The Library

Role in Cancer Prevention and/or therapy:
The following article shows sanguinarine  as a tumor suppressive agent through inhibition of cell growth, cell cycle arrest (prevents cell reproduction), and programmed cell death in prostate cancer, a sequel to this same group’s studies showing similar effects on skin cancer cells:

 
Sanguinarine causes cell cycle blockade and apoptosis of human prostate carcinoma cells via modulation of cyclin kinase inhibitor-cyclin-cyclin-dependent kinase machinery.

Adhami VM, Aziz MH, Reagan-Shaw SR, Nihal M, Mukhtar H, Ahmad N.

Department of Dermatology, University of Wisconsin Medical Science Center, 1300 University Avenue, Madison, WI 53706, USA.

Prostate cancer is the second leading cause of cancer-related deaths in males in the United States. This warrants the development of novel mechanism-based strategies for the prevention and/or treatment of prostate cancer. Several studies have shown that plant-derived alkaloids possess remarkable anticancer effects. Sanguinarine, an alkaloid derived from the bloodroot plant Sanguinaria canadensis, has been shown to possess antimicrobial, anti-inflammatory, and antioxidant properties. Previously, we have shown that sanguinarine possesses strong antiproliferative and proapoptotic properties against human epidermoid carcinoma A431 cells and immortalized human HaCaT keratinocytes. Here, employing androgen-responsive human prostate carcinoma LNCaP cells and androgen-unresponsive human prostate carcinoma DU145 cells, we studied the antiproliferative properties of sanguinarine against prostate cancer. Sanguinarine (0.1-2 micromol/L) treatment of LNCaP and DU145 cells for 24 hours resulted in dose-dependent (1) inhibition of cell growth [as evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay], (2) arrest of cells in G0-G1 phase of the cell cycle (as assessed by DNA cell cycle analysis), and (3) induction of apoptosis (as evaluated by DNA ladder formation and flow cytometry). To define the mechanism of antiproliferative effects of sanguinarine against prostate cancer, we studied the effect of sanguinarine on critical molecular events known to regulate the cell cycle and the apoptotic machinery. Immunoblot analysis showed that sanguinarine treatment of both LNCaP and DU145 cells resulted in significant (1) induction of cyclin kinase inhibitors p21/WAF1 and p27/KIP1; (2) down-regulation of cyclin E, D1, and D2; and (3) down-regulation of cyclin-dependent kinase 2, 4, and 6. A highlight of this study was the fact that sanguinarine induced growth inhibitory and antiproliferative effects in human prostate carcinoma cells irrespective of their androgen status. To our knowledge, this is the first study showing the involvement of cyclin kinase inhibitor-cyclin-cyclin-dependent kinase machinery during cell cycle arrest and apoptosis of prostate cancer cells by sanguinarine. These results suggest that sanguinarine may be developed as an agent for the management of prostate cancer.

One of the other principal alkaloids of Sanguinaria is chelidonine. The following study demonstrates yet another example of cell cycle arrest by which Sanguinaria canadensis is fighting cancer:

The effects of chelidonine on tubulin polymerisation, cell cycle progression and selected signal transmission pathways.

Panzer A, Joubert AM, Bianchi PC, Hamel E, Seegers JC.

Department of Physiology, University of Pretoria, South Africa.

Chelidonine is a tertiary benzophenanthridine alkaloid known to cause mitotic arrest and to interact weakly with tubulin. Our interest in chelidonine began when we found it to be a major contaminant of Ukrain, which is a compound reported to be selectively toxic to malignant cells. The effects of chelidonine in two normal (monkey kidney and Hs27), two transformed (Vero and Graham 293) and two malignant (WHCO5 and HeLa) cell lines, were examined. Chelidonine proved to be a weak inhibitor of cell growth, but no evidence for selective cytotoxicity was found in this study. It was confirmed that chelidonine inhibits tubulin polymerisation (IC50 = 24 microM), explaining its ability to disrupt microtubular structure in cells. A G2/M arrest results, which is characterised by abnormal metaphase morphology, increased levels of cyclin B1 and enhanced cdc2 kinase activity. Exposure of all cell lines examined to chelidonine leads to activation of the stress-activated protein kinase/jun kinase pathway (SAPK/JNK).

Editor’s note: Chelidonine is a tertiary benzophenanthridine alkaloid known to cause mitotic arrest and to interact weakly with tubulin.  The above study shows a separate mechanism of cell cycle arrest, which is one of the mechanisms found to be useful in treating rapidly dividing cancer cells. 

The next article is focused upon the anti-tumor properties of another major constituent in Sanguinaria, again focusing on a critical enzyme inhibition inducing cell cycle arrest:

Chelerythrine is a potent and specific inhibitor of protein kinase C.

Herbert JM, Augereau JM, Gleye J, Maffrand JP.

Sanofi Recherche, Toulouse, France.

The benzophenanthridine alkaloid chelerythrine is a potent, selective antagonist of the Ca++/phospholopid-dependent protein kinase (Protein kinase C: PKC) from the rat brain. Half-maximal inhibition of the kinase occurs at 0.66 microM. Chelerythrine interacted with the catalytic domain of PKC, was a competitive inhibitor with respect to the phosphate acceptor (histone IIIS) (Ki = 0.7 microM) and a non-competitive inhibitor with respect to ATP. This effect was further evidenced by the fact that chelerythrine inhibited native PKC and its catalytic fragment identically and did not affect [3H]- phorbol 12,13 dibutyrate binding to PKC. Chelerythrine selectively inhibited PKC compared to tyrosine protein kinase, cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase. The potent antitumoral activity of celerythrine measured in vitro might be due at least in part to inhibition of PKC and thus suggests that PKC may be a model for rational design of antitumor drugs.

Apoptosis:
Here is the earlier study demonstrating apoptosis induction by sanguinarine on epidermoid cancer cells from the same group that demonstrated the cell cycle arrest of prostate cancer cells with sanguinarine :

 
Activation of prodeath Bcl-2 family proteins and mitochondrial apoptosis pathway by sanguinarine in immortalized human HaCaT keratinocytes.

Adhami VM, Aziz MH, Mukhtar H, Ahmad N.

Department of Dermatology, University of Wisconsin, Madison, Wisconsin 53706, USA.

Sanguinarine, derived from the root of Sanguinaria canadensis and other poppy fumaria species, possesses strong antimicrobial, anti-inflammatory, and antioxidant properties. We earlier showed that sanguinarine kills human epidermoid carcinoma A431 cells via an induction of apoptosis [N. Ahmad et al., Clin. Cancer Res., 6: 1524-1528, 2000]. In this study, using immortalized human keratinocytes (HaCaT cells), we provide information about mechanism of the antiproliferative effect of sanguinarine. Sanguinarine [0.1 (M-2 (M)] treatment to HaCaT cells was found to inhibit in a dose-dependent manner the cell proliferation and induce apoptosis, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ELISA, respectively. Sanguinarine treatment also resulted in a significant cleavage of poly(ADP-ribose) polymerase in HaCaT cells. Because mitochondrial pathway is critical for the regulation of apoptosis, we studied the involvement and regulation of mitochondrial events in sanguinarine-mediated apoptosis of HaCaT cells. As shown by the immunoblot analysis, our data clearly demonstrated that sanguinarine  treatment to HaCaT cells resulted in a dose-dependent (a) increase in the level of Bax with a concomitant decrease in Bcl-2 levels and (b) increase in Bax/Bcl-2 ratio. Sanguinarine also resulted in significant increases in the proapoptotic members of Bcl-2 family proteins, i.e., Bak and Bid. This was accompanied by increase in (a) protein expression of cytochrome c and apoptotic protease-activating factor-1 and (b) activity and protein expression of caspase-3, caspase-7, caspase-8, and caspase-9. Taken together, our data showed the involvement of mitochondrial pathway and Bcl-2 family proteins during sanguinarine-mediated apoptosis of immortalized keratinocytes. We suggest that sanguinarine could be developed as a drug for the management of hyperproliferative skin disorders, including skin cancer.
More on the anticancer mechanism of apoptosis induced by sanguinarine :

 
Differential antiproliferative and apoptotic response of sanguinarine for cancer cells versus normal cells.

Ahmad N, Gupta S, Husain MM, Heiskanen KM, Mukhtar H.

Department of Dermatology, Case Western Reserve University, Cleveland, Ohio 44106, USA.

Sanguinarine, derived from the root of Sanguinaria canadensis, has been shown to possess antimicrobial, anti-inflammatory, and antioxidant properties. Here we compared the antiproliferative and apoptotic potential of sanguinarine against human epidermoid carcinoma (A431) cells and normal human epidermal keratinocytes (NHEKs). Sanguinarine treatment was found to result in a dose-dependent decrease in the viability of A431 cells as well as NHEKs albeit at different levels because sanguinarine-mediated loss of viability occurred at lower doses and was much more pronounced in the A431 carcinoma cells than in the normal keratinocytes. DNA ladder assay demonstrated that compared to vehicle-treated control, sanguinarine treatment of A431 cells resulted in an induction of apoptosis at 1-, 2-, and 5-microM doses. Sanguinarine treatment did not result in the formation of a DNA ladder in NHEKs, even at the very high dose of 10 microM. The induction of apoptosis by sanguinarine was also evident by confocal microscopy after labeling the cells with annexin V. This method also identified necrotic cells, and sanguinarine treatment also resulted in the necrosis of A431 cells. The NHEKs showed exclusively necrotic staining at high doses (2 and 5 microM). We also explored the possibility of cell cycle perturbation by sanguinarine in A431 cells. The DNA cell cycle analysis revealed that sanguinarine treatment did not significantly affect the distribution of cells among the different phases of the cell cycle in A431 cells. We suggest that sanguinarine could be developed as an anticancer drug.
Further clarification of Apoptosis (programmed cell death) induction as a mechanism of fighting cancer with sanguinarine:

Role of Bcl-2 family proteins and caspase-3 in sanguinarine-induced bimodal cell death.

Weerasinghe P, Hallock S, Tang SC, Liepins A.

Faculty of Medicine, Memorial University of Newfoundland, Canada. priyaw@mit.edu

Sanguinarine, a benzophenanthridine alkaloid, has anticancer potential through induction of cell death. We previously demonstrated that sanguinarine treatment at a low level induced apoptosis or programmed cell death (PCD) in the Bcl-2 low-expressing K562 human erythroleukemia cells, and that a high level induced blister cell death (BCD); whereas Bcl-2 overexpressing, sanguinarine-treated JM1 pre-B lymphoblastic cells displayed neither apoptosis nor BCD morphologies. Here, we report that sanguinarine-treated K562 cells, when analyzed by western blot, showed significant increase in expression of the pro-apoptotic Bax protein in apoptosis, but not in BCD. cDNA expression array of PCD in K562 cells failed to reveal the presence of Bax at the gene transcript level, which suggests that this cell death process does not require de novo protein synthesis. Treated JM1 cells, on the other hand, showed an increase in the expression of Bcl-2 protein in both forms of cell death, but failed to show Bax expression. The role of other members of the Bcl-2 family remained negligible. Caspase-3 activation was observed in apoptosis of K562 cells but not in BCD or in sanguinarine-treated JM1 cells. These results suggest that sanguinarine in K562 cells induces apoptosis through increasing Bax and activating caspase-3, whereas sanguinarine-induced BCD involves neither. These results also suggest that in JM1 cells, Bcl-2 may play a role in susceptibility of cells to induction of apoptosis and BCD.

