Cultural Experience

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[tabtext] 1.1 Ethics Paper [/tabtext]
[tabtext] 1.2 Abstract [/tabtext]
[tabtext] 1.3 Introduction [/tabtext]
[tabtext] 1.4  Methods [/tabtext]
[tabtext] 1.5 Data Analysis [/tabtext]
[tabtext] 1.6 Results [/tabtext]
[tabtext] 1.7 Results cont. [/tabtext]
[tabtext] 1.8 Discussion [/tabtext]
[tabtext] 1.9 Conclusion [/tabtext]
[tabtext] 1.10 References [/tabtext]
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More to Peru than Machu Picchu

What to expect of Peru?
My impressions of Peru previous to arriving here were based on the little information I had about the country. I was aware of Peru’s famous Machu Picchu and was warned about the health and safety precautions I should take. With this information I really did not know what to expect in terms of the culture of Peru. After one month of residing in Peru, I have found my experiences in the cities of Lima and Trujillo, and the town of Huanchaco to be fascinating.

Lima, Peru’s Mexico City.
The city of Lima became my first impression of Peru. When I exited the plane at the airport, I immediately felt the humid, warm weather. As our MHIRT research group was transported by a taxi cab from the Lima airport to our hotel, I played close attention to the architecture of the buildings and the designs of the streets. I thought to myself, I am in Mexico. Everything looked the same; I must be in Mexico. I had to repeat to myself, I am in Peru, not Mexico, but Peru. I am in South America. I was intrigued by this thought and although Lima is not representative of the entire country of Peru, I automatically felt a sense of comfort since I was surrounded by something that was new yet so familiar to me!

Lima, the largest city of the third world country of Peru, has some of the commodities one can find in a first world country. Although I did not have the opportunity to explore much of Lima, we visited the Larcomar mall which is built into a cliff by the shore. I was surprised to find stores such as Guess and others that had prices I could not afford myself. I had a similar experience with the malls in Trujillo. I asked myself, can people here afford this? They must. Otherwise those stores would not be there, right? These thoughts underlie my assumptions of Peru as a third world country. I find it interesting that citizens of Peru, a third world country, may be economically better off than some citizens, such as myself, from the United States which is considered a first world country.

Huanchaco, the place where the Pescadores and the voluntarios meet.
The beach town of Huanchaco seems to be taken over by tourists. The beach is filled with surf instructors and their students, vendors selling souvenirs and jewelry, and people enjoying the sun.

Huanchaco is foreigner friendly. Many of the businesses in Huanchaco are welcoming to foreigners with foods I recognize such as hamburgers, pizza, and the Desayuno Americano, an American breakfast which includes sunny side up eggs and coffee, offered by the Huanchaco Hostal café. Many of the restaurants and surf shops carry English names such as My Friend, Menu Land and One Wave. In addition to tourists and other foreigners, there seems to be many volunteers in Huanchaco. Local restaurants and even surf shops are also foreigner friendly in the way that they offer discounts to volunteers.

I have rarely seen Peruvians eating at the restaurants. In fact I always see foreigners only, mainly Europeans and Americans, and have begun to recognize some other foreigners who have been in Huanchaco for some time. Foreigners not only eat at the restaurants, but sometimes actually own them. I find this very interesting since these foreigners have decided to move from their native countries to live in Huanchaco permanently.

After one month of residing in Huanchaco, it is obvious that it is a tourist hot spot but I still wonder whether Huanchaco’s true culture is hidden. I feel like I am missing out on the native culture of this town in Peru, which is overshadowed by the tourists. Then again, this maybe the culture of Huanchaco since the people here are used to tourists and voluntarios, volunteers. With this idea in mind, I wonder what is beyond the beach? What are the neighborhoods like? What does a typical home in Huanchacho look like and how do families here live?

