Abstract
Tuberculosis has existed in Southeast Asia for thousands of years. Many traditional treatments involve herbal remedies. Over time, these traditional treatments have had the chance to become refined based on efficacy and safety. It was therefore hypothesized that plants that were used in the past and are still used today to treat symptoms associated with tuberculosis are more likely to contain anti-tubercular compounds than plants that have not been used continuously. To try to deduce which plants were used in Laos in the past, a collection of palm leaf manuscripts was studied and a list of plants used to treat symptoms associated with tuberculosis was compiled. Interviews were then conducted with contemporary healers to see if the same plants are still being used today. Plants that were found in the manuscripts and/or are presently used by healers were collected, extracted and were evaluated in an anti-tubercular assay. This paper presents the methods used to identify and collect plants used to treat symptoms indicative of tuberculosis, and the results of anti-TB assays to test for activity.
Introduction
Due to many circumstances, Laos has remained relatively secluded from the outside world. It is surrounded by China and Myanmar (formerly Burma) to the North, Vietnam to the East, Cambodia to the South, and Thailand to the West, and has been referred to as “the land in between” due to its position. The country covers 236,800 km2 (CitationCIA World Factbook, 2007), an area slightly larger than the size of Utah. Undeveloped forest covers more than 40% of the country, and 80% of the population lives in rural areas as subsistence farmers. This situation places most of the population into close contact with medicinal plants from forested areas. Therefore, in the event of health problems, it is less expensive and easier to turn to traditional herbal remedies than to visit a government clinic for medicine. The magnitude of traditional medicine use in Laos makes it an ideal place to study ethnomedicine.
The government recognizes the importance of traditional medicine (TM) in Laos, and has established the Traditional Medicine Research Center (TMRC) under the Ministry of Public Health. An important focus of their research is to gather and catalogue indigenous traditional medicine treatments, the greater part of which consists of herbal remedies, and to publish the results of their research to serve as a reference for use by contemporary healers and the wider public. As part of its function and mandate, the TMRC has established smaller traditional medicine units called provincial traditional medicine stations (TMSs) (CitationSoejarto et al., 1999). Initially, a TMS was established in each of 10 provinces. Each TMS is also affiliated with a traditional medicine hospital. Today, three more TM stations have been added, for a total of 13 TM stations in operation. The staff of a TMS work in direct contact with rural traditional healers who both give and receive information on treatment methods currently being used around the country.
Lao Traditional Medicine or “Ya Phurn Meuang Lao” dates back 4500 years (to the first group of Lao nation in southern part of present China), while the well documented practices started as early as the 14th century, after the establishment of the First Kingdom (Lane Xang Kingdom) (CitationViravong, 1973). While the earliest practices of traditional medicine in Lao PDR are still unclear, it is evident that Buddhist and Sino-Indian influences helped shape the traditional pharmacopoeia and practices used in traditional medicine. In addition, in many of the Buddhist temple-compounds, or wats found in every village in Lao PDR, young monks are trained in traditional medicine by older monks. Information about the uses of medicinal plants also appears in palm-leaf manuscripts located in the libraries of the larger wats, written several generations ago in old Lao and Pali, the language of the sacred Buddhist texts (CitationAbhakorn, 2000).
Lao palm leaf manuscripts date back to the 1400s CE. They are typically written on leaves of the palmyra palm (Borassus flabellifer L., Arecaceae), which are very thick and durable. The leaves are pressed, dried, and cut into uniform shapes, usually around 10 cm in width and range anywhere from 15 to 60 cm in length. The leaves are cleaned and sanded to get a uniform writing surface, then transcribed using a stylus of wood or metal. It has actually been hypothesized that the characters used in the manuscripts developed their rounded shapes because too many straight lines has a tendency to tear the leaves. As this scratching does not give any color, a mixture of soot and oil is then rubbed on to the leaves to make the characters stand out. The leaves are attached together with string, and are often bound between two flat pieces of wood. The length often indicates the type/subject of palm leaf manuscript, with medicinal manuscripts being around the length of a forearm, while religious manuscripts are typically the length of an entire arm.
