Ingelia White, University of Hawaii - Windward Community College, ingelia@hawaii.edu 
Lora Oshima, University of Hawaii - Windward Community College, oshimal001@hawaii.rr.com
Nelly D. Leswara, University of Indonesia, nellyleswara@yahoo.com

Abstract

Peperomia tetraphylla (G. Forst.) Hook. & Arn. (Piperaceae) or ala ala wai nui kane is a fleshy native Hawaiian herb reputed to cure feminine ailments in traditional Hawaiian medicine. A preliminary study on its antimicrobial activity, palatability and micropropagation techniques was conducted at the University of Hawaii – Windward Community College from May to December 2003. The entire plant, including stems, leaves and spikes was ground in 99.9% methanol with mortar and pestle, and centrifuged for 10 minutes at maximum speed to obtain its supernatant. Various concentrations in duplicates of crude methanolic extract of P. tetraphylla were prepared for pour plate and agar diffusion tests against Candida albicans, Escherichia coli and Staphylococcus aureus. Extract concentrations of 125 mg/mL (1.25 mg /10 µL) and 250 mg/mL (2.5 mg /10 µL) influenced the microbial growth. A highly significant colony reduction (72%) was shown in pour plates containing 125 mg/mL of extract for C. albicans, 57% for E. coli and 30% for S. aureus. The disks containing 2.5 mg /10 µL extract in agar diffusion plates yielded a 33 mm inhibition zone for C. albicans, 9 mm for E. coli and 10 mm for S. aureus. This study suggests the possible use of P. tetraphylla for treatment of vaginitis, cystitis and skin infections caused by those microorganisms. The plant can be safely ingested as a food medicine. In vitro culture of leaves, stems and seeds produced plantlets within 28 to 30 days.

Introduction

Peperomia tetraphylla (G. Forst.) Hook & Arn. (Piperaceae) or ala ala wai nui kane is a Native Hawaiian plant. It grows as a lithophyte or epiphyte on all the main Hawaiian islands except Niihau and Kahoolawe. It is a succulent plant with stems erect or ascending, 8 to 30 cm long. Leaves are opposite or whorled. Spike Inflorescence bears perfect flowers. Ovary bears 1 carpellate. There are 2 stamens, and stipules are absent (Wagner et al., 1990).

In many parts of the world, Peperomias are known to have been used as medicines. Arrigoni-Blank et al. (2002) reported the use of P. pellucida in Brazil to treat abscesses and as an antiedematogenic. P. pellucida has also been shown to have analgesic activity (Aziba et al., 2001). Wee (1992) mentioned P. pellucida popularity in the Philippines for use as a poultice for abscesses and boils. In Peru, P. galioides is known to heal wounds (Villegas et al., 1997). In the Marquesas, P. tetraphylla was used to treat skin diseases (Whistler, 1992) and P. membranacea has been used to treat venereal diseases in Hawaii (Bushnell et al., 1950).

Handy et al. (1934) reported a common use of Peperomia sp. in traditional Hawaiian medicines to treat uterine abnormalities and abnormal menses. Kaaiakamanu and Akina (1972) described a mixture of Peperomia sp. (ala ala wai nui pehu) with Euphorbia multiformis, coconut and Dryopteris cyatheoides used in a drink for “afflictions of female sex organs”; while Peperomia sp. known as ala ala wai nui pohina was combined with Euphorbia multiformis, Cyrtandra sp., sugar cane and Dryopteris cyatheoides as a drink to treat “swollen wombs”. Whistler (1992) indicated the use of P. tetrapylla in treating uterine problems.

Ala ala wai nui kane is the common name for all Peperomia species in Hawaii. The two Native Hawaiian Peperomias, P. degeneri and P.rockii are listed in the Hawaii State Statutes under Species of Concern (SOC). Based on the suspected pharmacological properties of many Peperomia sp. and their extensive use in traditional Hawaiian medicine, Peperomia tetraphylla was selected for this study since it is available in nature and is not listed in the Federal List of Threatened and Endangered Plants of Hawaii (Anonymous, 2006).

Antimicrobial activity testing of P. tetraphylla against Candida albicans, Escherichia coli and Staphylococcus aureus was conducted at the University of Hawaii - Windward Community College from May to December 2003. Since Piperaceae (pepper family) is known to be edible, palatability testing of P. tetraphylla was performed to determine its possible uses as a food medicine. Because P. tetraphylla is a minute, slow-growing plant and occurs only in small scattered populations in its natural habitats, micropropagation techniques were explored to cultivate and help protect this plant from being over collected, threatening its existence.

