same researcher establishes that Samento and Banderol kill all forms of Lyme very effectively, including biofilm.

[Guest guest]

same researcher establishes that Samento and Banderol kill all forms of Lyme

very effectively, including biofilm.

                             Shan

In Vitro Effectiveness of Samento and Banderol Herbal Extracts on the Different

Morphological Forms of Borrelia Burgdorferi

http://www.townsendletter.com/July2010/sapi0710.html

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. 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– 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;

( Banderol extract; © Samento + Banderol extracts. As a negative control,

0.25% ethanol was a used. *P- values & gt;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; ( Samento (1:300 dilution); © 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 & gt;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@...

Notes

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