Biological activities of an aboriginal herbal medicine, Piperaceae kadsura (Choisy) Ohwi, in Taiwan

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1 Biological activities of an aboriginal herbal medicine, Piperaceae kadsura (Choisy) Ohwi, in Taiwan Cheng-Hong Yang, Chang-You Pan, and Li-Yeh Chuang* Abstract In recent years, due to the popularity of people for the health conscious, the importance of food safety and quality, natural antioxidants and antimicrobial agents has become the focus of medical researches. This study investigated the biological activities of an aboriginal herbal medicine, Piperaceae kadsura (Choisy) Ohwi. The herbal medicine was extracted with 95% ethanol, and the crude extracts were then subjected to partition extraction based on the solvent polarity (hexane, diethyl ether, ethyl acetate). The various extracts were screened for total phenolic and flavonoids contents, antioxidant activity, and antibacterial activity. Evaluations of antioxidant activity included: DPPH radical scavenging ability, trolox equivalent antioxidant capacity, and reduction capacity. Antibacterial activity against the clinical antibiotic pathogens was screen using the disc diffusion method and minimum inhibition concentration. The results showed that the diethyl ether extracts had the highest total phenolic and flavonoids contents and exhibited significant antioxidant activity. Further, the diethyl ether extracts also revealed a broad and significant antibacterial spectrum against all of the test strains included Gram-positive and Gram-negative bacteria. The study indicates that the diethyl ether extracts of Piperaceae kadsura (Choisy) Ohwi have marked antioxidant and antibacterial activities. Keywords Piper kadsura (Choisy) Ohwi, antioxidant activity, antibacterial activity I. INTRODUCTION N recent years, due to medical improvement, many antibiotics Iwere discovered. Doctors commonly use antibiotics to cure diseases, but lack of proper management of their use, and even long-term abuse, have led to mutations in several bacteria and the emergence of anti-medicine genes. Therefore, stronger antibiotics have to be adopted. However, if this trend continues, a generation of multiply resistant and pan-drug-resistant bacteria will develop, and human beings may have no drugs to use. In Taiwan, the strains of Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii are the most serious resistance to antibiotics [1]. In our lives, many factors are able to cause radicals produced, such as the irradiation of radiation or UV and air pollution. Researchers have confirmed that radicals are associated with Cheng-Hong Yang, Department of Electronic Engineering, National Kaohsiung University of Applied Sciences, Kaohsiung, 807, Taiwan, R.O.C. Chang-You Pan, 2. Department of Chemical Engineering, I-Shou University, 840, Taiwan, R.O.C. Li-Yeh Chuang, Department of Chemical Engineering, I-Shou University, 840, Taiwan, R.O.C.(* chuang@isu.edu.tw ). many diseases, including heart disease, cancers and cataracts and others. Thus, many researches are focused on antioxidant and various health foods have appeared. Numerous foods contain chemically synthesized antioxidants, such as butylated hydroxytoluene (BHT) and active beta hydroxyl (BHA) and others. These antioxidants have been proven having side-effects on human beings [2], so natural substances with antioxidant effects are searching for the synthesized antioxidant substitutes. In recent years, lots of literatures have reported that many bioactivities in Chinese medicinal herbs possess antioxidant capacities [3]. In this study, we try to investigate antibacterial and antioxidant properties of Piper kadsura (Choisy) Ohwi. Piper kadsura (Choisy) Ohwi used in this study is an aboriginal herbal plant in Taiwan. The herbal plant is traditional used for getting rid of rheumatism, clearing meridians, arresting pain and mainly used in wind cold type of arthritis, pain in joints, spastic muscles, cold and pain in abdomen, edema [4]. II. PROCEDURE FOR PAPER SUBMISSION A. Taiwanese herbal medicines Piper kadsura (Choisy) Ohwi. was purchased from local folk medicine