Editor’s Note: The following two articles explore further the inverse relationship between proteins expressed by Bax and Bcl-2 genes and their relationship to the ability to induce apoptosis. This relates to one mechanism by which sanguinarine works against cancer cells.  We include these articles because they explain why in certain experimental cell lines there is resistance to treatment with sanguinarine.  Most cancer cells in the body do not exhibit excessive Bcl-2 expression and therefore, do not confer resistance to sanguinarine induction of apoptosis and therefore confer resistance to its use in treatment.

Sanguinarine induces bimodal cell death in K562 but not in high Bcl-2-expressing JM1 cells.

Weerasinghe P, Hallock S, Tang SC, Liepins A.

Faculty of Medicine, Memorial University of Newfoundland, Canada. priyaw@mit.edu

Our previous studies with low Bcl-2-expressing K562 cells have shown that, when treated with the putative anti-cancer drug sanguinarine, concentrations of 1.5 microg/ml induced the morphology of apoptosis or programmed cell death (PCD), while concentrations of 12.5 microg/ml induced a morphology of blister formation or blister cell death (BCD). To elucidate the possible role of Bcl-2 in this dual cell death modality induced by sanguinarine, K562 and the high Bcl-2-expressing JM1 cells were treated with sanguinarine concentrations of 1.5 microg/ml and 12.5 microg/ml respectively, and multiple parameters of their effects were studied using light and electron microscopy, terminal deoxynucleotidyl transferase (TdT) end-labeling, 51Cr release, trypan blue exclusion, propidium iodide exclusion, and annexin-V binding. In general, we found that, while K562 cells underwent PCD and BCD when treated with sanguinarine, JM1 cells failed to undergo either PCD or BCD under the same experimental conditions. Thus, the over-expression of anti-apoptotic Bcl-2 may have prevented sanguinarine from inducing PCD and BCD in JM1 cells. These results indicate that the resistance of JM1 cells to the alkaloid sanguinarine may have been due to an anti-BCD role played by Bcl-2, in addition to its widely reported anti-apoptotic role.

 
Bax, Bcl-2, and NF-kappaB expression in sanguinarine induced bimodal cell death.

Weerasinghe P, Hallock S, Liepins A.

Faculty of Medicine, Memorial University of Newfoundland, 300 Prince Phillip Drive, St. John's, Newfoundland, A1B 3V6, Canada.

The apoptosis related proteins Bax, Bcl-2, and NF-kappaB were analyzed in sanguinarine induced apoptosis and blister cell death (BCD) of K562 erythroleukemia cells and in sanguinarine treated high Bcl-2 expressing JM1 pre-B lymphoblastic cells, utilizing immunofluorescence-flow cytometry. Sanguinarine induced apoptosis of K562 cells was found to have increased Bax expression and decreased NF-kappaB, whereas BCD showed a decrease in Bax expression and an increase in NF-kappaB. In contrast, high Bcl-2 expressing JM1 cells, when exposed to the same concentrations (and duration) of sanguinarine that induced PCD and BCD in K562 cells, failed to show the respective morphologies while showing a concomitant increase in Bcl-2. Results from studies with K562 cells suggest that Bax is pro-apoptotic and also that NF-kappaB activation may be associated with BCD. Results from studies with JM1 cells suggest that Bcl-2 is anti-apoptotic and anti-BCD. Results from JM1 cells strengthen the assumption in the literature of the central role Bcl-2 plays in chemoresistance by assuming an anti-PCD role. These results also suggest that, in JM1 cells, Bcl-2 may further complicate chemoresistance by being anti-BCD in nature, in addition to its anti-PCD role. Copyright 2001 Academic Press.
The following article amplifies the role of chelerythrine inhibiting protein kinase C inducing apoptosis in fighting cancer using a type of leukemic cell line upon which to demonstrate:
.

Induction of apoptotic DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C.

Jarvis WD, Turner AJ, Povirk LF, Traylor RS, Grant S.

Department of Medicine, Medical College of Virginia, Richmond 23298-0230.

The present studies were undertaken to characterize further the potential role of protein kinase C (PKC) in the regulation of apoptosis in HL-60 promyelocytic leukemia cells. The capacity of acute exposure to specific and nonspecific pharmacological inhibitors of PKC to promote apoptotic DNA fragmentation was examined both quantitatively and qualitatively and correlated with effects on cellular differentiation and proliferation. Incubation of HL-60 cells for 6 h with chelerythrine and calphostin C (highly specific inhibitors that act at the regulatory domain) or H7 and gossypol (nonspecific inhibitors that act at the PKC catalytic domain) produced concentration-dependent increases in DNA fragmentation. Induction of DNA fragmentation by chelerythrine, calphostin C, and gossypol was biphasic, resulting in a sharp decline in effect at concentrations above 5 microM, 0.1 microM, and 100 microM, respectively, whereas maximal and more stable effects were observed in response to H7 (100 microM). A 6-h exposure to staurosporine, a nonspecific but potent PKC inhibitor, failed to induce DNA fragmentation at concentrations generally used to achieve maximal inhibition of enzyme activity (e.g., 50 nM) but promoted fragmentation at considerably higher concentrations (e.g., > or = 200 nM). In contrast, 6-h exposures to the nonspecific protein kinase inhibitor hypericin (0.1 to 100 microM) or to the nonspecific inhibitor of protein kinase A, HA1004 (50 microM), were without effect on DNA fragmentation. DNA obtained from cells exposed to chelerythrine (5 microM), calphostin C (100 nM), H7 (50 microM), gossypol (50 microM), and staurosporine (200 nM)--but not hypericin (25 microM)--exhibited clear evidence of internucleosomal DNA cleavage on agarose gel electrophoresis; moreover, these cells exhibited the classical morphological features of apoptosis (cell shrinkage, nuclear condensation, and the formation of apoptotic bodies). All of the PKC inhibitors that induced apoptosis, and one of the inhibitors that did not (hypericin), substantially inhibited HL-60 cell clonogenicity at the concentrations evaluated. None of the agents tested induced cellular maturation as assessed by nonspecific esterase and nitro-blue tetrazolium positivity. DNA fragments obtained from cells exposed to specific and nonspecific PKC inhibitors possessed predominantly 5'-phosphate termini, consistent with the action of a Ca(2+)-/Mg(2+)-dependent endonuclease. Finally, Northern blot analysis revealed that exposure to calphostin C at a concentration that induced apoptosis (100 nM) failed to alter expression of bcl-2, an oncogene known to block apoptosis in both lymphoid and myeloid leukemia cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Berberine is another major constituent in Bloodroot.  The following article describes its probable mechanism of fighting cancer is also induction of apoptosis by inducing DNA topoisomerase poisoning:

In vitro cytotoxicity of berberine against HeLa and L1210 cancer cell lines.

Kettmann V, Kosfalova D, Jantova S, Cernakova M, Drimal J.

Department of Pharmaceutical Analysis and Nuclear Pharmacy, Faculty of Pharmacy, Comenius University, Bratislava, Slovakia. kettmann@fpharm.uniba.sk

Previous studies on anti-cancer activity of protoberberine alkaloids against a variety of cancer cell lines were extended to human uterus HeLa and murine leukemia L1210 cell lines. Cytotoxicity was measured using in vitro techniques and cell morphology changes were examined by light microscopy in both cytostatic and cytocidal concentration ranges. The IC50 was found to be less than 4 microg/ml, a limit put forward by NCI for classification of the compound as a potential anti-cancer drug. The microscopy examination indicated that at cytocidal concentrations the HeLa and L120 cells died apoptotically. The comparative analysis revealed that berberine belongs to the camptothecin family of drugs characterized by the ability to induce DNA topoisomerase poisoning and hence apoptotic cell death. Although the cytotoxic potency of berberine was found to be several orders of magnitude lower compared to camptothecin, its significance may increase in future in view of the lack of unwanted side effects characteristic for camptothecin compounds currently in clinical use for treatment of cancer.
And further as to the induction of apoptosis of cancer cells, the mechanism of ellipticine’s mode of action is explored further below:

 
Induction of endoplasmic reticulum stress by ellipticine plant alkaloids.

Hagg M, Berndtsson M, Mandic A, Zhou R, Shoshan MC, Linder S.

Department of Oncology and Pathology, Cancer Center Karolinska, Karolinska Institute and Hospital, Stockholm, Sweden.

Anticancer drugs often show complex mechanisms of action, including effects on multiple cellular targets. Detailed understanding of these intricate effects is important for the understanding of cytotoxicity. In this study, we examined apoptosis induction by ellipticines, a class of cytotoxic plant alkaloids known to inhibit topoisomerase II. The potent ellipticine derivative 6-propanamine ellipticine (6-PA-ELL) induced rapid apoptosis in MDA-MB-231 breast cancer cells, preceded by a conformational change in Bak and cytochrome c release. Experiments using knock-out mouse embryo fibroblasts established that Bak was of particular importance for cytotoxicity. 6-PA-ELL increased the expression of the endoplasmic reticulum chaperones GRP78/BiP and GRP94, suggesting induction of endoplasmic reticulum stress. Induction of GRP78 expression was dependent on the endoplasmic reticulum stress response element (ERSE) of the GRP78 promoter. Examination of different ellipticine derivatives revealed a correlation between pro-apoptotic activity and the ability to induce GRP78 expression. Furthermore, 6-PA-ELL was found to induce splicing of the mRNA encoding the XBP1 transcription factor, characteristic of endoplasmic reticulum stress, and to induce activation of the endoplasmic reticulum-specific caspase-12 in mouse colon cancer cells. We finally demonstrate that 6-PA-ELL induces apoptotic signaling also in enucleated cells, consistent with the existence of a cytoplasmic target for this compound. Our data suggest that induction of endoplasmic reticulum stress may contribute to the cytotoxicity of ellipticines.

Editor’s note: We include a paper studying a substance called NF Kappa B, a nuclear transcription factor, whose activation results in inflammation, viral replication, and tissue growth modulation. A transcription factor means that it activates certain genes to transcribe their information to make messenger RNA specific for certain destructive proteins responsible for initiating inflammation, viral replication and/or cancer.  Sanguinarine inhibits NF-kappaB activation and reveals in so doing a possible mechanism for cancer prevention/treatment, fighting inflammation and inhibiting viral replication in viral infections.

 
Sanguinarine (pseudochelerythrine) is a potent inhibitor of NF-kappaB activation, IkappaBalpha phosphorylation, and degradation.

Chaturvedi MM, Kumar A, Darnay BG, Chainy GB, Agarwal S, Aggarwal BB.

Cytokine Research Section, Department of Molecular Oncology, the University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA. aggarwal@utmdacc.mda.uth.tmc.edu

The nuclear factor NF-kappaB is a pleiotropic transcription factor whose activation results in inflammation, viral replication, and growth modulation. Due to its role in pathogenesis, NF-kappaB is considered a key target for drug development. In the present report we show that sanguinarine (a benzophenanthridine alkaloid), a known anti-inflammatory agent, is a potent inhibitor of NF-kappaB activation. Treatment of human myeloid ML-1a cells with tumor necrosis factor rapidly activated NF-kappaB, this activation was completely suppressed by sanguinarine in a dose- and time-dependent manner. Sanguinarine did not inhibit the binding of NF-kappaB protein to the DNA but rather inhibited the pathway leading to NF-kappaB activation. The reversal of inhibitory effects of sanguinarine by reducing agents suggests a critical sulfhydryl group is involved in NF-kappaB activation. Sanguinarine blocked the tumor necrosis factor-induced phosphorylation and degradation of IkappaBalpha, an inhibitory subunit of NF-kappaB, and inhibited translocation of p65 subunit to the nucleus. As sanguinarine also inhibited NF-kappaB activation induced by interleukin-1, phorbol ester, and okadaic acid but not that activated by hydrogen peroxide or ceramide, the pathway leading to NF-kappaB activation is likely different for different inducers. Overall, our results demonstrate that sanguinarine is a potent suppressor of NF-kappaB activation and it acts at a step prior to IkappaBalpha phosphorylation.

