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
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