dealers. B. Test organisms a) S. aureus ATCC 6538P, A. baumannii ATCC 19606, P. aeruginosa ATCC 27853, P. aeruginosa ATCC and E. coli ATCC were purchased from the Food Industry Research and Development Institute in Taiwan. b) P. aeruginosa 717, A. baumannii 814, A. baumannii 817, E.coli 9005 UTI, S. aureus 908, S. aureus 985, Methicillin-Resistant S. aurens 331, Methicillin-Resistant S. aurens 2118, Oxacillin-Resistant S. aurens 220, Oxacillin-Resistant S. aurens 287 isolates were provided by Chiayi Christian Hospital in Taiwan. III. METHODS A. Preparation of crude extracts The dry herbs (Piper kadsura (Choisy) Ohwi) were minced by a shredder, and then 95% ethanol was added for extraction( herbs and ethanol ratio of 1 g:5 ml). The extraction 20

2 condition was set at 37 C, 200 rpm incubator for 24 shocks hours. The crude extracts were suction filtered and concentrated under reduced pressure machine, and then freeze-dried using the lyophilizer for 24 hours, the yield was calculated. B. Copyright Form The ethanolic crude extracts contain a variety of ingredients which were then purified by partition extraction based on the different polarity of organic solvents. In this study, three different organic solvents were used, including hexane, diethyl ether, and ethyl acetate. Each extraction fractions were collected and concentrated for the biological activity analysis. C. Analysis of antimicrobial activity The petri dish was prepared with a base layer of Muller Hinton (MH) agar (10 ml) and a top layer of 0.75 % MH agar ( 5 ml ), and then inoculated with 50 μl of each bacterial suspension ( 10 5 cfu/ml ). Paper discs (8 mm in diameter) were impregnated with 30 μl of crude extract (3 mg/disc), and placed on the inoculated plates. The plates were then incubated at 37 for 14 hr. The diameters of the inhibition zones (DIZ) were measured [5]. D. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay The crude extracts were diluted with methanol and then reacted with the DPPH solution. The mixture was shaken vigorously and left to stand at room temperature for 30 min in the dark. The absorbance at 517 nm of the reaction solution was measured by spectrophotometer [6]. Inhibition of the DPPH radicals was calculated using the following equation: DPPH radical scavenging rate = {1-[(experimental group-background group)/control group]} x100% E. Determination of total phenolics concentration An aliquot of 0.2 ml sample and gallic acid in distilled water were were added to test tubes containing 1 ml Folin-Ciocalteu reagent. After 8 min, 1 ml of 0.75g/mL Na 2 CO 3 solution was added and the mixture was allowed to stand for 2 h with intermittent shaking. Absorbance was measured at 760 nm [7]. The total phenolics were assessed by plotting the gallic acid calibration curve ( ppm ) was Y = X (where X = concentration of gallic acid equivalents expressed as gram of gallic acid per 100 g of dry weight of the plant material; Y = measured absorbance). The correlation coefficient was R 2 = F. Determination of total flavonoids concentration An aliquot of 1.0 ml sample and quercetin in methanol were added to test tubes containing 0.1 ml potassium acetate, aluminium nitrate and 3.8 ml methanol. After 40 min, the absorbance was determined at 415 nm [8]. The flavonoids were assessed by plotting the quercetin calibration curve ( ppm ) was Y = X (where X = concentration of quercetin equivalents expressed as milligrams of quercetin per 100 g of dry weight of the plant material; Y = measured absorbance). The correlation coefficient was R 2 = G. Reducing power test An aliquot of 75 μl sample were added to test tubes containing 75 μl 1% K 3 Fe (CN) 6 and Sodium phosphate buffer mixture, then incubated at 50 water bath. After cooling on ice, 75 μl of 10% TCA, 0.1% FeCl 3 and ddh 2 0 were added to 300 μl and completely mixed for 14 minutes, then determined the absorbance at 700 nm [9]. The slope of the plot represents the reduction capacity of the test herbal extracts. H. Trolox equivalent antioxidant capacity (TEAC) assay An aliquot of 20 μl sample and different concentrations of Trolox were added to 1 ml mm ABTS solution and