In Russia Anticancer properties of sanguinarine have been studied in elegant fashion.  Sanguinarine and ellipticine are very similar alkaloids as already discussed. In the first article below 4 different effects inside the cell give the compounds their anticancer attributes; however, we emphasize the process of DNA intercalation involved in the 4 effects noted in this abstract:

[Sanguinarine and ellipticine cytotoxic alkaloids isolated from well-known antitumor plants. Intracellular targets of their action]

[Article in Russian]

Faddeeva MD, Beliaeva TN.

Common molecular and cellular targets for alkaloids sanguinarine and ellipticine, isolated from well-known antitumor plants (as well as from their various natural and synthetic derivatives), have been studied and described. Sanguinarine and ellipticine are characterized by significant biological activities including a high antitumor potential. Among the important targets of their action the following are to be noted. 1. DNA and other double helical polynucleotides. Due to the ability of DNA-intercalation sanguinarine, ellipticine and some of their derivatives can modify the double helical structures and topological forms of polynucleotides. The results of these modifications in intercalative complexes manifest themselves in the inhibition of numerous enzymatic reactions, dependent on the structures and topological forms of DNA and other polynucleotides.  2. ATP synthesis in mitochondria. Most of DNA-intercalators, including sanguinarine and ellipticine, belong to a group of penetrating (hydrophobic) cations, which are accumulated near the external side of inner mitochondrial membranes during the membrane energization. They neutralize negative charges, arising just as the inner mitochondrial membranes become energized. By this neutralization of membrane charges the ATP synthesis in inhibited and the oxidative phosphorylation renders to be uncoupled. All studied DNA-intercalators under certain conditions uncouple the mitochondrial oxidative phosphorylation. Apparent correlation between the agents' ability for DNA-intercalation and for mitochondrial ATP synthesis inhibition seems to be determined by the importance for both types of reactions of molecule hydrophobicity and positive charges. 3. Cholinesterase systems. Sanguinarine, ellipticine and some of their derivatives, like other DNA-intercalators studied, inhibit also the enzymatic activities of cholinesterase systems due to hydrophobicity and positive charges of their molecules. 4. Sanguinarine (and chelerythrine), are also capable of inhibiting the biological activity of SH-dependent enzymes and proteins. Due to the reactivity of iminium groups in sanguinarine and chelerythrine molecules with nucleophilic reagents, e.g. thiol groups of enzymes and other proteins, the activities of SH-enzymes and proteins are inhibited. In particular, sanguinarine and chelerythrine inhibit enzymatic activity of some SH-dependent ATPases, including membrane-bound cation-transport ATPases. The earlier accumulated experience of the application in medicine of plant saps and extracts containing these alkaloids, and of the treatment of many diseases (including benign and malignant tumors) by isolated alkaloids may be explained, to a certain extent, by the inhibition of activities of the above mentioned cellular targets. The selective toxicity of these alkaloids for the number of transformed cells can be explained in the same manner.
Ellipticine is amplified as a DNA intercalating anti-cancer agent below, again referenced here as a major constituent in Sanguinaria canadensis:

Extending nature's leads: the anticancer agent ellipticine.

Garbett NC, Graves DE.

Department of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

The natural plant product ellipticine was isolated in 1959 from the Australian evergreen tree of the Apocynaceae family. This compound was found to be an extremely promising anticancer drug. The planar polycyclic structure was found to interact with DNA through intercalation, exhibiting a high DNA binding affinity (10(6) M (-1)). The presence of protonatable ring nitrogens distinguished ellipticine from other simple intercalators. Both monocationic and uncharged species were found to be present under physiological conditions. The positive charge stabilized the binding of ellipticine to nucleic acids, while the more lipophilic uncharged compound was shown to readily penetrate membrane barriers. The structural nature of these compounds offers a plausible basis for the implication of multiple modes of action, including DNA binding, interactions with membrane barriers, oxidative bioactivation and modification of enzyme function; most notably that of topoisomerase II and telomerase. Pharmacologically, a number of toxic side effects have been shown to be problematic, but the amenability of ellipticine towards systematic structural modification has permitted the extensive application of rational drug design. A number of successful ellipticine analogs have been designed and synthesized with improved toxicities and anticancer activities. More recently the synthetic focus has broadened to include the design of hybrid compounds, as well as drug delivery conjugates. Considerable research efforts have been directed towards gaining a greater understanding of the mechanism of action of these drugs that will aid further in the optimization of drug design.
Even though sanguinarine is not mentioned in the study below, the similarities between sanguinarine and ellipticine support the same mechanism of intercalation efficacy as illustrated in the following:

Interrelationship between affinity for DNA, cytotoxicity and induction of DNA-breaks in cultured L1210 cells for two series of tricyclic intercalators. Simplified analogues of ellipticine derivatives.

Pierson V, Pierre A, de Cointet P, Nguyen CH, Bisagni E, Gros P.

Sanofi Recherche, Toulouse, France.

The interrelationship between affinity for DNA, cytotoxicity and induction of single-strand DNA breaks in cultured L1210 cells was studied for 21 compounds belonging to two series of tricyclic intercalators: 1-amino-substituted 4-methyl-5H-pyrido[4,3-b]indoles (gamma CARB) and 1-amino-substituted 4-methyl-5H-pyrido[3',4':4,5]pyrrolo[2,3-c]pyridines (PPP), which are simplified analogues of ellipticine derivatives obtained by deletion of one cycle. Adriamycin, m-AMSA (4'-(9-acridinylamino) methanesulfon-m-anisidide), PZE (10-[diethylaminopropyl amino]-6-methyl-5H-pyrido[3',4':4,5]-pyrrolo[2,3-g] isoquinoline and RTE [( 1-(3-diethylaminopropylamino)-9-methoxy ellipticine, bimaleate) are used as reference compounds. The intercalation of these compounds into DNA was strongly suggested by three experimental observations: (i) the competitive inhibition of ethidium bromide intercalation, (ii) bathochromic and hypochromic effects on absorption spectra induced by DNA, and (iii) drug-induced increase of the DNA length, measured by viscosimetry. PPP derivatives are generally less cytotoxic and induce DNA breaks less efficiently than the gamma CARB ones, both in terms of maximum breakage frequencies and required drug concentrations. The most active compounds induced SSB in the DNA of L1210 cells, in a bell-shaped manner: the SSB frequency increased, rose to a maximum and then decreased as the drug concentrations increased. The maximum SSB frequencies induced by the most active compounds are of the same order as those of reference compounds Adriamycin and PZE. The structurally important requirements are essentially the same for both DNA breakage activity and cytotoxicity: (i) a N-CH3 in the 5-position, (ii) a CH3 in the 4-position, (iii) a hydroxy in the 8-position and (iv) the presence of an (aminoalkyl)amino side chain with preferentially a 3 carbon unit. There is no direct relationship between DNA affinity in vitro and induction of DNA breaks in cells, although a relatively high affinity seemed to be a necessary condition, since the most active compounds have the highest affinities and compounds having a very low affinity are totally inactive. The close correlation between cytotoxicity and extent of induction of DNA breaks suggests that these breaks may be in fact the lethal lesions responsible for cell death and thereby for the antitumor properties of these tricyclic intercalators.
The fact that both alkaloids, ellipticine and sanguinarine  are present in Sanguinaria canadensis, suggests the efficacy of Sanguinaria vs. breast cancer:

Ellipticine derivatives with an affinity to the estrogen receptor, an approach to develop intercalating drugs with a specific effect on the hormone-dependent breast cancer.

Delbarre A, Oberlin R, Roques BP, Borgna JL, Rochefort H, Le Pecq JB, Jacquemin-Sablon A.

In order to obtain breast tumor directed agents, we have prepared mixed compounds using estradiol or (E)-clomiphene as specific vectors of the breast tissue and a DNA intercalator from the ellipticine series as the cytotoxic agent. Among the newly synthesized ellipticine derivatives, only the 2-[3-aza-5-(3,17 beta-dihydroxy-1,3,5-estratrien-17 alpha-yl)-4-oxopentamethylene]ellipticinium bromide shows the desired properties, DNA intercalation and affinity for estrogen receptor. Competition experiments with estradiol on the hormone-dependent human MCF-7 breast cancer cell line demonstrate that a transport by the estrogen receptor system is not involved in the antitumor activity of derivative 24.
And this article as well:

Multimodal action of antitumor agents on DNA: the ellipticine series.

Auclair C.

Laboratoire de Biochimie-Enzymologie, INSERM U140, CNRS LA 147, Institut Gustave Roussy, Villejuif, France.

Most cytotoxic anticancer agents interact directly or indirectly with nuclear DNA, the ultimate target for this class of compounds. For a given type of drug both direct and indirect action at the DNA level usually causes various types of interference or damage. This multimodal mechanism of action is well illustrated by antitumor drugs in the ellipticine series which may bind to DNA through intercalation, may undergo covalent binding, may generate oxidizing species, and may interfere with the catalytic activity of topoisomerase II. The antitumor activity of these compounds may, therefore, result from alternative cytotoxic events. The present review summarizes information obtained with ellipticine compounds on the relation between the nature of the drugs' action on DNA and their cytotoxic and/or antitumor activity. The occurrence of topoisomerase-mediated DNA cleavage appears to be responsible for antitumor activity. The capability of the drugs to interfere with the action of topoisomerase II requires the presence of an oxidizable phenolic group on their structure. This feature (or a related one) is shared by all antitumor drugs acting on this enzyme.

This article on ellipticine specifically relates to the mechanism of anti-tumor efficacy based on intercalation and adducts formation, which we have discussed previously:

 
The anticancer drug ellipticine forms covalent DNA adducts, mediated by human cytochromes P450, through metabolism to 13-hydroxyellipticine and ellipticine N2-oxide.

Stiborova M, Sejbal J, Borek-Dohalska L, Aimova D, Poljakova J, Forsterova K, Rupertova M, Wiesner J, Hudecek J, Wiessler M, Frei E.

Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic. stiborov@natur.cuni.cz

Ellipticine is an antineoplastic agent, the mode of action of which is considered to be based on DNA intercalation and inhibition of topoisomerase II. We found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts. We now identified the ellipticine metabolites formed by human CYPs and elucidated the metabolites responsible for DNA binding. The 7-hydroxyellipticine, 9-hydroxyellipticine, 12-hydroxyellipticine, 13-hydroxyellipticine, and ellipticine N(2)-oxide are generated by hepatic microsomes from eight human donors. The role of specific CYPs in the oxidation of ellipticine and the role of the ellipticine metabolites in the formation of DNA adducts were investigated by correlating the levels of metabolites formed in each microsomal sample with CYP activities and with the levels of the ellipticine-derived deoxyguanosine adducts in DNA. On the basis of this analysis, formation of 9-hydroxyellipticine and 7-hydroxyellipticine was attributable to CYP1A1/2, whereas production of 13-hydroxyellipticine and ellipticine N(2)-oxide, the metabolites responsible for formation of two major DNA adducts, was attributable to CYP3A4. Using recombinant human enzymes, oxidation of ellipticine to 9-hydroxyellipticine and 7-hydroxyellipticine by CYP1A1/2 and to 13-hydroxyellipticine and N(2)-oxide by CYP3A4 was corroborated. Homologue modeling and docking of ellipticine to the CYP3A4 active center was used to explain the predominance of ellipticine oxidation by CYP3A4 to 13-hydroxyellipticine and N(2)-oxide.
More on DNA-intercalating anticancer mechanism of action:

[The toxicity of sanguinarine compared to a number of other DNA-tropic compounds for ethidium bromide-sensitive and -resistant transformed murine fibroblasts in culture]

[Article in Russian]

Beliaeva TN, Faddeeva MD, Sal'nikov KV, Ignatova TN.