Materials and methods

Preparation of plant material and extracts

Antimicrobial properties of methanolic extract of P. tetraphylla were tested with loop inoculated pour plate and agar diffusion methods. The whole plant, except roots, of P. tetraphylla were collected from a rocky cliff on the Old Pali Road on Oahu in May 2003. The plant was identified at the Kuhi Laau - Tropical Plant and Orchid Identification Facility, Windward Community College. A voucher specimen was preserved in wet herbarium bearing identification number WCC 717. A fresh specimen weighing 15.5 g was surface sterilized in 70% ethanol with two drops of surfactant, Tween 20 for 1 minute, then cut into 5 mm pieces aseptically. Using a sterilized mortar and pestle, plant pieces were mashed in 2 mL of methanol concentrate (> 99.9%). The portions were then transferred into sterile microtubes for centrifuging with the Eppendorf Centrifuge 5415C at maximum speed for 10 minutes. The crude methanolic extract (7750 mg/mL) was pipetted out into empty sterile microtubes as stock A, for use in a pour plate test. Stock B containing 1000 mg/mL crude methanolic extract was prepared by mashing and microcentrifuging 2 g fresh specimen in 2 mL methanol concentrate (> 99.9%) for use in the agar diffusion test. 

Loop inoculated pour plate

This experiment employed a modified loop inoculated pour plate protocol described in Beishir (1996 a). Pure cultures of C. albicans, E. coli and S. aureus in Tryptic Soy Broth (TSB) were each incubated at 30⁰C, 37⁰C and 37⁰C respectively for 24 hours. Diluted suspensions were made from each of these original inocula with a ratio of 1 : 1000 by taking one loopful (10 µL) inoculum to 9.99 mL new TSB. Thereafter, a loopful (10 µL) of each of these diluted microbial suspensions was transferred to test tubes containing 100 µL of stock A and 2.99 mL of melted agar maintained at 50⁰C. Sabouraud Dextrose Agar (SDA) melted agar was used for C. albicans, and Tryptic Soy Agar (TSA) for E. coli and S. aureus. Each tube now contained 250 mg/mL of crude methanolic extract of P. tetraphylla (PA). Each tube was rotated vigorously back and forth 50 times between the palms and the contents were poured into a sterile 35 mm x 10 mm Petri plate.

The same procedures were repeated for pour plate cultures containing 125 mg/mL methanolic extract (PB: 10 µL inoculum was transferred to a tube containing 50 µL of stock A and 3.04 mL of melted agar) and for pour plates without extract as a control (C: a tube containing 10 µL of inoculum in 3.09 mL of melted agar). All treatments were in duplicate. The plates were inverted and incubated at 30⁰C for C. albicans and 37⁰C for E. coli and S. aureus. The number of microorganisms growing on the surface of the agar and those embedded in the medium were counted after 48 hours of incubation. The percentages of growth on both treatments (PA and PB) were calculated based on the number of microorganisms grown in the control media (C) that has been standardized as 100% growth.

Agar diffusion

A modified Kirby-Bauer antimicrobial susceptibility test was used (Beishir, 1996 b). Stock B containing 1000 mg/mL P. tetraphylla crude methanolic extract (P1) was diluted to 500 mg/mL (P2), 250 mg/mL (P3) and 125 mg/mL (P4). Ten µL from each of these four extract concentrations was pipetted onto individual 6 mm sterile filter paper disks placed in closed sterile plates and allowed to dry for 30 minutes. Other sterile filter paper disks each soaked in 10 µL methanol concentrate (M) and sterile distilled water (W) were used as controls.

One hundred µL of each diluted microbial suspension (of 1 : 1000 ratio) containing C. albicans, E. coli and S. aureus was spread with a sterile glass rod over the surface of agar plates (85 mm x 15 mm). The plates were left undisturbed for 5 minutes allowing complete absorption of the suspension into the agar. C. albicans suspension was cultured on SDA. E. coli and S. aureus were each cultured on TSA. On agar plates were placed 6 paper disks each containing one of the following: 10 µL sterile distilled water (W), 10 µL methanol (M), 1.25 mg /10 µL of P4 extract (P4a), 2.5 mg /10 µL of P3 extract (P3a), 5 mg /10 µL of P2 extract (P2a) and 10 mg /10 µL of P1 extract (P1a). All treatments were in duplicate. Plates were inverted and incubated at 30⁰C for 48 hours for C. albicans and 37⁰C for 24 hours for E. coli and S. aureus. Inhibition zones were observed.

