aortic smooth muscle [29], and showed anti-platelet [30], vasorelaxant [31], antioxidant [32] and antitumor activities [33]. Particularly, isoliquiritigenin was very potent antioxidant toward LDL oxidation [24], as well as, xanthine oxidase inhibitor [25] and tyrosinase inhibitor [26]. It was first reported to inhibit proliferation and induce apoptosis of human hepatoma (Hep G2 cells) via p53-dependent pathway [34]. Inhibition of tumor necrosis factor-α (TNF-α) on the induction of ROS production and neutrophil adhesion to endothelial cell was also described [35]. Moreover, its biological activities including the decrease of prostaglandin E2 and nitric oxide production in RAW 264.7 mouse macrophages cell [36], potent antitumor activity and inhibition of prostate cancer cell [37] were documented.
The specific rotation of butin (4) was -87° and was first found in Hydnophytum formicarum Jack. Previously, butin was isolated from the seeds of Butea frondosa and was shown to possess postcoital antiimplantation and anticonceptive activities in the pregnant rats when administered orally during the implantation period [46]. Furthermore, butin was found in Vernonia anthelmintica Willd and in dried vine stem of Spatholobus suberectus Dunn [47], which showing the greatest (80.1%) anti-tyrosinase activity of human melanocytes.
It is well known that stigmasterol is a phytosterol isolated from soy beans and other plants. Stigmasterol showed antioxidative activity, determined by the thiocyanate method [65], and lipid antioxidant properties [66]. In addition, stigmasterol significantly suppressed HMG-CoA reductase activity leading to approximately 11% reduction in plasma cholesterol levels in Wistar and WKY rats feeding 0.5 % stigmasterol [67].
According to the reported biological activities of isoliquiritigenin (2), protocatechualdehyde (3), butin (4), butein (5) and stigmasterol (1) our results provide remarkable support for the use of Hydnophytum formicarum Jack. as traditional medicine, e.g. for treatment of diabetes, rheumatism and diarrhea. Furthermore, Hydnophytum formicarum Jack. can be considered as a new natural source of the bioactive flavonoid and phenolic compounds 2-5.

Conclusions
In conclusion, we have provided the experimental data to support the use of Hydnophytum formicarum Jack. as a herbal medicine. Significantly, the crude ethyl acetate extract exhibited potent antioxidative activity (DPPH assay), with IC 50 8.40 μg/mL, as well as antimicrobial activity. In addition, the other tested crude hexane, dichloromethane and methanol extracts show antimicrobial and antioxidative activities. The compounds 2-5 isolated from the ethyl acetate extract are effective antioxidants and antimicrobials. Although these compounds 2-5 were previously isolated from other plants, they have been first found in Hydnophytum formicarum Jack. by our group. Their structures have been fully established by 1D and 2D NMR. The compounds 3-5 were tested for antimicrobial and cytotoxic activities. The tested compounds were inactive against HuCCA-1 and KB cell lines, showing ED 50 >10 μg/mL. Protocatechualdehyde (3) completely inhibits the growth of P. shigelloides, with MIC ≤60 μg/mL. As a result, the Hydnophytum formicarum Jack. could serve as a new natural source enriched with potent antioxidative and antimicrobial agents. Our isolates 2-5 are potential lead compounds to be further structurally modified leading to new and effective therapeutics. Other biological activities of Hydnophytum formicarum Jack. are being investigated.

General
Melting points were determined on an Electrothermal melting point apparatus (Electrothermal 9100) and are reported without correction. 1 H-and 13 C-NMR spectra were recorded on a Bruker AM 400 instrument with a 400/100 MHz operating frequency using deuterochloroform or deuteromethanol solutions with tetramethylsilane as internal standard. Mass spectra were determined using a Finnigan MAT INCOS 50 mass spectrometer. Infrared spectra (IR) were obtained on Perkin Elmer System 2000 FTIR. Ultraviolet (UV) spectra were measured with Milton Roy Spectronic 3000 Array. Optical rotation was measured in methanol solution with sodium D line (590 nm) on JASCO DIP370 digital polarimeter. Column chromatography was carried out using silica gel 60 (0.063 -0.200 mm) and silica gel 60 (particle size less than 0.063 mm). Thin Layer Chromatography (TLC) and preparative Thin Layer Chromatography were carried out on silica gel 60 PF 254 (cat. No. 7747 E., Merck).

Plant material
Tubers of Hydnophytum formicarum Jack. were purchased from the "Chao krom pur" traditional drug store, Bangkok. It has been identified (BKF 135252) by The Forest Herbarium, Royal Forestry Department, Bangkok. A voucher specimen has been deposited at Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, Thailand.

Cell cultures
HuCCA-1 cells were established from chlolangiocarcinomas experimentally induced in hamsters. The cell lines were characterized and have been maintained in CRI laboratory ever since 1994 in Ham's F12 culture medium (GIBCO Laboratories, Grand Island NY) supplemented with 10% fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT), 100 U/mL penicillin and 100 μg/mL streptomycin. The KB cell lines, originally derived from epidermoid carcinoma of the floor of the oral cavity and commonly used as a reference laboratory standard for cytotoxicity assay, has been maintained in CRI laboratory in DMEM (Dulbecco's modified Eagle medium).

Extraction and Isolation Extraction
The air-dried tubers of Hydnophytum formicarum Jack. (4 kg) were ground and extracted with hexane (10 L × 7 days × 3), followed by filtration. The filtrates were combined and evaporated to dryness under reduced pressure to obtain crude hexane extract. Similary, the extraction was carried out using dichloromethane, ethyl acetate and methanol to give the corresponding dichloromethane, ethyl acetate and methanol extracts.