A natural DNA-intercalator plant benzo-c-phenanthridine alkaloid sanguinarine is more toxic for mouse transformed fibroblast L-cells in culture than synthetic DNA-intercalator ethidium bromide (EtB) and alkaloid berberine. Dimidium bromide is also an inhibitor of the L-cell growth. In assay conditions, growth of L-cells is stopped by 1.5 x 10(-5) M of sanguinarine. Lebr-625 cells, resistant to 25 micrograms/ml of EtB, have sanguinarine sensitivity close to that of L-cells, but Lebr-625 cells are resistant to dimidium bromide. Sanguinarine is more toxic for L-cells in culture than the anticancer drug cis-PtNH3)2Cl2. Trans-Pt(NH3)2Cl2 is less toxic for these cells. The strong toxicity of sanguinarine for L- and Lebr-625 cells in culture, as compared to other DNA-complexing drugs, seems to be associated with the wide range of potential cell targets for sanguinarine influence. Besides the inhibition of nucleic acid metabolism reactions, characteristic of DNA-intercalators, and disruption the mitochondrial ATP synthesis, also characteristic of organic heterocyclic cationic molecules of DNA-intercalators, sanguinarine can modify the thiol groups of enzymes including SH-sensitive membrane-bound Na+, K(+)-ATPase of cerebral cortex and Ca2(+)-ATPase of skeletal muscle sarcoplasmic reticulum fragments.

Evidence for why DNA intercalation is important pharmacologically and why sanguinarine works on fighting cancer proliferation:

[DNA intercalators: their interaction with DNA and other cell components and their use in biological research]

[Article in Russian]

Faddeeva MD, Beliaeva TN.

DNA intercalators include aromatic heterocyclic compounds of various chemical classes with profound biological activities. The flat molecules of these ligands intercalate between base pairs of DNA right-handed helix, lengthening and unwinding this structure at the intercalation sites. Lerman first postulated the intercalation model for complexes of native DNA with acridine derivatives. The structures of intercalative complexes were further confirmed by the X-ray diffraction method. Besides, other physico-chemical criteria of DNA intercalation are as following: the increase in the contour length of duplex DNA; unwinding of supercoils from natural supercoiled covalently closed duplex DNA; the increase in Tm of DNA in the complexes with ligands. The changes of spectral properties of bounded ligands are also observed for DNA-intercalating agents. Various experimental methods are based on changes in the properties of nucleic acid structures and ligands due to DNA intercalation, including the fluorescent determination of nucleic acid structures and quantities; fluorescent assays of activities of various enzymes involved in nucleic acid metabolism; chromosome identification according to their fluorescent banding patterns; separation of nucleic acid topological forms, and many other methods. The inhibition of reactions of DNA replication, transcription, topoisomerization and of enzymatic degradation by DNA intercalators represents an important consequence of DNA structure modification due to intercalation. Besides, as hydrophobic cations DNA intercalators uncouple the oxidative phosphorylation in mammalian cell mitochondria. There are some other protein and phospholipid targets for DNA-intercalators in vivo. The intracellular distribution of these agents appear to be a very complicated selective process. These data point to the importance of application of DNA intercalators in pharmacology.
Vascular endothelial growth factor (VEGF) inhibitor:

Editor’s note: New blood vessel formation is an important part of inflammation as well as cancer growth.  Suppression of angiogenesis, therefore, is a very important attribute of sanguinarine:

Suppression of angiogenesis by the plant alkaloid, sanguinarine.

Eun JP, Koh GY.

Department of Neurosurgery, Chonbuk National University Hospital, Jeonju 560-180, Republic of Korea.

Sanguinarine is a benzophenanthridine alkaloid derived from the root of Sanguinaria canadensis. Its principal pharmacologic use is in dental products where it has antibacterial, antifungal, and anti-inflammatory activities that reduce gingival inflammation and supragingival plaque formation. Angiogenesis is indispensable for inflammation, and most angiogenesis is dependent on vascular endothelial growth factor (VEGF). However, the effect of sanguinarine on angiogenesis is not known. In the present study, we examined the effect of sanguinarine on VEGF-induced angiogenesis in vitro and in vivo. Interestingly, sanguinarine markedly suppressed VEGF-induced endothelial cell migration, sprouting, and survival in vitro in a dose-dependent manner at nanomolar concentrations. Furthermore, sanguinarine potently suppressed blood vessel formation in vivo in mouse Matrigel plugs and the chorioallantoic membrane of chick embryos. Our biochemical assays indicated that sanguinarine strongly suppressed basal and VEGF-induced Akt phosphorylation, while it did not produce any changes in VEGF-induced activation of ERK1/2 and PLCgamma1. Therefore, we conclude that sanguinarine is a potent antiangiogenic natural product, and its mode of action could involve the blocking of VEGF-induced Akt activation. Thus, in addition to antibacterial, antifungal, and anti-inflammatory activities, sanguinarine has a novel antiangiogenic role.
Sanguinarine proved effective against multidrug resistant human cervical cancer cells by inducing apoptosis at low doses and blister cell death from higher concentrations, suggesting further exploration of this in resistant tumors to current chemotherapeutics

 
The alkaloid sanguinarine is effective against multidrug resistance in human cervical cells via bimodal cell death.

Ding Z, Tang SC, Weerasinghe P, Yang X, Pater A, Liepins A.

Division of Basic Sciences, Faculty of Medicine, Memorial University of Newfoundland, 300 Prince Philip Drive, St. John's, NF, Canada A1B 3V6.

Sanguinarine, a benzophenanthrine alkaloid, is potentially antineoplastic through induction of cell death pathways. The development of multidrug resistance (MDR) is a major obstacle to the success of chemotherapeutic agents. The aim of this study was to investigate whether sanguinarine is effective against uterine cervical MDR and, if so, by which mechanism. The effects of treatment with sanguinarine on human papillomavirus (HPV) type 16-immortalized endocervical cells and their MDR counterpart cells were compared. Trypan blue exclusion assays and clonogenic survival assays demonstrated that MDR human cervical cells are as sensitive as their drug-sensitive parental cells to death induced by sanguinarine. Upon treatment of both types of cells with sanguinarine, two distinct concentration-dependent modes of cell death were observed. Treatment with 2.12 or 4.24 microM sanguinarine induced death in most cells that was characterized as apoptosis using the criteria of cell surface blebbing, as determined by light and scanning electron microscopy, and proteolytic activation of caspase-3 and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP), as detected by Western blot analysis. However, 8.48 and 16.96 microM sanguinarine caused a second mode of cell death, oncosis, distinguished by cell surface blistering, and neither caspase-3 activation nor PARP cleavage. This study provides the first evidence that sanguinarine is effective against MDR in cervical cells via bimodal cell death, which displays alternative mechanisms involving different morphologies and caspase-3 activation status.

Contrasting opinion: More on anti-cancer, especially as topical preparation combined with zinc, a contrasting opinion:

 
Consequences of using escharotic agents as primary treatment for nonmelanoma skin cancer.

McDaniel S, Goldman GD.

Division of Dermatology, University of Vermont College of Medicine, Fletcher-Allen Health Care, Burlington, VT 05401, USA.

BACKGROUND: The use of escharotic or caustic pastes to treat skin cancer is based on the centuries-old observation that selected minerals and plant extracts may be used to destroy certain skin lesions. Zinc chloride and Sanguinaria canadensis (bloodroot) are 2 agents that are used as part of the Mohs chemosurgery fixed-tissue technique. The use of escharotics without surgery has been discredited by allopathic medicine but persists and is promoted among alternative practitioners. Patients may now purchase "herbal supplements" for the primary self-treatment of skin cancer, and physicians will see patients who elect this therapy for their skin cancers. OBSERVATIONS: We reviewed the history of escharotic use for skin disease and performed an Internet search for the availability and current use of escharotics. Our search located numerous agents for purchase via the Internet that are advertised as highly successful treatments for skin cancer. We report 4 cases from our practice in which escharotic agents were used by patients to treat basal cell carcinomas in lieu of the recommended conventional treatment. One patient had a complete clinical response, but had a residual tumor on follow-up biopsy. A second patient successfully eradicated all tumors, but severe scarring ensued. A third patient disagreed with us regarding his care and was lost to follow-up. One patient presented with a nasal basal cell carcinoma that "healed" for several years following treatment elsewhere with an escharotic agent but recurred deeply and required an extensive resection. The lesion has since metastasized. CONCLUSIONS: Escharotic agents are available as herbal supplements and are being used by patients for the treatment of skin cancer. The efficacy of these agents is unproven and their content is unregulated. Serious consequences may result from their use. Conventional medicine has an excellent track record in treating skin cancer. Physicians should recommend against the use of escharotic agents for skin cancer, and the Food and Drug Administration should be given the authority to regulate their production and distribution.
Another contrasting opinion to sanguinarine as a general anticancer drug

 
Sanguinarine-induced apoptosis is associated with an early and severe cellular glutathione depletion.

Debiton E, Madelmont JC, Legault J, Barthomeuf C.

UMR-INSERM U-484, Rue Montalembert, BP 184, 63005, Clermont-Ferrand, France.

PURPOSE: The quaternary benzophenanthridine alkaloid sanguinarine exhibits a broad range of activity, including cytotoxicity against various human tumour and normal cell lines. Here, we examined its potency as an anticancer drug. METHODS: The differential cytotoxicity against cancer versus normal cells was assessed in vitro by two fluorimetric assays (RRT and Hoechst 33342 dye DNA assays, respectively) in a panel of human solid cancer cell lines and a human fibroblast primary culture. The ability to induce apoptosis was demonstrated in PC3 human prostatic adenocarcinoma cells by analysis of morphological changes, internucleosomal DNA fragmentation, cellular poly(ADP-ribose) polymerase cleavage and caspase 3/7 activation. Production of reactive oxygen species was evaluated by the 2',7'-dichlorofluorescin diacetate assay. Depletion of cellular glutathione content was assessed with the monochlorobimane assay. RESULTS: Sanguinarine markedly inhibited the growth of all tested cells (IC(50) 0.9-3.3 microM) without differential cytotoxicity against normal versus cancer cells. In PC3 cells, continuous treatment with 5 microM sanguinarine induced an early (within 10 min) cellular reduced glutathione depletion insensitive to dithiothreitol or N-acetylcysteine treatment, followed by a caspase 3/7-dependent apoptotic response within 2 h. Complementary assays suggested that the glutathione depletion was initiated by direct reactivity of sanguinarine with reduced glutathione. CONCLUSIONS: Taken together, these results show that (1) sanguinarine exhibits no specificity for cancer cells, and (2) its strong cytotoxicity is probably due to a rapid apoptotic response induced by an early and severe glutathione-depleting effect. They also suggest that the clinical usefulness of this alkaloid as an anticancer drug is limited.
Immune system modulation: This next article exemplifies a mechanism by which Sanguinaria canadensis is an immune system modulator by its impact on PhosphoLipase D (PLD) activation, which plays a myriad of different roles in immune system activation:

Chelerythrine and other benzophenanthridine alkaloids block the human P2X7 receptor.

Shemon AN, Sluyter R, Conigrave AD, Wiley JS.

Department of Medicine, The University of Sydney at Nepean Hospital, Penrith, NSW, 2751, Australia.