then dark stand for 10 minutes. The absorbance was determined at 734 nm [10]. Antioxidant capacity of the natural herbal extracts was determined based on the calibration curve of the different concentrations of Trolox. A trolox solution (final concentration ppm ) was used for calibration of the standard curve was Y = X (where X = concentration of Trolox equivalents expressed as milligrams of gallic acid per 100 g of dry weight of the plant material; Y = measured absorbance) and the correlation coefficient was R 2 = IV. RESULTS A. Analysis of antimicrobial activity This study used various extracts of Piper kadsura (Choisy) Ohwi, selected 5 standard strains and 10 clinical antibiotic resistance strains to conduct antibacterial activity test. As shown in Table I, the extracts of diethyl ether revealed a highest antimicrobial activity against all of the test strains. For some of the strains, the antimicrobial activities of the diethyl ether extracts are even higher than the antibiotic tetracycline. Compare to all of the extracts, the crude extracts presented a broader antibacterial spectrum than other extracts, but the extracts of diethyl ether showed a most significant antibacterial activities among all of the extracts. B. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay The results of DPPH radical scavenging rate revealed that the scavenging ability increased with the concentration of extract increased (Fig.1). As shown in Table 2, the extracts of diethyl ether showed a best scavenging rate among all of the extracts with IC 50 value of 9.47 ppm. The aqueous extract had worse scavenging rate with IC 50 value of ppm. Compared it to BHT (IC 50 value of ppm) and Vit.C (IC 50 value of 7.94 ppm), the radical scavenging ability of the diethyl ether extracts is better than the synthesized antioxidant and close to the nature antioxidant. 21

3 TABLE IV THE TOTAL FLAVONOIDS CONCENTRATION (TFC) OF THE EXTRACTS FROM PIPER KADSURA (CHOISY) OHWI Fig. 1 Free radical scavenging ability of the extracts from Piper kadsura (Choisy) Ohwi bhy DPPH assay TABLE II IC50 VALUES OF THE EXTRACTS FROM PIPER KADSURA (CHOISY) OHWI BY DPPH ASSAY DPPH IC 50 (ppm) BHT ± 1.80 Vitamin C 7.94 ± 0.69 Crude ± 0.36 Hexane ± 0.13 Diethyl ether 9.47 ± 0.38 Ethyl acetate ± 1.45 Water ± 4.99 C. Determination of total phenolics concentration (TPC) The results of TPC are showed in Table 3. The extracts of diethyl ether had the highest TPC ( g Gallic acid/100 g DW), fallowing by the extracts of hexane, crude, ethyl acetate, and aqueous with 78.87, 64.87, 29.26, 2.96 g Gallic acid/100 g DW, respectively. Compared the results of TPC and antioxidant activity, the TPC had positively correlated with radical scavenging ability. TABLE III THE TOTAL PHENOLICS CONCENTRACTION (TPC) OF THE EXTRACTS FROM PIPER KADSURA (CHOISY) OHWI TPC (g Gallic acid /100 g DW) Crude ± 0.21 Hexane ± 0.28 Diethyl ether ± 0.07 Ethyl acetate ± 0.12 Water 2.96 ± 0.04 D. Determination of total flavonoids concentration (TFC) The result of TFC showed that all of the test extracts from Piper kadsura (Choisy) Ohwi have similar total flavonoids contents (Table IV). The extracts of diethyl ether revealed a substantial higher TFC than other extracts. Compared the results of TFC and antioxidant activity, there is no significant correlation between TFC and radical scavenging ability. Flavonoid (g Quercetin / 100 g DW) Crude ± Hexane ± Diethyl ether ± Ethyl acetate ± Water ± 0.01 E. Reducing power test The results of reductive capacity tests revealed that the extracts of ethyl acetate have superior reducing power with approximate abs/10-3 ppm (Table V). The aqueous extracts indicated a lowest reducing capacity (Fig. 2); it means that the components responsible for the reductive capacity are at middle to high polarity. Compared the results of reducing power and radical scavenging ability, it suggested that the active components are different for both antioxidant mechanisms. TABLE V. REDUCING POWER OF THE EXTRACTS FROM PIPER KADSURA (CHOISY) OHWI Reducing Power (abs / 10-3 ppm) BHT 2.9 Vitamin C 21.9 Crude 9.5 Hexane 1.6 Diethyl ether 11.2 Ethyl acetate Water 0.4 Fig. 2 Reductive capacity of the extracts from Piper kadsura (Choisy) Ohwi F. Trolox equivalent antioxidant capacity (TEAC) assay As shown in Table 6, the results of TEAC revealed that extracts of Piper kadsura (Choisy) Ohwi had similar antioxidant capacities (38 mmol Trolox / 100 g DW) except the aqueous extracts (6 mmol Trolox / 100 g DW). Compared the results of DPPH assay and TEAC method, the antioxidant activites are contributed from different active substances. 22