Isolation
The crude hexane extract (15 g) was purified by column chromatography on silica gel. Elution was conducted initially with hexane gradually enriched with acetone. All fractions were collected, monitored by TLC and combined as appropriate. The solvents were evaporated to dryness in vacuo to afford 14 fractions (H1 -H14). Fractions H9 and H10, eluted by 3-5% acetone-hexane, were combined and evaporated to give green gum containing white crystals (2.7 g). This mixture was further separated by column chromatography over silica gel using gradient elution (hexane-acetone) to give a white solid. Recrystallization of the solid from hexane-ethyl acetate gave stigmasterol (1, 496 mg), m.p. 152-153 o C.
The crude dichloromethane extract (20 g) was subjected to silica gel column. Initial elution with hexane then gradually increasing with acetone and finally enriched with methanol gave fractions which were collected and combined according to TLC chromatogram. The solvents were evaporated to dryness to afford 18 fractions (C1 -C18). Fractions C9 and C10 (2.31 g) was repeatedly isolated by silica gel column. Elution with increasing polarity (hexane-acetone) afforded white amorphous compound which was recrystallized from hexane-ethyl acetate to give stigmasterol (1, 77 mg), m.p. 152-153 o C.
The crude ethyl acetate extract (59 g obtained from 10 kg of Hydnophytum formicarum Jack. which was extracted as described) was isolated by silica gel column. Elution with hexane, then gradually increasing polarity with acetone and finally enriched with methanol were performed. All fractions were collected and combined as usual using TLC monitoring. The solvents were evaporated to dryness to give 11 fractions (E1 -E11). Three selected main fractions (E1 -E3) were isolated and purified.
Fraction E2 (6.75 g) obtained from elution with 30% acetone-hexane, was further purified by silica gel column chromatography. Elution with increasing amounts of methanol in dichloromethane, then purification by preparative TLC using 3% methanol-dichloromethane as a developing solvent afforded a semi-solid. Recrystallization from methanol-ethyl acetate gave 3,4-dihydroxy-benzaldehyde Fraction E3 (2.26 g of brown solid obtained from 40% acetone-hexane elution) was rechromatographed on a silica gel column. Elution with 6% methanol-dichloromethane afforded a brown gum, which was further purified by preparative TLC on silica gel developing five times with 6% methanol-dichloromethane to provide two bands. The lower band (R f = 0.40) was recrystallized from acetone-methanol to give 2- (3,4- Biological evaluation: Antimicrobial assay [19] Antimicrobial activity of the crude extracts and isolates 3-5 was investigated using the agar dilution method [19]. Briefly, the tested compounds dissolved in either CH 2 Cl 2 or MeOH were individually mixed with Müller Hinton (MH) broth to obtain a final volume of 2 mL. Two-fold dilution was prepared and the solution was then transferred to the MH agar solution to yield the final concentrations ranging from 256-4 μg/mL. Twenty seven strains of microorganisms (listed below), cultured in MH broth at 37°C for 24 h, were diluted with 0.9 % normal saline solution to adjust the cell density of 10 8 CFU/mL.
The organisms were inoculated onto each plate using a multipoint inoculator and further incubated at 37°C for 18-48 h. Compounds which possessed high efficacy to inhibit bacterial cell growth were analyzed.
Antioxidative assay [20,21] Two methods were used, DPPH and SOD assays. The antioxidative activity of the crude extracts was elucidated by DPPH radical scavenging assay [20]. When DPPH (a stable purple color) reacts with an antioxidant compound, it is reduced to yield a light-yellow color of diphenylpicrylhydrazine. Changes of the color can be spectrophotometrically measured. In this study, experiment was initiated by preparing 0.2 mM solution of DPPH in methanol. One mL of this solution was added sample solution (1 mg/mL dissolved in methanol, 0.5 mL). After 30 min, absorbance was measured at 517 nm and the percentage of radical scavenging activity was calculated from the following equation: % Radical scavenging = (1-Abs.sample/Abs.cont)×100 where Abs.cont is the absorbance of the control reaction and Abs.sample is the absorbance in the presence of sample.
The SOD activity was assayed by measuring inhibition of the photoreduction of nitro blue tetrazolium (NBT) [21]. The indirect assay is comprised of several reactions: the photochemically excited riboflavin was first reduced by methionine into a semiquinone, which donated an electron to oxygen to form the superoxide source. The superoxide readily converted NBT into a purple formazan product. In this regard, the SOD activity was inversely related to the amount of formazan formed.
Cytotoxic assay [22] Cytotoxic activity was determined by a slightly modified quantitative cytotoxicity assay from other investigators [22]. Briefly, the confluent cell monolayers were trypsinized and diluted with appropriate culture medium to a final concentration of 3×10 5 cells/mL. Portions (100 μL) containing approximately 3×10 4 cells were distributed into 96-well flat-bottomed tissue culture plates and incubated overnight at 37 o C in a humidified 5% CO 2 incubator. Solutions (100 μL) containing different concentrations of tested compounds 3 -5 (0.001 -10 μg/mL) or taxol (0.012 -1.2 μg/mL) were added to each well and the plates were incubated as above for an additional 48 h. After the incubation, each well was washed (x 3) with phosphate-buffered saline (PBS, pH 7.2) and then stained with crystal violet. After the excess dye was removed, the stained cells were lysed with 100 mM HCl (100 μL) in absolute methanol and the optical density was determined by a microtitre plate reader (Titertek, Multiskan MCC/340) set to read at a wavelength of 540 nm. All tests were carried out in quadruplicate and the mean value was calculated. The activity was expressed as ED 50 (the effective dose that inhibits 50% of cell growth).