Extracellular ATP can activate a cation-selective channel/pore on human B-lymphocytes, known as the P2X7 receptor. Activation of this receptor is linked to PLD stimulation. We have used ATP-induced 86Rb+ (K+) efflux to examine the effect of benzophenanthridine alkaloids on P2X7 channel/pore function in human B-lymphocytes. 2 Both ATP and the nucleotide analogue 2'-3'-O-(4-benzoylbenzoyl)-ATP (BzATP) induced an 86Rb+ efflux, which was completely inhibited by the isoquinoline derivative 1-(N,O-bis[5-isoquinolinesulphonyl]-N-methyl-l-tyrosyl)-4-phenylpiperazine (KN-62), a potent P2X7 receptor antagonist. 3 The benzophenanthridine alkaloid chelerythrine, a potent PKC inhibitor, inhibited the ATP-induced 86Rb+ efflux by 73.4+/-3.5% and with an IC50 of 5.6+/-2.3 microm. Similarly, other members of this family of compounds, sanguinarine and berberine, blocked the ATP-induced 86Rb+ efflux by 58.8+/-4.8 and 61.1+/-8.0%, respectively. 4 Concentration-effect curves to ATP estimated an EC50 value of 78 microm and in the presence of 5 and 10 microm chelerythrine this increased slightly to 110 and 150 microm, respectively, which fits a noncompetitive inhibitor profile for chelerythrine. 5 Chelerythrine at 10 microm was effective at inhibiting the ATP-induced PLD stimulation in B-lymphocytes by 94.2+/-21.9% and the phorbol 12-myristate 13-acetate-induced PLD stimulation by 68.2+/-7.4%. 6 This study demonstrates that chelerythrine in addition to PKC inhibition has a noncompetitive inhibitory action on the P2X7 receptor itself.
Editor’s note: The above article is included as evidence in depth and in general as to why Sanguinaria is an immune modulator.  The emphasis is on the myriad of immune and endocrine functions mediated by activation of P2X7 Receptor and PLD stimulation.
Immune modulation: Even though antibiotic potency is well demonstrated, there appears to be a dose dependent impact on the phagocytic neutrophil behavior (white blood cells that are a part of the kill mechanism of host defense against bacteria by the immune system), the consequences of which leave open questions.  However, this next article does point out how there is immune modulation by Sanguinaria.

Effects of sanguinarium, chlorhexidine and tetracycline on neutrophil viability and functions in vitro.

Agarwal S, Piesco NP, Peterson DE, Charon J, Suzuki JB, Godowski KC, Southard GL.

Division of Oral Biology and Dental Surgical Sciences, University of Pittsburgh School of Dental Medicine, PA 15261, USA.

The effectiveness of an ideal antimicrobial agent depends on its ability to kill microbes with minimal toxicity to host cells. Depending on the treatment regimen, antimicrobial agents come into contact with host cells for various intervals of time. Sanguinarium (SANG), chlorhexidine (CHX) and tetracycline (TET) are 3 antimicrobial agents frequently used in the management of periodontal infections. However, their effects on host immune cells during different treatment regimens are not known. Due to their ability to serve as the first line of host defense against microbial infections, we have compared the effects of these antimicrobial agents on human neutrophil functions and viability. The results show that SANG is not lytic to neutrophils from peripheral blood or crevicular fluid, at all concentrations tested. However, exposures of neutrophils to very low concentrations of SANG (0.001%) inhibits neutrophil chemotaxis, oxidative metabolism and degranulation within 5 min. Increasing the exposure time results in a similar inhibition of neutrophil functions, albeit at 50-100 fold lower concentrations of SANG. CHX rapidly disrupts the cell membrane of both crevicular and peripheral blood neutrophils at concentrations above 0.005% within 5 min, and inhibition of all neutrophil functions is due to its lytic properties. While TET is least toxic to neutrophils, a dose dependent inhibition of neutrophil functions is dependent on the calcium concentrations of the cellular environment, and is observed only above 0.04% or higher concentrations in the absence of calcium. The data suggest that a critical cumulative concentration of these drugs is essential for their toxicity and inhibition of neutrophil functions. Therefore, both the length of exposure and the dose of the drug both are critical while considering the effectiveness of SANG, CHX or TET in the treatment of infections. Furthermore, due to differences in their mechanisms of action, the consequences of their effects on neutrophils may have significant bearing on tissue pathology as well as on their therapeutic efficacy.
Editor’s note: Although the above data and interpretations are reasonable, with Sanguinaria at least, we have additional evidence that the oxidative burst response of neutrophils that is responsible for killing bacteria is unaffected, and that the reduction in chemotaxis and some of the other behaviors may modulate an anti-inflammatory effect to reduce the often over reaction induced by pro-inflammatory cytokines present in bacterial infection.  This article above does not take that into account.
The following article on cyclosporin A, an anti-inflammatory used in advanced rheumatoid arthritis does the same thing to neutrophils as sanguinarine , and therefore, highlights one reason why sanguinarine  may be proving a useful anti-inflammatory immune modulator:

 
Inhibition of neutrophil responses by cyclosporin A. An insight into molecular mechanisms.

Spisani S, Fabbri E, Muccinelli M, Cariani A, Barbin L, Trotta F, Dovigo L.

Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy.

OBJECTIVE: Cyclosporin A (CsA) is an effective agent in rheumatoid arthritis (RA), slowing joint damage progression. Its therapeutic effect on T lymphocytes has been studied extensively, but there is little information available about neutrophils, the cells responsible for a substantial proportion of inflammation. A study was performed to investigate the in vitro effects of CsA on neutrophil functions triggered by several agonists and determine whether the drug could counteract the binding of formyl-methionyl-leucyl-phenylalanine (fMLP) to its receptor and/or modulate changes in the intracellular Ca(2+) concentration ([Ca(2+)]i). METHODS: CsA was added to neutrophils 5-50 min before the incubation steps for neutrophil function assays (chemotaxis, superoxide anion production, lysozyme release), calcium measurements and receptor binding experiments. RESULTS: CsA appeared to be particularly effective in lowering chemotaxis, superoxide anion production and lysozyme release induced by different agonists. However, it did not significantly affect either basal or agonist-stimulated neutrophil [Ca(2+)]i and the interaction between fMLP and its receptor. CONCLUSIONS: Because of its in vitro inhibition of neutrophil functions, CsA appears to have considerable potential as an anti-inflammatory drug. Moreover, as it is also a potent immunosuppressive agent, it may reduce the progression of joint damage in RA. More work remains to be done to clarify the molecular mechanism of CsA action on neutrophils.

Editor’s note: Some of the effects of cyclosporin A have been documented in some of the articles in this library to modulate the response of neutrophils to inflammatory stimulation.  The same results have been attributed to cyclosporin A as for sanguinarine.  Therefore it is reasonable to consider this type of immunomodulation as useful to use in auto-immune inflammatory conditions like Rheumatoid arthritis.

Anti-inflammatory: The following is a study that shows why sanguinarine, as an immune modulator of neutrophil behavior, may have anti-inflammatory implications because of its impact on neutrophil behavior:

The effect of sanguinarine on human peripheral blood neutrophil viability and functions.

Agarwal S, Reynolds MA, Pou S, Peterson DE, Charon JA, Suzuki JB.

Department of Periodontics, University of Maryland Dental School, Baltimore.

Human polymorphonuclear cell (PMN) viability, morphology, adherence, chemotaxis, oxidative metabolism, degranulation and phagocytosis were evaluated following treatment with sanguinarine (SANG). SANG was noncytotoxic to PMNs at all concentrations tested (0.31-200 microM). SANG entered the PMNs rapidly without altering the membrane fluidity and localized in the nuclear matrix. SANG (1.56-6.21 microM) inhibited chemotaxis, chemokinesis and adhesion in a dose-dependent manner, with a complete inhibition at 6.2 microM concentration. Concentrations of SANG up to 1.56 microM did not affect PMN oxidative burst; however, higher concentrations were found to inhibit basal as well as PMA-induced superoxide anion generation. The effect of SANG was time- and dose-dependent, and could be reversed if the PMNs were exposed to 12.5 microM or lower concentrations of SANG for less than 5 min. Autologous serum increased the tolerance of PMNs to SANG. Exogenous Ca2+ or Mg2+ did not alter the SANG-mediated inhibition of PMN functions. Treatment of PMNs with 3.12 microM or higher concentrations of SANG also resulted in inhibition of PMN degranulation and phagocytosis. The results suggest that SANG-mediated inhibition of PMN functions, without cytolysis or resultant release of inflammatory mediators, may have clinical implications.
Additional insight into anti-inflammatory mechanism of Sanguinaria canadensis is contained in the following article, in which Sanguinaria extract among other rich alkaloid containing plant tinctures were used and is shown to inhibit the 5-lipoxigenase/COX -2 enzyme system, which generates the pro-inflammatory mediators responsible for inflammation.

 
Dual inhibition of 5-lipoxygenase/cyclooxygenase by a reconstituted homeopathic remedy; possible explanation for clinical efficacy and favourable gastrointestinal tolerability.

Jaggi R, Wurgler U, Grandjean F, Weiser M.

VitaPlant AG, Benkenstrasse 254, 4108 Witterswil, Switzerland. rjaeggi@vitaplant.ch

OBJECTIVE: In order to elucidate potential anti-inflammatory activities of Zeel comp. N and its constituents, the inhibition of the synthesis of Leukotriene B4(LTB4) and Prostaglandin (PGE2) by 5-lipoxygenase (5-LOX) and cyclo-oxygenase 1 and 2 (COX 1 and 2) respectively were examined in vitro. MATERIALS: Human HL-60 cells, differentiated for 6-8 days with DMSO (1.2% v/v) were used for the 5-LOX assay. The COX activity assays were carried out with purified enzymes, COX 1 (ram seminal vesicles), COX 2 (sheep placenta)

and with human THP-1 cells, differentiated for 24 h with PMA (50 nM). METHODS: LTB4 and PGE2 production in the 5-LOX and COX assays respectively were determined by enzyme linked immunoassays. RESULTS: A reconstituted Zeel comp. N combination as well as its constituent mother tinctures of Arnica montana, Sanguinaria canadensis and Rhus toxicodendron (Toxicodendron quercifolium) showed distinct inhibitory effects on the production of LTB4 by 5-LOX (IC50 values of 10, 20, 2 and 5 microg/ml respectively) and on the synthesis of PGE2 by COX 1 (IC50 values of 50, 80, 40 and 20 microg/ml respectively) and COX 2 enzymes (IC50 values of 60, 110, 50 and 20 microg/ml respectively). The mother tincture of Solanum dulcamara inhibited the production of PGE2 by COX 1 (IC50 40 microg/ml) and COX 2 (IC50 150 microg/ml) but not production of leukotriene LTB4 by 5-LOX. CONCLUSIONS: The observed dual inhibition of both LOX- and COX-metabolic pathways may offer an explanation for the reported clinical efficacy and the favorable gastrointestinal tolerability of the original remedy Zeel comp. N.
More on the anti-inflammatory mechanisms applicable to Sanguinaria alkaloids:

Benzophenanthridine alkaloids of Chelidonium majus; I. Inhibition of 5- and 12-lipoxygenase by a non-redox mechanism.

Vavreckova C, Gawlik I, Muller K.

Institute of Medical Chemistry, Palacky University, Czech Republic.

The benzophenanthridine alkaloids sanguinarine and chelerythrine of Chelidonium majus, L. (Papaveraceae), are potent inhibitors of 5-lipoxygenase in polymorphonuclear leukocytes and 12-lipoxygenase in mouse epidermis, while the activity of soybean lipoxygenase is not influenced. The extract of the herb of Ch.majus also inhibits the 5-LO enzyme. Chelidonine, which cannot form pseudobases, is inactive against LO enzymes. Pro- and antioxidant actions of benzophenanthridine alkaloids can be excluded from the lack of deoxyribose degradation, reactivity against free radicals and inhibition of lipid peroxidation, suggesting that the inhibitory effects against LO enzymes appear to be due to specific enzyme interaction rather than a nonspecific redox mechanism.

