In Vitro Effectiveness of Samento and Banderol Herbal Extracts on the Different Morphological Forms of Borrelia Burgdorferi



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July 2010 



     

   


 

In Vitro Effectiveness of Samento and Banderol Herbal Extracts on 

the Different Morphological Forms of Borrelia Burgdorferi

 

 

by Akshita Datar, Navroop Kaur, Seema Patel, David F. Luecke, and Eva Sapi, PhD 

Lyme Disease Research Group 

University of New Haven  

 

Abstract 



 

A tick-borne, multisystemic disease, Lyme borreliosis caused by the spirochete Borrelia 

burgdorferi has grown into a major public health problem during the last 10 years. The primary 

treatment for chronic Lyme disease is administration of various antibiotics. However, relapse 

often occurs when antibiotic treatment is discontinued. One possible explanation for this is that 

B. burgdorferi become resistant to antibiotic treatment, by converting from their vegetative 

spirochete form into different round bodies and/or into biofilmlike colonies. There is an urgent 

need to find novel therapeutic agents that can eliminate all these different morphologies of B. 

burgdorferi. In this study, two herbal extracts, Samento and Banderol, as well as doxycycline 

(one of the primary antibiotics for Lyme disease treatment) were tested for their in vitro 

effectiveness on several of the different morphological forms of B. burgdorferi (spirochetes, 

round bodies, and biofilmlike colonies) using fluorescent, darkfield microscopic, and BacLight 

viability staining methods. Our results demonstrated that both herbal agents, but not doxycycline, 

had very significant effects on all forms of B. burgdorferi, especially when used in combination, 

suggesting that herbal agents could provide an effective therapeutic approach for Lyme disease 

patients.  

Borrelia burgdorferi, the primary causative agent of Lyme disease, is a spirochetal bacterium that 

can adopt different inactive forms, such as cystic and granular forms (round bodies), as well as 

colonylike aggregates both in vivo and in vitro, in the presence of unfavorable conditions such as 

exposure to the antibiotics commonly used for treating Lyme borreliosis.

1-4

 Unfortunately, when 



B. burgdorferi is in these inactive forms, conventional antibiotic therapy will not destroy the 

bacteria.

3

 Still to date, the frontline treatment for Lyme disease is administration of 



pharmaceutical antibiotics such as doxycycline, minocycline, clarithromycin, penicillin G, and 

ceftriaxone.

4,5

 Many studies have shown that in spite of continued and high-dose antibiotic 



therapy, chronic Lyme disease is not treated successfully in many cases.

6

 Also, in the absence of 



ongoing antibiotic treatment, relapse is common.

7,8


 This means that even after antibiotic 

treatment, the host immunity fails to prevent recurrence.

8

 One possible explanation for this 



clinical observation is the presence of different morphological forms of B. burgdorferi, which 


mayprotect it from the antibacterial therapy. Soon after treatment, relapse is observed, most 

likely because the B. burgdorferi can revert to the spirochetal form. Furthermore, the cost of 

antibiotic treatment, especially when administered intravenously, is substantial. Antibiotic 

therapy may also cause multiple undesirable side effects.

9

 Thus, there is an urgent need for 



novel, more efficient, and more cost-effective treatment approaches that can efficiently eliminate 

all forms of B. burgdorferi.  

 

There is an alternative clinical treatment option gaining wide use, called Cowden Condensed 



Support Program, that utilizes several herbal extracts designed to eliminate microbes in Lyme 

disease patients. Richard Horowitz, MD, president of the International Lyme and Associated 

Diseases Educational Foundation (ILADEF), has prescribed this protocol for over 2000 of his 

patient and reports that it has been effective for more than 70% of them. The two herbal agents 

from the Cowden Condensed Support Program selected for this study are Samento (a pentacyclic 

chemotype of Cat's Claw [Uncaria tomentosa] that does not contain tetracyclic oxindole 

alkaloids), with reported antibacterial and antiviral properties, and Banderol (Otoba sp.), known 

to have antibacterial, antiprotozoal and anti-inflammatory effects.