Palatability

P. tetraphylla leaves and stems were tasted raw in green salads, boiled in teas, and  cooked in soups and fritters to test their palatability.

Micropropagation

The entire plant was submerged in 50% Versa Clean liquid detergent for 20 minutes. All leaves, stems bearing 2 opposite nodes, and 3 mm³ meristematic tissues were dissected aseptically and sterilized individually in a 10% Clorox solution for 20 minutes, followed by dipping in 5% Clorox solution for 10 minutes and then rinsed with sterile water.

The excised leaves (for aseptic leaf culture) and stem cuttings (for aseptic stem culture) were planted individually in Murashige - Skoog (MS) solid media. The meristems were placed in MS liquid media on a Brinkmann OrbiMix 3010 shaker with a constant agitation of 125 rpm. Seeds (for aseptic seed culture) were wrapped in bibulous paper for submersion in 50% Versa Clean liquid detergent for 20 minutes. They were subsequently sterilized in a 10% Clorox solution for 20 minutes, then rinsed with sterile water. The paper wrapper was opened to retract the seeds for transfer into MS solid media containing 0.5% Plant Preservative Mixture (PPM) and 2% Nystatin.

All aseptic cultures were done in 5 replications. They were maintained at a 23⁰ +  2⁰ C temperature with a relative humidity of 60% and a light intensity of 15 foot candles provided by cool white fluorescent lights. Tissue differentiation and proliferation were observed.

As a comparison, a side study on a traditional stem propagation was conducted. Stem cuttings bearing three internodes were rinsed in tap water and kept in a sealed beaker in the laboratory. Other stem cuttings were directly planted in a peatmoss and vermiculite mixture under a mist system in the greenhouse.

Statistical analysis

Data were analyzed using a Two-way ANOVA test for the block designs with replications. The statistical significance of the differences in the treatments (extract concentrations) for three types of microorganisms and their interactions were assessed by comparing the calculated F and the critical F values at levels of P < 0.05 for pour plate tests and of P = 0.001 for agar diffusion tests.

Results

The result from pour plate cultures revealed that the P. tetraphylla crude methanolic extract indeed influenced the growth of C. albicans, E. coli and S. aureus. A decrease in microbial growth was observed with treatments of 250 mg/mL (PA) and 125 mg/mL (PB) (Table1). The application of 125 mg/mL (PB), however, significantly reduced the growth of C. albicans by 72%, E. coli by 57% and S. aureus by 30% (Fig. 1).

Computation using a Two-way ANOVA reveals a very significant difference in the treatments (PA, PB, or C) at a critical F value (P < 0.05) of 4.3, the calculated F value of 14.2, and P-value of 0.0021. A moderate significant difference in the type of microorganisms receiving these treatments results at a critical F value (P = 0.05) of 4.3, the calculated F value of 6.4, and P-value of 0.0189. There is little or no significant difference in the interaction between these two extract concentrations and the type of microorganisms as shown in its critical F value (P = 0.05) of 3.6, the calculated F value of 2.1, and P-value of 0.16. This is confirmed by a consistent reduction of microbial growth occurring among those three microorganisms treated with either PA or PB.
 

Fig. 1. Percentages of microbial growth in pour plates with and without P. tetraphylla methanolic extracts. A decrease in microbial growth occurred in all microorganisms treated with extracts PA and PB. An optimum concentration is 125 mg/mL. This result is statistically significant (P < 0.05). See text for details.

Results from the agar diffusion test also show that the P. tetraphylla crude methanolic extract is most effective against C. albicans. As seen in Fig. 2, only the disks containing extract concentrations between 1.25 mg /10 µL (P4a) and 5 mg /10 µL (P2a) inhibited the growth of all microorganisms. An optimum concentration of 2.5 mg /10 µL (P3a), however, produced the largest inhibition zone of 33 mm in C. albicans, 9 mm in E. coli and 10 mm in S. aureus (Fig. 3). Control treatments of distilled water (W) or methanol concentrate (M) had no effect on the growth of these microorganisms.

A Two-way ANOVA analysis for the agar diffusion test shows highly significant differences in the effects of treatments on the microorganisms and also their interactions. With P-value of 0.0005, the calculated F values for treatments (= 412) is significantly larger than its critical F value of 6.8 at P = 0.001 (u = 5 df, v = 18 df); the calculated F value for the microorganisms (= 118) is also larger than its critical F value of 10.4 at P = 0.001 (u = 2 df, v = 18 df); and the calculated F value for interactions between treatments and microorganisms is 118, with critical F value of > 3.7 at P = 0.001 (u = 10 df, v = 18 df).