More on the selectivity of the alkaloids in Sanguinaria canadensis to suppress unregulated proliferation of skin cells called keratinocytes makes it a candidate for anti-tumor therapeutics of skin cancer and for hyperproliferative skin disorders like eczema and psoriasis the following paper studies a plant related to Sanguinaria canadensis, but focuses on the two most active alkaloids in Sanguinaria:

Benzophenanthridine alkaloids of Chelidonium majus; II. Potent inhibitory action against the growth of human keratinocytes.

Vavreckova C, Gawlik I, Muller K.

Institute of Medical Chemistry, Palacky University, Olomouc, Czech Republic.

The benzophenanthridine alkaloids sanguinarine and chelerythrine of Chelidonium majus, L. (Papaveraceae), were tested for their action against the growth of human keratinocytes. Cell proliferation was independently determined by directly counting the cells and in the colorimetric MTT assay. Sanguinarine potently inhibited cell growth with an IC50 of 0.2 microM. Chelidonine, the main alkaloid of Ch. majus, was much less efficient. The extract of the herb of Ch. majus was also effective with an IC50 of 1.9 microM, calculated on the basis of its alkaloid content in terms of chelidonine. Keratinocytes were further tested for their susceptibility for the action of sanguinarine and chelerythrine on plasma membrane integrity, which resulted in a twofold increase in lactate dehydrogenase (LDH) activity as compared to controls. On the other hand, the activity of the extract of the herb of Ch. majus was due to cytostatic rather than cytotoxic effects, as LDH release was unchanged as compared to controls.
This abstract as well as the one above highlights the potential of sanguinarine’s usefulness in treating eczema, psoriasis and skin cancers.

 
Activation of prodeath Bcl-2 family proteins and mitochondrial apoptosis pathway by sanguinarine in immortalized human HaCaT keratinocytes.

Adhami VM, Aziz MH, Mukhtar H, Ahmad N.

Department of Dermatology, University of Wisconsin, Madison, Wisconsin 53706, USA.

Sanguinarine, derived from the root of Sanguinaria canadensis and other poppy fumaria species, possesses strong antimicrobial, anti-inflammatory, and antioxidant properties. We earlier showed that sanguinarine kills human epidermoid carcinoma A431 cells via an induction of apoptosis [N. Ahmad et al., Clin. Cancer Res., 6: 1524-1528, 2000]. In this study, using immortalized human keratinocytes (HaCaT cells), we provide information about mechanism of the antiproliferative effect of sanguinarine. Sanguinarine [0.1 (M-2 (M)] treatment to HaCaT cells was found to inhibit in a dose-dependent manner the cell proliferation and induce apoptosis, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ELISA, respectively. Sanguinarine treatment also resulted in a significant cleavage of poly (ADP-ribose) polymerase in HaCaT cells. Because mitochondrial pathway is critical for the regulation of apoptosis, we studied the involvement and regulation of mitochondrial events in sanguinarine-mediated apoptosis of HaCaT cells. As shown by the immunoblot analysis, our data clearly demonstrated that sanguinarine treatment to HaCaT cells resulted in a dose-dependent (a) increase in the level of Bax with a concomitant decrease in Bcl-2 levels and (b) increase in Bax/Bcl-2 ratio. Sanguinarine also resulted in significant increases in the proapoptotic members of Bcl-2 family proteins, i.e., Bak and Bid. This was accompanied by increase in (a) protein expression of cytochrome c and apoptotic protease-activating factor-1 and (b) activity and protein expression of caspase-3, caspase-7, caspase-8, and caspase-9. Taken together, our data showed the involvement of mitochondrial pathway and Bcl-2 family proteins during sanguinarine-mediated apoptosis of immortalized keratinocytes. We suggest that sanguinarine could be developed as a drug for the management of hyperproliferative skin disorders, including skin cancer.
One of the first uses for sanguinarine derived from Bloodroot was in Viodent toothpaste.  At the end of the library the reader is directed to the FDA document on the safety and efficacy of the compound.  Here, however, a contrasting view regarding safety of sanguinarine in a dentifrice:

Sanguinaria-related leukoplakia: epidemiologic and clinicopathologic features of a recently described entity.

Allen CL, Loudon J, Mascarenhas AK.

College of Dentistry, Ohio State University, Columbus, Ohio, USA.

An association between the use of Viadent toothpaste and/or mouthwash and the development of leukoplakia oral mucosal lesions has been described recently. Discontinuing the Viadent products may result in resolution of the leukoplakia, although frequently this is not the case. In order to corroborate the earlier study and to provide further insight regarding the clinical features of this process, a case-control study was conducted. A significant association was seen between the use of Viadent products and the development of oral leukoplakia. Furthermore, leukoplakias affecting sites other than the buccal vestibule also were associated with the use of these products.
More on leukoplakia and Sanguinaria:

 
Sanguinaria-associated oral leukoplakia: comparison with other benign and dysplastic leukoplakic lesions.

Eversole LR, Eversole GM, Kopcik J.

University of the Pacific and Pathology Consultants of New Mexico, Department of Pathology and Medicine, University of the Pacific School of Dentistry, San Francisco, CA 94115, USA.

OBJECTIVE: This study was undertaken to compare and contrast biomarkers and ploidy data from maxillary gingiva leukoplakias associated with dentifrices and mouthrinses containing the herbal compound Sanguinaria with other forms of oral benign and premalignant mucosal keratosis. STUDY DESIGN: Representative archived specimens of benign keratosis, sanguinaria-associated keratosis, and keratosis with dysplasia were used for computerized image analysis and biomarker immunohistochemical assays to assess ploidy, DNA content, and p53 and proliferating cell nuclear antigen immunoreactivity of nuclei. RESULTS: DNA content was significantly higher and higher numbers of cell populations with hyperploid nuclei were encountered in the dysplastic group than in the other two groups (P <.001). Sanguinaria-associated keratosis did not harbor significant numbers of p53-expressing nuclei, yet it showed a significant elevation in proliferating cell nuclear antigen-labeled nuclei in total, in the basal layer, and in the spinous layer in comparison with benign keratoses (P <.001). In addition, 1.5% of the Sanguinaria-associated leukoplakia epithelial cell population was characterized by nuclei with a greater than 4-fold increase in DNA content. CONCLUSIONS: Sanguinaria-associated keratoses show some marker and image analysis profiles similar to those of non-Sanguinaria dysplastic lesions of the lip and mucosa. Preparations containing Sanguinaria should be avoided until the risk for malignant transformation is determined.
Opposition to contrasting opinion on leukoplakia and sanguinarine:

 
Viadent usage and oral leukoplakia: a spurious association.

Munro IC, Delzell ES, Nestmann ER, Lynch BS.

CanTox Health Sciences International, Inc., Mississauga, Ontario, L5N 2X7, Canada.

Oral rinse and toothpaste products (Viadent) containing Sanguinaria extract have been shown through extensive clinical trials to be effective against plaque build-up and gingivitis. To establish safety, a comprehensive research program was conducted, including a series of clinical studies and a number of animal studies to evaluate acute, subchronic, and chronic toxicity, and the potential for irritation of mucosal tissues. In 1990 and 1993, an Expert Panel reported on reviews of these data and concluded that Viadent products are safe for their intended use. Despite the large database of information to support the safety of Viadent products, Damm et al. (1999) recently raised the possibility that their usage may be causally associated with development of oral leukoplakia. However, a critique of this recent report shows that it does not fulfill criteria for establishing causation. In particular, the study does not show that exposure to Viadent preceded the onset of leukoplakia, it does not demonstrate dose-response or biological plausibility, and it suffers from selection and information bias and from potential confounding. Furthermore, upon critical evaluation, the Damm et al. (1999) report on a case-series is inconsistent with the weight of available clinical evidence showing that Sanguinaria extract-containing oral health care products cause no cytotoxic or significant irritant effects in the oral mucosa in human studies of up to 6 months duration. The animal data similarly do not support a causal association between Viadent usage and oral leukoplakia in humans. These data demonstrate that Sanguinaria extract and whole Viadent formulations are without significant irritation potential and have no effects on the oral mucosa, even in studies with life-long dietary exposure to Sanguinaria extract. The mutagenicity and genotoxicity data do not indicate that Sanguinaria extract or its components are genotoxic in vivo. The results of 2 GLP-compliant rat oncogenicity studies provide no evidence of any carcinogenic effect of Sanguinaria extract. In conclusion, the available clinical and animal data provide no support for and in fact argue strongly against the hypothesis that the use of Viadent toothpaste and/or oral rinse products may be causally associated with the development of leukoplakia in humans. Copyright 1999 Academic Press.

Natural antibiotic properties:

Antimicrobial action of sanguinarine.

Godowski KC.

Sanguinarine is a benzophenanthridine alkaloid derived from rhizomes of Sanguinaria canadensis L. (bloodroot). It is a cationic molecule which converts from an iminium ion form at pH less than 6 to an alkanolamine form at pH greater than 7. Sanguinaria extract is composed of sanguinarine and five other closely related alkaloids. The safety profile of both sanguinarine and Sanguinaria extract provide a broad margin for their safe use in oral health products. Sanguinarine has broad antimicrobial activity as well as antiinflammatory properties. In vitro studies indicate that the anti-plaque action of Sanguinaria is due to its ability to inhibit bacterial adherence to newly formed pellicle, its retention in plaque being 10-100 times its saliva concentration, and due to its antimicrobic properties. The MIC of sanguinarine ranges from 1 to 32 micrograms/mL for most species of plaque bacteria. Long term use of Sanguinaria-containing toothpaste and oral rinse products does not predispose users to detrimental shifts in oral flora. Electron microscopic studies of bacteria exposed to sanguinarine demonstrate that bacteria aggregate and become morphologically irregular. Sanguinarine-containing slow release polymer systems are currently being developed for use in periodontitis treatment applications.
More on antibiotic properties:

The evaluation of forty-three plant species for in vitro antimycobacterial activities; isolation of active constituents from Psoralea corylifolia and Sanguinaria canadensis.

Newton SM, Lau C, Gurcha SS, Besra GS, Wright CW.

The School of Pharmacy, University of Bradford, BD7 1DP, West Yorkshire, UK.

Extracts from forty-three plant species were selected on account of reported traditional uses for the treatment of TB and/or leprosy. These were assayed for antimycobacterial activities. A simple in vitro screening assay was employed using two model species of mycobacteria, M. aurum and M. smegmatis. Crude methanolic extracts from three of the plants, C. mukul, P. corylifolia and S. canadensis, were found to have significant antimycobacterial activity against M. aurum only (MIC=62.5 microg/ml). Bioassay guided fractionation led to the isolation of two known benzophenanthridine alkaloids, sanguinarine (1) and chelerythrine (2), from the roots S. canadensis and the known phenolic meroterpene, bakuchiol (3) from the seeds of P. corylifolia. The fractionation of the resin of C. mukul lead to a decrease in antimycobacterial activity and hence further work was not pursued. Compound (2) was the most active against M. aurum and M. smegmatis (IC(50)=7.30 microg/ml [19.02 microM] and 29.0 microg/ml [75.56 microM], respectively). M. aurum was the most susceptible organism to all three compounds. No significant difference in antimycobacterial activity was observed when the two alkaloids were tested for activity in media of differing pH values. The activities of the pure compounds against M. aurum were comparable with those against M. bovis BCG with compound (2) being the most active (M. bovis BCG, IC(50)=14.3 microg/ml [37.3 microM]). These results support the use of these plants in traditional medicine.
Note:    Mycobacteria are the genus of tuberculosis bacteria

What is good for gums, gingivitis caused by bacteria suggests same mechanisms may be useful in other applications of bloodroot derivatives:

Antimicrobial action of sanguinarine.