10-12

 Both herbal agents are 



used during the first two months of Cowden Condensed Support Program, then in rotation with 

other antimicrobials for the duration of this 6-month protocol. 

 

In this study, we evaluated these natural antimicrobial herbal extracts as well as doxycycline 



(one of the primary pharmaceutical antibiotics for Lyme disease treatment) for their potential 

effects on the different forms of B. burgdorferi.  

 

The infectious B31strain of B. burgdorferi used in this study, obtained from American Type 



Tissue Collection(ATCC# 35210), was culturedin 5% CO2 at 34 oC, in Barbour–Stoener–Kelly 

H (BSK H) medium supplemented with 6% rabbit serum (Sigma, St. Louis, Missouri) to 

midlogarithmic stage (2 × 107 cells/ml). Samento and Banderol were obtained from Nutramedix 

LLC (Jupiter, Florida). Doxycycline was obtained from Sigma. A wide range of concentrations 

of Samento and Banderol were initially tested to determine the effective concentrations (1:100–

1:1000 dilutions). For doxycycline, a concentration 10× higher than the reported minimum 

bactericidal concentration (250 µg/ml) was used.

13

 Triplicate test tubes containing BSK H 



medium, with and without the appropriately diluted antimicrobial agents, were inoculated with a 

final density of 5 × 106 cells/ml of the test organism. 

 

Direct cell counting methods with Petroff-Hausser counting chambers and morphological studies 



using fluorescent and darkfield microscopic techniques, as well as LIVE/DEAD BacLight 

Bacterial Viability Assay (Life Technologies Corp, Carlsbad, California), were utilized to assess 

the effect of the antimicrobial agents. For statistical analyses, one sample paired T-test was 

performed using NCSS statistical software (NCSS LLC, Kaysville, Utah). 

Samento & Banderol Herbal Extracts 

 



Figure 1A 

 

Figure 1B 



 

Figure 1C 

 



Figures 1: The in vitro susceptibility of the spirochete and round-body forms of the B31 B. 

burgdorferi to Samento and Banderol extracts and to doxycycline (250 µg/ml) for 96 hours' 

treatment period using direct cell counting and darkfield morphological evaluation methods. (A) 

Samento extract; (B) Banderol extract; (C) Samento + Banderol extracts. As a negative control, 

0.25% ethanol was a used. *P- values >0.05 indicates statistical significance. 

 

In the first set of experiments, we tested the in vitro susceptibility of the spirochete and round-



body forms of the B. burgdorferi B31 strain to Samento and Banderol extracts for 96 hours, then 

direct cell counting and darkfield morphological evaluation methods were used to measure the 

effects of the antimicrobial agents. For both herbal extracts, the dilution of 1:400 most efficiently 

eliminated both the spirochetal and round-body forms (Figure 1A and 1B). However, when we 

used the combination of Samento and Banderol extracts, 1:300 dilution showed the most 

effectiveness, and this concentration was chosen for further study (Figure 1C). As a negative 

control, 0.25% ethanol treatment was also included in all experiments, because these herbal 

extracts contain ~25% ethanol to transport the nutrients into the cells and for stability. 

In these experiments, we also compared the effect of Samento and Banderol with doxycycline, 

the most common antibiotic treatment agent for Lyme disease treatment in a 96-hour treatment 

period. Our results showed that doxycycline (250 µg/ml) was very effective in eliminating the 

spirochetal form of B. burgdorferi, but it significantly increased the round-body forms. 

Comparing this doxycycline data with that of the herbal extracts, Banderol and the combination 

of Samento and Banderol (1:300) were more efficient in eliminating both the spirochetal and 

round-body forms of B. burgdorferi in vitro (Figures 1A–C).  