I was excited to go into a humble home in Huanchaco where I purchased charcoal. The home was small and very similar to a humble home I would find in my parents’ home town in Mexico. I was fascinated to see how guinea pigs are raised like chickens. I saw four guinea pigs, one cat, and one dog feeding on the same scraps of food off the floor. I figured I would probably never see a rodent and a cat sharing food anywhere else!

My home away from home.
The Huanchaco Hostal, our hostel, is very comforting and home-like. It is located by the Plaza de Armas, Huanchaco’s plaza. Surrounding our hostel are several homes that seem to be uninhabited. This area of Huanchaco seems lonely. My guess is that vacant houses may be owned by foreigners who are not currently here. Besides the lonely nature of the Plaza de Armas, the area is more private and quiet than the beach front area. I find this enjoyable since I can balance the beach’s noise and fun with a peaceful stay at the hostel.

Where are you from?
A typical conversation I tend to have with the Peruvian people involves them asking me about my nationality and my reason for being in Peru. Most people assume that I am either a volunteer or a tourist. Interestingly I have been asked if I was Peruvian, Australian, or White. When I speak Spanish and I say that I am American, I am then usually questioned about that as well. I find myself having to explain that I grew up speaking Spanish since I am Mexican-American.

My struggle with Peruvian Food
Although I find Peruvian food to be delicious, I struggle with the availability of vegetables and other lean foods. In order to avoid becoming sick, I find myself having to be conscious and wise about what I eat. For example, the strange idea of having a salad without lettuce has become a normal request that I make on a daily basis. Not having to worry about food safety, especially with raw vegetables, is something I have taken for granted living in the United States.

Surprising attitudes towards medicinal plants and curanderos
In addition to the Peruvian culture, other Latin American cultures rely on home remedies as a form of healthcare that includes the use of medicinal plants. In my experience volunteering at the Natividad Medical Center in Salinas, California, I observed how pediatricians discouraged parents from using herbs to treat their children. In the United States, Western Medicine disapproves of such practices. I did not expect this to be true for Peru as well.

I became involved with the MHIRT Peru project which entails the study of the medicinal plants used by curanderos in Peru. I had a preconceived idea of Peruvians’ attitudes towards curanderos and their use of medicinal plants. I assumed that most or all Peruvians would be aware of these practices. I was surprised to find this idea to be false when I had the opportunity to listen to Dr. Sharon’s talk at UPAO. Dr. Sharon shared his research on curanderos in Peru to a class of medical students. The medical students seemed skeptical of the use of medicinal plants and seeking curanderos for healthcare. This observation made me realize that not all Peruvians share the same beliefs, especially students who will be practicing Western Medicine.

Universidad Nacional de Trujillo
My weekday routine consists of taking a daily bus to Trujillo, a city approximately 40minutes from Huanchacho. I find the bus cobradores, the bus personnel who charge the bus fare, to be very attentive. The cobradores shout out their destination and will usually make eye contact with people who are taking the bus. If someone who wishes to catch the bus is at a distance, the cobrador will confirm that they wish to take that bus and will stop and wait for the person to get on. The cobradores also help young children and the elderly when they need assistance getting on the bus. I appreciate the attentive nature of the cobradores.

Our MHIRT group works at the Universidad Nacional de Trujillo (UNT), National University of Trujillo. The university is similar to other universities in the States in that it has multiple colleges that offer a variety of majors. Scientific research is not common at UNT. In fact, our lab is currently the only lab equipped to conduct research. Our lab is the best there is, according to Peruvian standards.

I was shocked to learn that there are no regulations in regard to lab work. For example, there are no regulations for the disposing of chemicals and broken glassware. One professor mentioned how Peruvian scientists are given “bioconfianza”, trust with their science projects, and are not constrained by bioethics regulations.

Why I am here
My future career goals entail using research as a means of working with small rural communities. One of my most valuable experiences in conducting research in Peru has been learning about the difficulty of carrying out our research in a place where scientific research is not common. Our lab efforts are often challenged by our limited lab equipment and the few science professionals who are willing to collaborate, among many other things. I am curious to compare the resources available in Mexico, since I hope to work there in the future.