Godowski KC.

Sanguinarine is a benzophenanthridine alkaloid derived from rhizomes of Sanguinaria canadensis L. (bloodroot). It is a cationic molecule which converts from an iminium ion form at pH less than 6 to an alkanolamine form at pH greater than 7. Sanguinaria extract is composed of sanguinarine and five other closely related alkaloids. The safety profile of both sanguinarine and Sanguinaria extract provide a broad margin for their safe use in oral health products. Sanguinarine has broad antimicrobial activity as well as antiinflammatory properties. In vitro studies indicate that the anti-plaque action of Sanguinaria is due to its ability to inhibit bacterial adherence to newly formed pellicle, its retention in plaque being 10-100 times its saliva concentration, and due to its antimicrobic properties. The MIC of sanguinarine ranges from 1 to 32 micrograms/mL for most species of plaque bacteria. Long term use of Sanguinaria-containing toothpaste and oral rinse products does not predispose users to detrimental shifts in oral flora. Electron microscopic studies of bacteria exposed to sanguinarine demonstrate that bacteria aggregate and become morphologically irregular. Sanguinarine-containing slow release polymer systems are currently being developed for use in periodontitis treatment applications.

By virtue of its effects on the sodium/potassium pump of cell wall/membrane active transport in yeast, some yeast organisms will be killed by sanguinarine:

 
Sanguinarine induces K+ outflow from yeast cells expressing mammalian sodium pumps.

Scheiner-Bobis G.

Institut fur Biochemie und Endokrinologie, Fachbereich Veterinarmedizin, Justus-Liebig-Universitat Giessen, Germany. Georgios.Scheiner-Bobis@vetmed.uni-giessen.de

Sanguinarine, an alkaloid from Sanguinaria canadensis, has no effect on the yeast Saccharomyces cerevisiae at concentrations of up to 225 microM. Yeast cells become sensitive to sanguinarine and lose cytosolic K+ in a time- and concentration-dependent manner when they express the mammalian Na+,K+-ATPase (sodium pump). Dose-response studies show that sanguinarine induces K+ outflow from cells expressing wild-type sodium pumps with an EC50 of 29.3+/-1.2 microM. A similar effect with a comparable EC50) of 26.8+/-1.3 microM is obtained with cells expressing an Asp369Ala mutant of the sodium pump alpha1 subunit. Since this sodium pump mutant does not hydrolyze ATP, it can be excluded that the observed sanguinarine-induced outflow of K+ is an active ion transport process. Ouabain inhibits the sanguinarine effect at concentrations higher than 1 mM. In contrast, proscillaridin A inhibits the sanguinarine-induced K+ outflow from cells expressing the wild-type sodium pump with an IC50 of 48.9+/-1.3 microM. A similar IC50 of 52.2+/-3.0 microM is obtained with cells expressing the Asp369Ala mutant. These data, together with the fact that sanguinarine inhibits the binding of [3H]ouabain to microsomes prepared from yeast cells expressing the sodium pump with an IC50 of 94.5+/-4.3 microM, all indicate that sanguinarine specifically targets the sodium pump, and that the observed K+ outflow is tightly associated with the presence of the enzyme.
The application of sanguinarine and its antibiotic and anti-inflammatory properties to oral health is highlighted in this next article:

Antimicrobial action of sanguinarine.

Godowski KC.

Sanguinarine is a benzophenanthridine alkaloid derived from rhizomes of Sanguinaria canadensis L. (bloodroot). It is a cationic molecule which converts from an iminium ion form at pH less than 6 to an alkanolamine form at pH greater than 7. Sanguinaria extract is composed of sanguinarine and five other closely related alkaloids. The safety profile of both sanguinarine and Sanguinaria extract provide a broad margin for their safe use in oral health products. Sanguinarine has broad antimicrobial activity as well as antiinflammatory properties. In vitro studies indicate that the anti-plaque action of Sanguinaria is due to its ability to inhibit bacterial adherence to newly formed pellicle, its retention in plaque being 10-100 times its saliva concentration, and due to its antimicrobic properties. The MIC of sanguinarine ranges from 1 to 32 micrograms/mL for most species of plaque bacteria. Long term use of Sanguinaria-containing toothpaste and oral rinse products does not predispose users to detrimental shifts in oral flora. Electron microscopic studies of bacteria exposed to sanguinarine demonstrate that bacteria aggregate and become morphologically irregular. Sanguinarine-containing slow release polymer systems are currently being developed for use in periodontitis treatment applications.

Zinc enhances the antibiotic potency of sanguinarine in oral mouthwash suggesting enhancement in other applications desirous of enhancing antibiotic potency:

Interactions of sanguinarine and zinc on oral streptococci and Actinomyces species.

Eisenberg AD, Young DA, Fan-Hsu J, Spitz LM.

Department of Oral Biology, Eastman Dental Center Rochester, N.Y.

Sanguinaria extract, which contains benzophenanthridine alkaloids, has been used as a folk medicine for many years. Minimum inhibitory and minimum bactericidal concentrations (MIC and MBC values) for sanguinarine were determined for common and etiologically important plaque bacteria. Because the efficacy of sanguinarine is believed to be enhanced by zinc, isobolograms were assessed to determine their mode(s) of interaction. Hydrogen ion concentration influenced the inhibitory activity of both sanguinarine and zinc. For sanguinarine, at the optimum pH (6.5), MIC values were 4 or 8 micrograms/ml for Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguis, Actinomyces viscosus and Actinomyces naeslundii. MIC values were 0.125-0.50 mmol Zn/ml. MBC values ranged from 1 to 8 mmol Zn/ml at pH 5.5. Isobologram data revealed that sanguinarine and zinc interacted synergistically. Viadent oral rinse, which contained 300 micrograms Sanguinaria extract/ml and 0.2% zinc chloride (14.9 mmol Zn/l), was inhibitory to all strains tested. MIC values were 1 or 2% (ml Viadent oral rinse/100 ml aqueous solution) for all strains except A. viscosus for which the MIC value was 12% (vol/vol).
An aspect of contrasting results as pertains to sanguinarine not doing well against yeast organisms:

In vitro antifungal properties of mouthrinses containing antimicrobial agents.

Giuliana G, Pizzo G, Milici ME, Musotto GC, Giangreco R.

Department of Periodontology, University of Palermo, School of Dentistry, Italy.

The purpose of this study was to investigate the in vitro antifungal properties of seven commercial mouthrinses containing antimicrobial agents. These included cetylpyridinium chloride (CPC), chlorhexidine digluconate (CHX), hexetidine (HEX), sanguinarine (SNG), and triclosan (TRN). The minimum fungicidal concentration (MFC) against six species of yeasts was determined by a broth macrodilution method. The kill-time of mouthrinses at half the concentration of the commercial formulations was also determined. MFCs were achieved with each mouthrinse, except the SNG-containing mouthrinse, against all the organisms being tested. However, the CPC-containing mouthrinse appeared more active than the other products (P < 0.001). There were no significant differences in MFC values among CHX mouthrinse products, once adjusted for initial concentration differences (P = 0.1). Kill-times of mouthrinses containing either CHX or CPC were less than or equal to 180 seconds with all the species of yeasts, and no significant differences were found among these products (P = 0.18). On the other hand, mouthrinses containing either TRN or HEX did not show a lethal effect on Candida albicans, Candida parapsilosis, or Candida guilliermondii. No kill-times were achieved with the SNG-containing mouthrinse. These results suggest that mouthrinses containing antimicrobial agents might represent an appropriate alternative to conventional antifungal drugs in the management of oral candidiasis. However, the effectiveness of antimicrobial mouthrinses as antifungal agents needs to be evaluated in further clinical trials.

Although the two alkaloids mentioned in this next study came from another plant, they are principal alkaloids of Sanguinaria canadensis.  This study shows them to be effective against Candida albicans, a yeast.

 
Two antimicrobial alkaloids from Bocconia arborea.

Navarro V, Delgado G.

Laboratorio de Microbiologia, Centro de Investigacion Biomedica del Sur, Instituto Mexicano del Seguro Social, Mor., Mexico.

Two alkaloid constituents of Bocconia arborea showed considerable antimicrobial activity against gram-positive and gram-negative bacteria and Candida albicans. Bioactivity-guided fractionation with thin-layer bioautography led to the isolation of the two known benzophenanthridine alkaloids, dihydrochelerythrine and dihydrosanguinarine. (Editor’s note: These two are in Sanguinaria canadensis) The minimal inhibitory concentration was determined for each compound using a twofold serial dilution assay. The structures of these compounds were determined by 1H and 13C NMR analyses.
Antiviral Properties:
Antiviral properties of Sanguinaria may be partially explained by findings in this next article:

HIV-1 and HIV-2 reverse transcriptases: a comparative study of sensitivity to inhibition by selected natural products.

Tan GT, Miller JF, Kinghorn AD, Hughes SH, Pezzuto JM.

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois, Chicago.

One hundred and fifty six pure natural products, which had previously been tested against HIV-1 reverse transcriptase, were evaluated for HIV-2 reverse transcriptase inhibitory activity. Compounds that lacked effect in the HIV-1 reverse transcriptase system were found also to be inactive against HIV-2 reverse transcriptase. However, compounds belonging to the benzophenanthridine and protoberberine classes of alkaloids, certain flavonoids, the iridoid, fulvoplumierin, and the ansamycin antibiotic, daunomycin, exhibited similar potencies in both enzyme systems. In contrast, HIV-2 reverse transcriptase was observed to be four-fold more sensitive toward the inhibitory effects of the ipecac alkaloids, O-methylpsychotrine sulfate heptahydrate and psychotrine dihydrogen oxalate. Such differences in susceptibilities to inhibitors may indicate subtle dissimilarities in enzyme structure and function.

The susceptibility of H. pylori to Sanguinaria suggests it is useful as an agent to prevent and/or treat chronic gastritis and chronic or recurrent ulcer disease caused by this organism:

 
In vitro susceptibility of Helicobacter pylori to isoquinoline alkaloids from Sanguinaria canadensis and Hydrastis canadensis.

Mahady GB, Pendland SL, Stoia A, Chadwick LR.

Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, USA. mahady@uic.edu

Methanol extracts of the rhizomes of Sanguinaria canadensis, and the roots and rhizomes of Hydrastis canadensis, two plants used traditionally for the treatment of gastrointestinal ailments, were screened for in vitro antibacterial activity against 15 strains of Helicobacter pylori. The rhizome extracts, as well as a methanol extract of S. canadensis suspension-cell cultures inhibited the growth of H. pylori in vitro, with a MIC50 range of 12.5-50.0 microg/ml. Three isoquinoline alkaloids were identified in the active fraction. Sanguinarine and chelerythrine, two benzophenanthridine alkaloids, inhibited the growth of the bacterium, with an MIC50 of 50.0 and 100.0 microg/ml, respectively. Protopine, a protopine alkaloid, also inhibited the growth of the bacterium, with a MIC50 of 100 microg/ml. The crude methanol extract of H. canadensis rhizomes was very active, with an MIC50 of 12.5 microg/ml. Two isoquinoline alkaloids, berberine and beta-hydrastine, were identified as the active constituents, and having an MIC50 of 12.5 and 100.0 microg/ml, respectively. Copyright 2003 John Wiley & Sons, Ltd.
Possible Cardiac benefits in heart failure resembling digitalis like effect of increasing the muscular force of contraction:

Sanguinarine: a positive inotropic alkaloid which inhibits cardiac Na+,K+-ATPase.

Seifen E, Adams RJ, Riemer RK.