In the next set of experiments, we evaluated the effect of the different antimicrobial agents on 

biofilmlike colonies of B. burgdorferi. The cultures were treated as described above for 96 hours 

and stained with BacLight fluorescent viability stains, which can help visualize the effects of the 

antimicrobial agents on the bacterial cells (Figure 2). The green fluorescent stain (SYTO 9, with 

excitation/emission maxima of about 480/500 nm) colors healthy bacteria that have intact 

membranes, thus staining live cells; and the red dye (propidium iodide with excitation/emission 

maxima of about 490/635 nm) colors bacteria with damaged membranes, by displacing the green 

dye, thus staining dead cells. 

 

Figures 2: BacLight viability staining of B31 strain of B. burgdorferi after 96-hour treatment 



using SYTO 9 green-fluorescent nucleic acid stain (live cells) and propidium iodide, a red-

fluorescent nucleic acid stain (dead cells). (A) Control; (B) Samento (1:300 dilution); (C) 

Banderol (1:300 dilution); (D) Samento + Banderol (1:300 dilution); (E) Doxycycline (250 

µg/ml). All images are taken at 40× magnification.  




Figure 2A 

 

Figure 2B 



 

Figure 2C

 



Figure 2D

   


In the absence of antimicrobial agents, B. burgdorferi is forming biofilmlike colonies (Figure 

2A) with mainly live bacterial cells. In the presence of Samento extract (1:300), the colonies 

were significantly smaller and less organized (Figure 2B), but they did stain with green dye, 

indicating that live cells remained. In the presence of Banderol extracts, the size of colonies did 

not show any reduction; however, the cells inside the colonies are >90% dead. 

 

In the presence of both herbal extracts, no sign of any colony formation was observed in the 



cultures, but we found evidence of a few individual nonmotile but green spirochetes and round 

bodies. In the presence of doxycycline (250 µg/ml), the average colony size was increased and 

contained mainly live round-body forms.  

In this study, our working hypothesis was that for an efficient therapy, we have to find 

antimicrobial agents that can eliminate all the forms of B. burgdorferi. During the course of 

Borrelia infection, the bacteriumcan shift among the different forms, converting from the 

spirochete form to the others when presented with an unfavorable environment and reverting to 

the spirochete when the condition is again favorable for growth.

1-4

 To successfully eradicate B. 



burgdorferi, antimicrobial agents should eliminate all those forms, including the spirochetes, 

round bodies, and biofilmlike colonies.  

Here we have provided evidence that two natural antimicrobial agents (Samento and Banderol 

extracts) had significant effect on all three known forms of B. burgdorferi bacteria in vitro. We 

have also demonstrated that doxycycline, one of the primary antibiotics used in the clinic to treat 

Lyme disease, only had significant effect on the spirochetal form of B. burgdorferi.

5

 

 



 


Figure 2E 

 

 



Our later results might provide some explanation for why relapse is so common after 

discontinuing antibiotic therapy. For example, some of the recent reports on animal experiments 

demonstrated that although pharmaceutical antibiotics are effective in ameliorating disease, the 

infection may persist even after seemingly effective therapy, which suggested that Borrelia may 

remain viable even after antibiotic administration.

14-15


 If those pharmaceutical antibiotics only 

eliminate one form of this bacterium, the other forms could be the source of the persistent 

disease. 

 

The other very important fact needs to be considered for an effective treatment for Borrelia 



infection: this bacterium typically has a life span ranging from several weeks to six to eight 

months; therefore, it may take six to eight months for even one generation of Borrelia to become 

exposed to the antimicrobial for elimination.

16

 Since the herbal extracts like Samento are 



reported to be nontoxic, they can be safely taken daily for the long period of time necessary to 

thoroughly eradicate Borrelia from an infected body.

17

 

In summary, our study has provided in vitro research data on a novel treatment approach using 



herbal antimicrobial agents to efficiently eradicate B. burgdorferi, the Lyme disease bacterium.  