Fig. 2. Average inhibition zones (mm) on agar diffusion plates treated with disks containing various P. tetraphylla methanolic extract concentrations, water (W) and methanol concentrate (M). Inhibition zones are seen in all microorganisms treated with extract concentrations between 1.25 mg /10 µL (P4a) and 5 mg /10 µL (P2a). An optimum concentration 2.5 mg /10 µL (P3a) is most effective against C. albicans. Results are highly significant at P = 0,001. See text for details.
 

                     C. albicans                                                    E.coli                                                            S. aureus

Fig. 3. The largest zones of inhibition on each of agar diffusion plates for C. albicans, E. coli and S. aureus at optimum methanolic concentration of 2.5 mg/10 µL (P3).

P. tetraphyllla was ingested to test its palatability in green salads, teas, soups and fritters. It was discovered that ingesting as little as 2 g of P. tetraphylla leaves and stems causes a slight numbing of the lips and a mild laxative reaction. It tastes watery, bland and crunchy.

Due to the sticky nature of P. tetraphylla seeds, it was necessary to add an antifungal Nystatin and Plant Preservative Mixture (PPM) to the MS media. One hundred percent of seeds germinated within 28 days. Eighty percent of leaf cultures produced adventitious roots within 30 days. The formation of 3 to 4 young plantlets around the leaf took place a month later. In vitro stem cultures gave a 40% success rate. Their adventitious leaves appeared within 30 days, which were followed by root initiations in about a month. Contamination was the biggest challenge in the meristem culture of P. tetraphylla, due to difficulties in handling this miniature plant, which lacks protective leaf primordia. Only 20% of these tissues proliferated and survived in 3 months. Further studies are encouraged to pursue this type of micropropagation.

Although traditional stem cuttings produced roots readily in approximately 7 days, they did not produce plantlets in a great number or as fast as in the in vitro leaf culture.

Discussion and Conclusion

P. tetrapylla has been shown to have antimicrobial properties. It is a powerful antifungal against C. albicans and a bactericidal for E. coli and S. aureus. Two different optimum extract concentrations were obtained to produce maximum inhibitory effects for microorganisms grown in pour plates and on agar diffusion plates. A lower extract concentration of 125 mg/mL or 1.25 mg /10 µL (PB) was most effective in inhibiting the growth of microbes dispersed in pour plates; while a higher concentration (250 mg/mL) was less effective. This might be explained by the fact that E. coli and S. aureus produce defense mechanisms such as capsules (Anonymous, 2002 and 2005) or, in the case of C. albicans, blastoconidia and chlamydoconidia (Tortora et al., 2006) in response to environmental anomalies (i.e. high chemical concentration). E. coli is remarkably responsive to chemical signals (Anonymous, 2002) as noted in Fig. 1, showing a greater difference in growth between PA (250 mg/mL) and PB (125 mg/mL) than C. albicans whose conidia/spores do not exhibit extreme chemical tolerance (Tortora et al., 2006). An extract concentration of 125 mg/mL (1.25 mg /10 µL) perhaps would be the most effective treatment for systemic applications. Since a higher concentration of 2.5 mg /10 µL (P3a) was required to inhibit the confluent growth of microbes on agar diffusion plates, this concentration might be more effectively used for localized or topical treatments.

This study supports the possible effectiveness of ala ala wai nui kane or P. tetraphylla as used in traditional Hawaiian medicine for treatment of uterine problems and other feminine ailments. Perhaps this traditional medicinal approach in selecting and determining P. tetraphylla for treatment of feminine ailments was based on the Doctrine of Signature, due to the phallic appearance of its spike. Those ailments are categorized as candidiasis and vaginitis caused by C. albicans, urinary tract infections and cystitis caused by E. coli, skin infections and scalded skin syndrome caused by S. aureus (Tortora et al., 1998).

Many plants in the Piperaceae family are consumed by humans, including black pepper (Piper nigrum), betel pepper (Piper betle), lolot (Piper lolot), kava (Piper methysticum) and Peperomia pellucida. Staples and Kristiansen (1999) reported the consumption of P. pellucida as green salads in the Philippines and as blanched vegetables in Thailand. It is also used in teas in Indonesia (Zakaria and  Mohd, 1992). Carper (2000) indicated that foods are full of pharmacological agents and for thousands of years, foods with distinct pharmacological properties have been regarded as potent medicines. This study suggests the possibility of commercialization of medicinal and palatable P. tetraphylla as a food medicine in promoting health.