Buddhist monks in Laos have been regarded as healers for many centuries, and have kept records of their treatments in palm leaf manuscripts. Many of these manuscripts are recorded in scripts that are known only by monks or lay-people who have studied them for many years. The TMRC and the National Library of Laos have hundreds of these medical manuscripts that have yet to be translated into a modern language. There is a vast amount of information waiting to be uncovered.
In the recent past, the use of palm leaf manuscripts has declined and many are becoming subject to mould and insects before being recopied. In recognition of the amount of information at risk of being lost, Dara Kanlaya headed up the Palm Leaf Manuscript Preservation (PLMP) Project in 1989 (CitationAbhakorn, 1996). This project allowed researchers to travel to rural areas of the country with microfilm equipment to capture images of important manuscripts without removing them from their respective community wats (temples). The microfilm is kept at the National Library of Laos, which currently holds around six thousand actual bundles of palm leaves dating back to 1465 along with more than 1030 rolls of microfilm containing more than 54,000 palm leaf manuscript fascicles from more than 830 wats.
The TMRC holds an ample library about traditional medicines, including a collection of medical palm leaf manuscripts. Many of these manuscripts have been translated from their original scripts into the modern Lao language, and some have been translated into English. It has been one of the goals of the International Cooperative Biodiversity Group (ICBG) project, a collaborative research between the University of Illinois at Chicago (UIC), government institutions in Vietnam, and the TMRC in Laos, to explore past and contemporary traditional medicinal plant treatments as a potential source of biologically active compounds for pharmaceutical development (CitationSoejarto et al., 1999, Citation2006). In this search, the information contained in the palm leaf manuscripts represents therapeutic practices of the past.
One of the disease targets in the ICBG project is tuberculosis (TB). Although TB is conventionally treated using modern antibiotics such as isoniazid, rifampin, pyrazinamide and ethambutol, the causative agent, Mycobacterium tuberculosis, has developed strains resistant to conventional antibiotics. Additionally, in countries such as Vietnam and Laos, modern drugs are often expensive and difficult to obtain for much of the population. There is an urgent need for alternative treatments. The aim of this research is to identify some of the herbal remedies used in contemporary and past traditional medical practices for the treatment of tuberculosis in Laos, and to investigate their effectiveness.
Materials and methods
Ethnographic field work
Two months of ethnographic fieldwork were conducted. During this time, palm leaf manuscripts, the majority of which were written in the Tham script to transcribe the Lao language, were perused for indications of tuberculosis. Because entries in these manuscripts were categorized according to symptoms, the manuscripts were investigated for symptoms of tuberculosis (chronic cough, cough with weight loss, cough with blood, cough with fever). The treatments for these entries were recorded, and a list of plants used in the treatments was compiled.
Field interviews were conducted in different regions of the country, where traditional healers were asked a series of questions about their familiarity with tuberculosis and the plants they use for treatment. Before beginning the interview, the healer was told about the goal of the project and was given the option to decline the interview or to withhold any information they did not feel comfortable giving. All of the interviews were conducted in the Lao language and digitally recorded, under a protocol approved by the UIC Institutional Review Board (IRB Protocol number 2007-0369). These field interviews yielded a second list of plants. The two lists were then compared, and the plants cited most frequently in the manuscripts and by healers were prioritized for collection. However, while in the field, if a plant was recognized that had been cited in the manuscripts, or that is presently being used by a healer for TB treatment, it was also collected.
Plant material collected and taxonomic identification
A team of healers and trained investigators from the TMRC and the ICBG project (UIC) carried out fieldwork to locate and collect the plants in both lists mentioned above. Samples of 500-1000 g (dry weight), namely the plant part(s) as indicated in the treatment, were collected. Each sample was supported by a set of fully annotated voucher herbarium specimens. Samples were dried in the sun using nylon mesh bags either in the field or at the TMRC’s facilities. Herbarium specimens were identified by comparison with identified specimens in the collection holdings of the TMRC Herbarium and the John G. Searle Herbarium of the Field Museum of Natural History in Chicago, as well as by using standard floristic treatises, including Pham Hoang Ho’s Cayco Vietnam (1999-2003). A set of voucher herbarium specimens has been deposited at each of the two institutions.