 

Corresponding Author 



Eva Sapi, PhD 

University of New Haven  

Department of Biology and Environmental Sciences  

300 Boston Post Road  

West Haven, Connecticut 06516  

esapi@newhaven.edu

 

 

Notes 



1.   Gruntar I, Malovrh T, Murgia R, Cinco M. Conversion of Borrelia garinii cystic forms to 

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2.   Brorson Ø, Brorson SH. In vitro conversion of Borrelia burgdorferi to cystic forms in spinal 

fluid, and transformation to mobile spirochetes by incubation in BSK-H medium. Infection. 

1998;26:44–50. 

3.   Miklossy J, Kasas S, Zurn AD, McCall S, Yu S, McGeer PL. Persisting atypical and cystic 

forms of Borrelia burgdorferi and local inflammation in Lyme neuroborreliosis. J 

Neuroinflammation. 2008;25:5–40. 

4.   Brorson Ø, Brorson SH, Scythes J, MacAllister J, Wier A, Margulis L. Destruction of 

spirochete Borrelia burgdorferi round-body propagules (RBs) by the antibiotic tigecycline. Proc 

Natl Acad Sci U S A. 2009 Nov;106(44):18656–18661. 

5.   Burrascano J. Advanced topics in Lyme disease. In: Managing Lyme Disease. 15th ed. 

2005:1–33. 

6.   Krause PJ, Foley DT, Burke GS, Christianson D, Closter L, Spielman A. Reinfection and 

relapse in early Lyme disease. Am Trop Med Hyg. 2006;75(6):1090–1094.  

7.   Klempner M, Linden MD, Hu T, J Evans J, et al. Two controlled trials of antibiotic 

treatment in patients with persistent symptoms and a history of Lyme disease. N Engl J Med. 

2001;345:85–92. 

8.   Horowitz R. Classical and integrative medical approaches in chronic Lyme disease: new 

paradigms in diagnosis & treatment. 8th Annual International Lyme and Associated Diseases 

Society (ILADS) Conference; 2007 October.  

9.   Matsuura T, Shimizu Y, Fujimoto H, et al. Minocycline-related lupus. 

Lancet.1992;340:1553. 

10. Ccahuana-Vasquez RA, Santos SS, Koga-Ito CY, Jorge AO. Antimicrobial activity of 

Uncaria tomentosa against oral human pathogens. Braz Oral Res. 2007 Jan-Mar;21(1):46–50. 

11. Valerio LG Jr, Gonzales GF. Toxicological aspects of the South American herbs cat's claw 

(Uncaria tomentosa) and Maca (Lepidium meyenii): a critical synopsis. Toxicol Rev. 

2005;24(1):11–35. 

12. Weniger B, Robledo S, Arango GJ, et al. Antiprotozoal activities of Colombian plants. J 

Ethnopharmacol. 2001 Dec;78(2–3):193–200. 

13. Baradaran-Dilmaghani R, Stanek G. In vitro susceptibility of thirty Borrelia strains from 

various sources against eight antimicrobial chemotherapeutics. Infection. 1996 Jan–

Feb;24(1):60–63. 

14. Bockenstedt LK, Mao J, Hodzic E, et al. Detection of attenuated, noninfectious spirochetes 

in Borrelia burgdorferi-infected mice after antibiotic treatment. J Infect Dis. 2002; 186:1430–

1437. 


15. Barthold SW, Hodzic E, Imai DM, Feng S, Yang X, Luft BJ. Ineffectiveness of tigecycline 

against persistent Borrelia burgdorferi. Antimicrob Agents Chemother. 2010 Feb;54(2):643–

651. 

16. Samuels DS and Radolf JD. Borrelia: Molecular Biology, Host Interaction and Pathogenesis. 



Caister Academic Press; 2010. 

17. Reinhard K-H. Uncaria tomentosa (Willd.) D.C.: Cat's claw, Una de Gato, or Saventaro. J 

Alt Comp Med. 1999;5:143–151. 

 

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