Although 100% seeds germinated from aseptic seed culture, the resulting plants grew very slowly. In vitro leaf culture is recommended because it produces a large number of plantlets in a relatively short time. Once the plantlets have grown into mature plants in the greenhouse, they can be further propagated by traditional stem cuttings. This will help to insure the protection of P. tetraphylla in its natural habitat.

Acknowledgements

This experiment was supported by a grant from the United States Department of Agriculture - Cooperative State Research, Education and Extension Service (USDA-CSREES) FY 2002. The corresponding author wishes to thank the Experimental Program to Stimulate Competitive Research (EPSCoR) for travel funding to present this paper at the International Conference on Natural Products: Chemistry, Technology and Medicinal Perspectives in Almaty, Kazakhstan on October 8 - 11, 2003.

References

Anonymous., 2002. Todar’s online textbook of bacteriology. Kenneth Todar University of Wisconsin. Madison Department of Bacteriology. http://www.textbookofbacteriology.net/e.coli.html

­­­_________., 2005. Todar’s online textbook of bacteriology. Kenneth Todar University of Wisconsin. Madison Department of Bacteriology. http://www.textbookofbacteriology.net/staph.html

Anonymous., 2006. Threatened and endangered plants of Hawaii. http://www.hawaii.gov/dlnr/dofaw/pubs/TEplant.html

Arrigoni-Blank, M., Oliveira, R., Mendes, S., Silva, P., Antoniolli, A., Vilar, J., Cavalcanti, S., Blank, A., 2002. Seed germination, phenology, and antiedematogenic activity of Peperomia pellucida (L.) H.B.K. BMC Pharmacol. 2, 12.

Aziba, P.I., Adedeji. A., Ekor, M., Adeyemi, O., 2001. Analgesic activity of Peperomia pellucida aerial parts in mice. Fitoterapia 72, 57 - 58.

Beishir, Lois., 1996 a. Loop-Iinoculated pour plates. In: Microbiology in Practice. Addison Wesley Longman Inc., pp.89 – 92.

Beishir, Lois., 1996 b. Effects of antibiotics. In: Microbiology in Practice. Addison Wesley Longman Inc., pp. 291 – 294.

Bushnell, O.A., Fukuda, M., Makinodan, T., 1950. The anti-bacterial properties of some plants found in Hawaii. Pac. Sci. 4, 167 - 183.

Carper, J., 2000. The Food Pharmacy. Pocket Books, pp. 7.

Handy, E.S.C., Pukui, M.K., Livermore, K., 1934. Outline of Hawaiian physical therapeutics. Bernice P. Bishop Museum Bull. 126, 21 - 25.

Kaaiakamanu, D.M., Akina, J.K., 1972. Hawaiian Herbs of Medicinal Value. Pacific Book House, Honolulu, pp. 13 - 14.

Staples, G.W., Kristiansen, W.S., 1999. Ethnic Culinary Herbs. University of Hawaii Press, pp. 79.

Tortora, G.J., Funke, B.R., Case, C.E., 1998. Microbiology - An Introduction. Benjamin Cummings Publishing Co. , pp. 562 - 669.

______________________________., 2006. Microbiology - An Introduction. Pearson Benjamin Cummings Publishing Co. , pp. 347 - 348.

Villegas, L.F., Fernandez, I.D., Maldonado,H., Torres, R., Zavaleta, A., Vaisberg, A.J., Hammond, G.B., 1997. Evaluation of the wound - healing activity of selected  traditional medicinal plants from Peru. J. Ethnopharmacol. 55, 193 - 200.

Wagner, W.L., Herbst, D.R., Sohmer, S.H., 1990. Manual of the Flowering Plants of Hawaii. University of Hawaii Press, pp. 1017 - 1036.

Wee, Y.C., 1992. A Guide to Medicinal Plants. Singapore Science Centre, Singapore, pp. 117.

Whistler, W.A., 1992. Polynesian Herbal Medicine. National Tropical Botanical Garden, Hawaii, pp. 181 - 182.

Zakaria, M., Mohd, M.A., 1992. Tumbuhan dan Perubatan Tradisional. Fajar Bakti, Indonesia, pp. 119.