Extraction
Extraction of samples was performed at the pharmacognosy laboratories of the TMRC in Vientiane. A 25 g sample of dried plant material was soaked in 250 mL of 90% ethanol and repeated twice, using 200 mL of ethanol of the same concentration. The samples were condensed using a Heidolph Laborota 4000 rotavapor. From each plant, 2.5 g of extract was transferred into plastic bottles and transported to the University of Illinois at Chicago (UIC).
Test microorganisms
Plant extracts were tested against the following microorganisms: Mycobacterium tuberculosis H37Rv (ATCC 27294), Mycobacterium smegmatis (ATCC 607), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 29213), and Candida albicans (ATCC 10231). All test organisms were cultured at 37°C until log phase. Cultures were then harvested; aliquots were made into 1.5 mL screw-cap tubes, and stored at -80°C until use.
Antimicrobial assays
Crude plant extracts were weighed and dissolved in dimethyl sulfoxide (DMSO) to make a stock concentration of 10 mg/mL. Primary bioassays were tested at 100 μg/mL at the laboratories of the Institute for Tuberculosis Research (ITR) at UIC, using the Microplate Alamar Blue Assay (MABA) and M. tuberculosis (H37Rv) (CitationCollins & Franzblau, 1997; CitationFranzblau et al., 1998). Extracts that exhibited 85% inhibition or higher were submitted for minimum inhibitory concentration (MIC) testing.
A primary bioassay was also conducted against S. aureus, E. coli, C. albicans, and M. smegmatis to test the extracts for tuberculosis specificity. S. aureus and E. coli were tested according to a modified protocol described by NCCLS documents M7-A2 and M100-S3 (CitationNCCLS, 1990; CitationNCCLS, 1991). C. albicans was tested using a modified protocol described by NCCLS document M27-A2 (CitationNCCLS, 2002). M. smegmatis was tested using MABA (CitationCollins & Franzblau, 1997; CitationFranzblau, et al., 1998).
Cytotoxicity assay
Cytotoxic effects of plant extracts were determined by using VERO cells (ATCC CCL-81) and rifampin as a positive control. The testing protocol followed the methods used by CitationFalzari et al. (2005). The IC50 values were calculated for crude extracts that exhibited at least 85% inhibition. The selectivity index was calculated by taking the IC50 value and dividing it by the MIC value: SI = IC50/MIC.
Results
Sixty-nine plants associated with treatments of respiratory illness were found from 29 entries in the manuscripts. Of these, four plants were cited twice and three plants were cited three times. Twenty-three interviews were conducted in three different regions of the country, leading to a list of 185 plants used by contemporary healers. All healers interviewed were male with an average age of 61.43 years, ranging from 36 to 85. The average number of years of formal schooling (in the French education system) reported was 3.64 years, ranging from no conventional formal schooling at all through completion of a university degree. Four healers asserted that they were educated at local Buddhist temples, 12 were living in Bolikhamxai province, one in Vientiane province, and 10 in Champasak province.
Of a total list of 206 plants, 11 plants were both named by healers and cited in the manuscripts. Sixty-six total plants were collected, 15 of these were cited in the manuscripts and 54 were named by traditional healers; 7 of these were both found in the palm leaf manuscripts and named by a healer. Fifty-one plants were taxonomically identified to the species level.
Upon submission to the TB bioassay, 4 plants showed inhibition above 90% and 3 showed inhibition above 85% (). Of the six plants that had inhibition above 85%, only one reported a selectivity index of greater than ten (). This extract also showed significant activity against S. aureus, E. coli, C. albicans, and M. smegmatis ().
Table 1. Percentage inhibition of crude EtOH extracts from plants used to treat different types of cough in Laos.
Table 2. In vitro selectivity for active ethanol. extracts from plants used to treat cough.