My cultural experiences in Peru have been very positive. I have enjoyed my stays in Lima, Trujillo and especially in Huanchaco. Peru is definitely a place I would like to come back to in the future and I would definitely recommend that others come to visit. In addition to learning science, this experience has been intellectually stimulating in many ways.

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A Comparison for the Efficacy of Bacterial Growth Inhibition Assays Conducted in TheSummers of 2011 and 2012.

ABSTRACT
This study tests the antimicrobial activity of the individual plants Menta spicata L., Pimpinella anisum, and Spartium junceum L as well as the plant mix Menta spicata L.: Pimpinella anisum via bacterial growth assays. Curanderos in Northern Peru use mixtures of these plants among many others plants to treat diseases thought to be caused by infectious agents (Bussman et al., 2007). Based on results from the previous year, quicker and more efficient methods for assays have been developed and carried out. Ethanol plant extracts were used in twenty experiments. These experiments demonstrated improved precision according to the lower% relative standard deviation values obtained in 2012 compared to those obtained in 2011.
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INTRODUCTION
This study tests the antimicrobial activity of the individual plants Menta spicata L., Pimpinella anisum, and Spartium junceum L as well as the plant mix Menta spicata L.: Pimpinella anisum via bacterial growth assays. Curanderos in Northern Peru use mixtures of these plants among many others plants to treat diseases thought to be caused by infectious agents (Bussman et al., 2007). Based on results from the previous year, quicker and more efficient methods for assays have been developed and carried out. Ethanol plant extracts were used in twenty experiments. These experiments demonstrated improved precision according to the lower% relative standard deviation values obtained in 2012 compared to those obtained in 2011.

Curanderos in Northern Peru rely on numerous plant species for their medicinal properties (Bussman et al., 2010). These healers often use mixtures of medicinal plants instead of individual plants, totreat diseases that are believed by western medicine to be infectious diseases. Previous studies have examined the phytochemical as well as antimicrobial properties of selected plants used by Peruvian curanderos (Bussman et al., 2009, 2010). This research project aims to quantitatively measure the antimicrobial activity of individual plants and their mixtures.

Prior efforts by the summer 2011 research group included the design of a reproducible bacterial growth assay that measures the antimicrobial activity of plant extracts. Results in 2011demonstrated varying percent relative standard deviation values, (%RSD), that ranged from2%RSD to 95%RSD for some of the experimental assays analyzed (Simpson, 2011). Instability in the extract during storage at -20ºC accounted for some of the variability in the %RSD values. This made it difficult to quantitatively compare IC50 values, values at which 50% of the bacterial growth is inhibited, for different extract preparations. Also, given the different trend lines need to obtain the best fit for each experiment, a single curve model could not be applied to all experimental data. In response to these problems, in 2012 methods for extract preparation and storage were modified. In addition, the bacterial growth assay was expanded for region of activity in order to obtain data that will be useful for curve fitting.
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METHODS
A total of 20 bacterial growth assay experiments using ethanol extracts of the individual plants Menta spicata L., Pimpinella anisum, and Spartium junceum L as well as the mix Mentha spicata: Pimpinella anisum were considered for this study. Plants were obtained in the Market at Trujillo and identified by local botanists. Voucher specimens are stored at the Herbarium in Trujillo and at the Missouri Botanical Gardens in the USA.

Extract Preparation: In 2011 and 2012 50g plants were macerated for 7 days, filtered through what man filter paper, and rotary evaporated to dryness. In 2011 the extract was resuspended in 20 mls boiling water, sterilized using syringe filters, concentration determined by dry weight, and the extract was stored for 3-4 weeks at -30ºC. In 2012 the amount of dried extract needed was resuspended in smaller volumes of boiling water, filter sterilized, concentration determined by dry weight and the solution stored at -30ºC for no more than one week before all assays were done.