4 TABLE VI. TROLOX EQUIVALENT ANTIOXIDANT CAPACITY (TEAC) OF THE EXTRACTS FROM PIPER KADSURA (CHOISY) OHWI TEAC ( mmol Trolox / 100 g DW) Crude ± 0.22 Hexane ± 0.00 Diethyl ether ± 0.10 Ethyl acetate ± 0.10 Water ± 1.21 V. CONCLUSION In this study, an aboriginal herbal medicine, Piper kadsura (Choisy) Ohwi, in Taiwan was investigated for the antioxidant and antimicrobial activities. The experimental showed that the extracts of diethyl ether revealed a highest antioxidant with the scavenging ability of free radicals and with a highest content of total phenolic compounds among all the test extracts. Morover, the diethyl ether extracts also showed a significant antibacterial activities against the antibiotic resistant strains. In the future works, it might be valuable to isolated and determine the active component from the diethyl ether extracts of Piper kadsura (Choisy) Ohwi. In conclusion, this study indicates that the extracts of diethyl ether from Piper kadsura (Choisy) Ohwi has the potential to be developed as a natural antioxidant and antibacterial agent. REFERENCES [1] C.Y. Hsu, Antioxidant activity of extract from Polygonum aviculare L., Biol. Res., vol. 39, pp , [2] S. Fejes, A. Blazovics, A. Lugasi, E. Lemberkovics, G. Petri, A. Kery, In vitro antioxidant activity of Anthriscus cerefolium L. (Hoffm.) extracts, J Ethnopharmacol, vol. 69, pp , [3] N. Pellegrini, R. Re, M. Yang, C. A. Rice-Evans, Screening of dietary carotenoids and carotenoid-rich fruit extracts for antioxidant activities applying the 2, 2'-azobis(3-ethylenebenzothiazoline-6-sulfonic) acid radical cation decolorization assay, Methods Enzymol., vol. 299, pp , [4] Piper kadsura (Choisy) Ohwi [5] W. U. Li, L. U. Ning, Preliminary studies on antifungal activity of Illicium verum, J. Hen. Agr. Sci., vol. 12, pp , [6] M.A. Gyamfi, M. Yonamine, Y. Aniya, Free-radical scavenging action of medicinal herbs from Ghana Thonningia sanguinea on experimentallyinduced liver injuries, Gen. Pharmacol., vol. 32, pp , [7] T. Yokozawa, C.P. Chen, E. Dong, T. Tanaka, G.I, Nonaka, I. Nishioka, Study on the inhibitory effect of tannins and flavonoids against the 1, 1-diphenyl-2-picrylhydrazyl radicals, Biochem. Pharmacol., vol. 56, pp , [8] N. Pellegrini, D, Del Rio, B. Colombi, M. Bianchi, F. Brighenti, Application of the 2, 2'-azobis(3-ethylenebenzothiazoline-6-sulfonic acid) radical cation assay to a flow injection system for the evaluation of antioxidant activity of some pure compounds and beverages, J. Agric. Food Chem., vol. 51, pp , [9] M.M.I. Nieva, M.I. Isla, A.R. Sampietro, M.A. Vattuone, Comparison of the free radical-scavenging activity of propolis from several regions of Argentina, J. Ethnopharmacol., vol. 71, pp , [10] C.Y. Hsu, Y.P. Chan, J. Chang, Antioxidant activity of extract from Polygonum cuspidatum, Biol. Res.,vol. 40, pp ,

5 TABLE I ANTIMICROBIAL ACTIVITIES OF THE EXTRACTS FROM PIPER KADSURA (CHOISY) OHWI Disc inhibition zone (mm) Strains Crude Hexane Diethyl ether Ethyl acetate Water Tetracycline (1000 mg / ml) (100 mg / ml) (100 mg / ml) 100(mg / ml) 100(mg / ml) 100(mg / ml) P. aeruginosa ATCC ± ± ± ± 0.34 P. aeruginosa ATCC ± ± ± ± 0.25 S. aureus ATCC 6538P 21.1 ± ± ± ± ± 1.09 A. baumannii ATCC ± ± ± ± ± 0.41 E.coli ATCC ± ± ± ± 0.34 P. aeruginosa ± ± ± ± ± ± 0.37 A. baumannii ± ± ± ± ± ± 0.17 A. baumannii ± 0.99 * * * * 20.6 ± 0.0 E.coli 9005 UTI ± ± ± ± 0.40 S. aureus ± ± ± ± ± 1.00 S. aureus ± 0.71 * * * * ± 1.77 Oxacillin-Resistant S. aurens ± 0.71 * * * * 19.9 ± 1.40 Oxacillin-Resistant S. aurens ± ± ± ± ± ± 0.54 Methicillin-Resistant S. aurens ± ± ± ± 1.06 Methicillin-Resistant S. aurens ± 0.35 * * * * ± 1.06 : no active; *: no detected 24