Discussion and conclusions
Other plants in the Southeast Asian region have also been investigated and shown to possess anti-TB properties. One example is found in a study conducted by CitationSoe et al. (2006) who tested five unnamed anti-TB ethnobotanicals from Myanmar, a country bordering on Laos. Five of these plants showed in vitro activity against M. tuberculosis H37Rv, and were given to patients with multi-drug resistant (MDR) TB who had not shown satisfactory response to the conventional first- or second-line anti-TB drugs for at least two years. After three months of administration of the extracts, in addition to the conventional second-line drugs, 13 of 15 of these patients had sputum conversion to negative.
Other ethnobotanicals containing anti-TB properties in Asia include Justicia adhatoda Nees (Acanthaceae), used in Nepal and India. This plant, also known as Adhatoda vasica contains the anti-TB compound vasicine in the leaves and led to the development of bromhexine and ambroxol, semi-synthetic derivatives added to cough syrups (CitationGrange, 1996). The rhizome of Alpinia galanga (L.) Willd. (Zingiberaceae), containing a high concentration of the active compound 1’acetoxy chavicol acetate (ACA) (CitationGautam, 2007), is used commonly as a spice in India and Thailand, and has shown activity against streptomycin-resistant mycobacteria (CitationChopra, 1957).
For plant species listed in our present study, previous findings include anti-TB testing on other Polyalthia species. The plant showing the highest percentage inhibition and selectivity index, P. longifolia Thw., has been cited as active against M. fortuitum and M. smegmatis using the disc diffusion method (CitationFaizi, 2003). P. cerasoides (Roxb.) Benth. & Hook. f. ex Bedd., has produced three anti-TB compounds (CitationKanokmedhakul, 2007), and P. evecta Finet & Gagnep. has also shown activity against M. tuberculosis (CitationKanokmedhakul, 2006).
While some interesting plants were cited in the manuscripts or by contemporary healers, there were no plants that were both mentioned in the manuscripts and by healers that showed greater than 80% inhibition of M. tuberculosis. This does not support the original hypothesis, but the small sample size could be misleading. The translation of more medical manuscripts and input from more contemporary healers should give a clearer indication of whether there are clear links between past and present healing systems.
All healers interviewed said that if they suspected that a member of their family had TB, they would first treat it with traditional herbal remedies. If that didn’t work, 10 out of 23 healers said they would turn to a biomedical clinic. Eight healers estimated that they had treated an average of 212 patients for cough, with one well-known 85-year-old healer claiming to have treated around 1,000 people for cough, indicating the numbers of people who turn to traditional healers before government-sponsored clinics. This means that if a herbal treatment is working, tuberculosis will most likely not be diagnosed or reported at a clinic.
Difficulties encountered in our study included different plant species grouped under the same local name, as well as the same plant species having different names in different regions of the country. This could obscure the correlation of plant names from healers to those in the manuscripts if the trend was the same at the time when the manuscripts were written. For future research, the healer will lead the researchers to the plants so that there will be less confusion about the taxonomic identification of plants presently used.
Because the specific symptoms of tuberculosis vary from patient to patient, and to concretely diagnose tuberculosis requires equipment not likely to be found in rural settings where the majority of the population reside and seek primary health care, the term for cough was used to try to include treatments for anything that might be pulmonary tuberculosis. This allowed for many treatments to be included that may have nothing to do with tuberculosis.
Extracts (infusion or decoction) used by healers were derived from single plants, but there were no treatments designed by healers that contained only one plant. This research, therefore, did not look at the effectiveness of the whole treatments, but of smaller parts of the treatments. In the future, it could be beneficial to observe and obtain the actual herbal preparation from the healer to be tested in the bioassays, which could allow active synergistic effects to be detected.
Acknowledgements
We would like to thank Professor Justin McDaniel at the University of California, Riverside for linguistic advice, and to Professor Norman R. Farnsworth at the University of Illinois at Chicago for access to the Napralert database. We also express thanks to Marcelle Hon at the Institute for Tuberculosis Research at UIC for assistance in IC50 analyses. We are indebted to traditional healers of Laos and monastic and lay people at Buddhist temples throughout Laos who helped to direct us. The study was financially supported by the Soejarto Senior University Scholar Fund, as well as ICBG Grant 2-U01-TW001015-09-10, through funds from the US National Institutes of Health, the US National Science Foundation, and Foreign Agricultural Service of the United States Department of Agriculture.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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