Bacterial Growth Assay (BGA): Each extract solution was serial diluted in Mueller Hinton Broth media and aliquots of each dilution placed into 4 tubes. 100ul of a 10 -5 dilution from a fresh bacterial overnight growth sample of either E. coli or S. aureus was added. A full growth control, containing broth and bacteria, and a no growth control, containing broth, bacteria, and an antibiotic, were set up. These were all incubated, at 37º C for 18 to 24 hours, centrifuged at 3500 x RPM for 6 minutes, resuspended in 3 mls 0.9% Saline solution, vortexed and read at an OD600 on a spectrophotometer. In 2012, shorter length tubes were used to allow a tube to be placed in every spot of the centrifuge. In addition, metal caps were substituted for the cotton and gauze plugs previously used. Also, each media solution made was not reused after the day it was opened. Unlike last year, this summer the tubes containing the initial serial dilutions were incubated and used as additional controls.

The first experiment for each extract was done with 1: 8 serial dilutions to determine the concentration at which growth inhibition occurred. A second assay was set up for plant extracts that showed antimicrobial activity using 2:1 dilutions. These expanded assays were set up according to the two concentrations at which we saw a break of no bacterial growth to bacterial growth in the first screening experiment.
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DATA ANALYSIS
For most experiments the absorbance values were converted to percent growth using the average absorbance of the full growth control tubes. For some experiments, in which the control tubes did not reach full growth, the percent growth was determined by using the tube with the highest growth, or highest absorbance.

The percent relative standard deviation, (%RSD), for each set of tubes in each experiment was determined using the average (AVG) and standard deviation (SD) of the OD 600 values as shown below:

%RSD = (SD /AVG) *100

%RSD values less than or equal to 10% were used as indicators of precision ( Karnes etal., 1993).
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RESULTS
Screening and Expansion of Menta spicata L: Experiments #78 & #79
The screening experiment, experiment #78, demonstrated the antimicrobial activity of the plant Menta spicata L. The percent bacterial growth for the expanded assay of the same extract,experiment 79, is shown in Figures 1a and 1b. The raw data for experiment #79 is shown in Table 1. A break between tubes in column 1 (OD ?? 0) and tubes in column 2 (OD ?? 0.1) is seen in Table 1.

Eight of the 20 experiments considered were screening assays while the remainder 12 experiments were expanded assays. Fifteen out of twenty experiments had %RSD values of less than 10% in at least 50% of its tubes as listed in Table 2. As shown in Figure 2, a total of 120 tubes were studied. 65% of the tubes, 78 tubes, had %RSD values lower than 10%. 15% of the tubes had %RSD values between 11 and 20%. 20% of the tubes, 24 tubes, had %RSD values higher than 20%.

Figure 1a: This graph shows the individual data curves of experiment #79.
Figure 1b: This graph takes the average of the four curves shown in Figure 1a and the curvewas fit with a quadratic trend line. Data is presented as the mean and error bars represent the SD.

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Experiment #79: Expanded Assay of Menta spicata L

Table 1: Raw data for Experiment #79 is shown. *Note symbols: -ve control = negative control +ve control = positive control AB = antibiotic

EtOH Plant Extract Experiments Conducted

Table 2: This table lists the experiments that were considered aswell as the percentage of tubes with %RSD values lower than 10%.

Individual %RSD Values for Plant Extract Experiments Conducted

Figure 2: This graph shows thedistribution of individual %RSD values inthe 20 experiments considered. Only thelowest %RSD valued tubes, 96 out of 120total tubes, are represented.

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DISCUSSION
While twelve expanded assays were completed, only experiment #79 demonstrated a break in the growth of bacteria. The data is shown in Table 1. This experiment was an expansion of experiment #78 which demonstrated antimicrobial activity between the final extract concentrations of 13,350 ug/ml and 3,340 ug/ml. Experiment #79, which is an expansion of #78showed a break between the first tubes with the final extract concentrationsof7,980 ug/ml and5,320 ug/ml (Table 1). This range fits within the range determined from the screening. Although the expanded experiments for most ethanol extracts that demonstrated antimicrobial activity were not successful, experiments #78 and #79 show that this two part assay is promising. It has potential to create sufficient data necessary for precisely determining the concentration at which 50% of the bacterial growth is inhibited. In some cases there was insufficient extract for the expanded assays and these showed all growth. Other expansions had all growth or no growth at all. Because the expanded experiments did not always cover the range of changing antibacterial activity, the OD of the screening experiments was also determined to use for curve fitting analysis. It is possible to visually inspect the tubes to determine where the activity diminishes in the serial dilution; however, if OD values are not determined no further analysis can take place.

Issues with the full growth controls of some of the experiments were faced and percent bacterial growth values were normalized. Since, these controls did not reach full growth of the bacteria; they could not be used to determine the percent bacterial growth of the experimental tubes. The problem with the full growth controls was solved by using a more concentrated fresh bacterial culture. A 10-5 dilution instead of a 10-9 dilution was used. When considering %RSDvalues, the absorbance (OD values) instead of the percent bacterial growth values were considered. This ensured consistency when comparing % RSD among all of the experiments.

In comparison to 2011, the %RSD values for the ethanol extracts experiments of 2011were lower. These lower values indicate improved precision. These improvements may be explained by the improved sterility obtained by using metal caps and fresh media. %RSD values greater than 100% were obtained. For example, in table 1, we see that the first set of tubes had a%RSD of 120%. In the same table we see that the OD values were 0.0.0.01, and 0.01. Since values are very close we can say that the standard deviation is low and a %RSD of 120 is not accurate for this case. This is worth noting since %RSD is only accurate when bacterial growth is greater than a certain amount of bacteria that is currently unknown. The data obtained this year for the screening experiments can be used to obtain IC50 values and is the topic of another paper by Cindy Lau from this year’s MHIRT group (Lau, 2012)
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CONCLUSION
With improvements in sterility, which included the use of metal caps and fresh media, the issue of contamination was solved and more precise data was obtained. Expansion assays should be continued and sufficient amounts of extract for the plants studied should be prepared for acomplete study if the activity of the fully dried extract at -20ºC is not stable. Results from 2012 serve as preliminary results. In the future, a larger data set should be generated and % RSDvalues should be compared to those of 2012.
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REFERENCES
1. Lau, Cindy. The Effectiveness of Individual Plant Extract vs. Mixed Extracts on E. coliand S. aureus. MHIRT Science Paper 2012.

2. Simpson, Rachel. The Antimicrobial Activity of Medicinal Plants Used by TraditionalHealers of Northern Peru to Treat Infectious Disease. MHIRT Poster Presentation 2011.

3. Bussmann, Rainer W et al. Herbal Mixtures in Traditional Medicine in Northern Peru.Journal of Ethnobiology and Ethnomedicine, 6(10), March 2010.

4. Bussmann, Rainer W et al. Minimum inhibitory concentrations of medicinal plants usedin Northern Peru and antibacterial remedies. Journal of Ethnopharmacology 132 (1),2010.

5. Bussmann,Rainer W et al.Phyto-Chemical Analysis of Peruvian Medicinal Plants.Arnaldoa 16 (1): 105 – 110, 2009.

6. Bussmann, Rainer and Sharon Douglas. Plants of the Four Winds: The Magic andMedicinal Flora of Peru. S.N., 1 edition, 2007.

7. Karnes, H. Thomas and March, Clark. Precision, Accuracy, and Data Acceptance Criteriain Biopharmaceutical Analysis. Pharmaceutical Research. 10(10):1420-1426, 1993.
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