In isolated, isometrically contracting left guinea pig atria, sanguinarine, a benzophenanthridine alkaloid from the papaveracea Sanguinaria canadensis, produced a concentration-dependent positive inotropic effect. Between 2.3 x 10(-6) M and 6.5 x 10(-5) M, sanguinarine increased contractility by 108% which was comparable to the maximal inotropic effect of ouabain. Within the same concentration range, sanguinarine caused inhibition of Na+, K+-ATPase isolated from guinea pig myocardium. 100% inhibition of Na+,K+,ATPase activity occurred at 1 x 10(-4) M sanguinarine. The I50 for enzyme inhibition and the ED50 for the inotropic action of sanguinarine were the same (6-6.5 x 10(-6) M) indicating that both effects may be causally related.

Sanguinarine may also be anti-arrhythmic and therefore beneficial to cardiac function:

Effect of sanguinarine on ventricular refractoriness.

Whittle JA, Bissett JK, Straub KD, Doherty JE, McConnell JR.

The purpose of this study was to determine the effect of the Na+K+ATPase inhibitor sanguinarine on the refractory period of the porcine ventricle. Measurements of ventricular refractoriness and strength-interval curves were obtained before and after 0.5 to 4.0 mg/kg sanguinarine. The ventricular refractory period was found to be prolonged at all doses studied from a control of 276 +/- 12 msec to 318 +/- 24 msec at 4.0 mg/kg (p < 0.01). The strength-interval curve was shifted to increased values for ventricular refractoriness from 0.5 to 16.0 ma. No depression of arterial pressure or left ventricular dp/dt was observed. This study shows that sanguinarine prolongs ventricular refractoriness. This property may be useful in the treatment of ventricular arrhythmias.

Editor’s note:  Further study of this property is warranted.

The following article is included merely to summarize the historical place of Sanguinaria canadensis and to provide a source to further explore the chemistry of the assorted constituents obtained from the rhizome of the plant:

The history, chemistry and pharmacokinetics of Sanguinaria extract.

Harkrader RJ, Reinhart PC, Rogers JA, Jones RR, Wylie RE 2nd, Lowe BK, McEvoy RM.

Atrix Laboratories, Inc.

Sanguinaria extract is a mixture of benzophenanthridine alkaloids derived from Sanguinaria canadensis L. (bloodroot). This mixture of alkaloids has a long history of use in tinctures and expectorants in pharmaceutical products. The purity of Sanguinaria extract is well defined. The chemistry and biochemistry of these alkaloids, including the dynamic equilibrium between acid and base forms, and pharmacokinetics of Sanguinaria extract shall be presented when this extract is incorporated into a dentifrice or oral rinse formulation.

Agronomics of Sanguinaria

This section is included to highlight the importance of the variance in growth conditions as pertains to the effect on alkaloid quality and concentration in the mature rhizomes.  The best and highest concentrations of the active alkaloids in bloodroot come from Western North Carolina.  Another reason for including this section is the definition it provides to the reader about the nature of this remarkable medicinal plant:

Environmental and genotypic influences on isoquinoline alkaloid content in Sanguinaria canadensis.

Salmore AK, Hunter MD.

Institute of Ecology, University of Georgia, Athens 30602, USA.

In a common garden, we investigated genetic and environmental influences on alkaloid production using Sanguinaria canadensis as a model. Nutrient and shade regimes were applied to replicated clones over one growing season, and induction of alkaloid production in bloodroot was tested on a whole-plant basis using jasmonic acid as an elicitor. Alkaloid concentrations increased with decreasing light intensity and fertilizer levels. Induction was not achieved by foliar application of jasmonic acid. Genetic influences represented by clone effects may be indicated by variation in alkaloid concentration by clone, but this experimental design did not allow us to distinguish genetic from pre-experiment environmental influences on the rhizomes.

Growth characteristics of Sanguinaria canadensis L. cell suspensions and immobilized cultures for production of benzophenanthridine alkaloids.

Rho D, Chauret N, Laberge N, Archambault J.

Biotechnology Research Institute, National Research Council Canada, Montreal, Qc.

Sanguinaria canadensis L. plants were harvested from a local forest and calli were initiated from leaf explants. The production of benzophenanthridine alkaloids (i.e. sanguinarine, sanguilutine, sanguirubine, chelerythrine, chelilutine and chelirubine) by S. canadensis cell grown in modified B5 and IM2 media was compared to the alkaloid content of rhizomes. Sanguinarine accounted for approximately 80% of the total alkaloid content of cultured cells (1.3%, g g-1) while sanguinarine and sanguirubine accounted for 70% of rhizome alkaloids (9.0%, g g-1). Sanguinarine, chelirubine and chelerythrine were the only known alkaloids detected in cultured S. canadensis cells. Maximum alkaloid production of cultures performed using B5 medium, containing half the original nitrate concentration, was observed following extracellular nitrate and sugar depletion. The scale-up of this culture was successfully performed in a 2-1 immobilization bioreactor. The consumption of sugar and nitrate as well as the oxygen (OTR) and carbon dioxide (CTR) transfer rates of the immobilized cell culture were monitored for 15 days. The maximum sugar and nitrate consumption rates were 1.8 g l-1 per day and 2.3 mM per day respectively. The maximum OTR and CTR of the immobilized cell culture were 0.8 mmol O2 l-1 h-1 and 0.95 mmol CO2 l-1 h-1 respectively. The sanguinarine yield of this culture reached 1.0% based on biomass dry weight (g g-1 dw) by day 15.

Elevational trends in defense chemistry, vegetation, and reproduction in Sanguinaria canadensis.

Salmore AK, Hunter MD.

Institute of Ecology, University of Georgia, Athens 30602, USA.

Evaluation of biotic interactions along geographic gradients reveals that pressure on plant populations by herbivores and pathogens increases as latitude decreases, and is accompanied by a parallel increase in the number and toxicity of alkaloid-bearing plants. We compared rhizome alkaloid content with plant reproductive and vegetative characters in Sanguinaria canadensis (Papaveraceae) along an elevational gradient over two growing seasons to ascertain 1) if alkaloid production in bloodroot varies among populations and systematically with elevation, and 2) if there exists a correlation between isoquinoline alkaloid, vegetative and reproductive production. In general, alkaloid content in bloodroot rhizomes declines with elevation, increases with rhizome water content, varies by site, and fluctuates seasonally with plant growth and reproduction. Alkaloid content was positively correlated with vegetative and reproductive effort with few exceptions. Analysis of total protopine and benzophenanthridine alkaloid concentrations revealed generally similar patterns as those of individual alkaloid concentrations, although significant differences did appear between individual alkaloid concentrations.

How to enhance alkaloid content in Sanguinaria canadensis:

Missouri Botanical Garden, St. Louis 63110.

An elicitation protocol, resulting in the accumulation of sanguinarine in suspension cultures of Papaver bracteatum, was assessed for induction of the same alkaloid in Sanguinaria canadensis. Although only a trace constituent of P. bracteatum plants, sanguinarine is a major alkaloid (1-3% dry wt) of S. canadensis rhizomes. By combining hormonal deprivation for various intervals and a 3-day fungal (Verticillium dahliae) elicitation, benzophenanthridine alkaloid accumulation was induced in S. canadensis cell suspensions. Chelirubine content increased (0.1-1.3% dry wt) consistently in elicited cell cultures while chelerythrine (0.01-0.10% dry wt) and sanguinarine (0-0.02% dry wt) levels were considerably less. Alkaloid accumulation always occurred upon removal of hormone but only at certain time intervals in the log phase upon fungal elicitation. Levels of dopamine, a precursor of the alkaloids, fluctuated over the incubation period, but displayed a 2- to 6-fold increase in cell suspensions grown without hormone. In some experiments dopamine accumulated to levels > 20% dry wt, and these increases were enhanced by the addition of fungal elicitor. Although the same fungal elicitor induces benzophenanthridines in taxonomically related S. canadensis and P. bracteatum, it did not elicit the accumulation of the same alkaloid in the two different plant cultures.
And similarly the next article exploits the use of hormonal deprivation to enhance one of the active alkaloids in Sanguinaria canadensis:

 
Isolation and Characterization of S-Adenosyl-L-Methionine: Tetrahydroberberine-cis-N-Methyltransferase from Suspension Cultures of Sanguinaria canadensis L.

O'Keefe BR, Beecher C.

Department of Medicinal Chemistry and Pharmacognosy, Program for Collaborative Research in the Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, Illinois 60612.

As part of a continuing study of the induction of alkaloid biosynthesis, we report the isolation to homogeneity and characterization of S-adenosyl-L-methionine: tetrahydroberberine-cis-N-mehtyltransferase from suspension cultures of Sanguinaria canadensis that were induced to produce alkaloids by hormone depletion. This enzyme catalyzes the stereospecific transfer of a methyl group from S-adenosyl-L-methionine to the tertiary nitrogen of the protoberberine alkaloid tetrahydroberberine (canadine). The enzyme was purified 315-fold by ammonium sulfate precipitation, gel permeation chromatography, affinity dye chromatography, and both diethylaminoethyl and Mono-Q ion-exchange chromatography. The enzyme was further purified to an optimum specific activity of 225 nkat/mg of protein (3500-fold) and electrophoretic homogeneity by native polyacrylamide gel electrophoresis (PAGE). In contrast to previous reports with partially purified enzyme, the isolated protein was found to have a pH optimum of 7.0, a temperature optimum of 25 to 30[deg]C, and an isoelectric point of 5.1. Furthermore, the molecular weight of the homogeneous protein was found to be 39,000 by sodium dodecyl sulfate-PAGE. The homogeneous enzyme preferred tetrahydroberberine over all other substrates tested, showing an apparent Km of 2.1 [mu]M, but also showed partial activity with tetrahydrojatrorrhizine and tetrahydropalmatrubine.

Another way to enhance alkaloid production that may well be applied to soil enrichment:

Quercetin-induced benzophenanthridine alkaloid production in suspension cell cultures of Sanguinaria canadensis.

Mahady GB, Beecher CW.

Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago 60612.

Addition of micromolar concentrations of quercetin or rutin to suspension cell cultures of Sanguinaria canadensis L. (bloodroot) induced the biosynthesis of sanguinarine and chelerythrine in a dose-dependent manner. In contrast, related compounds: baicalein, naringin, naringenin, catechin, caffeic acid and benzoic acid displayed very weak inductive activity. Of the two active flavonoids, quercetin was the most effective for inducing benzophenanthridine alkaloid biosynthesis, with doses of 100 microM increasing alkaloid production over 375% as compared to negative controls. Quercetin's inductive effects were similar to that of an elicitor derived from fungus Penicillium expansum (PE-elicitor). Suppression of quercetin and PE-induced alkaloid biosynthesis by low doses of actinomycin D (5 micrograms/ml, alpha-amanitin (20 micrograms/ml), or cycloheximide (1 microgram/ml) demonstrate a requirement for both RNA and de novo cytoplasmic protein synthesis and suggest that alterations in gene expression are involved in the inductive mechanism. Furthermore, quercetin-induced alkaloid biosynthesis was significantly reduced by pretreatment of the cells with the calcium chelator, EGTA (3 mM), or the calcium channel inhibitor, verapamil (100 microM), suggesting that this process was calcium dependent.
Possible benefit of sanguinarine in Agriculture as an herbivoricide (pest control) which would be non-toxic to plants and to their ultimate use as foods and wines, etc.:

 
Biochemical activities of berberine, palmatine and sanguinarine mediating chemical defence against microorganisms and herbivores.

Schmeller T, Latz-Bruning B, Wink M.

Institut fur Pharmazeutische Biologie, Universitat Heidelberg, Germany.

The alkaloids berberine, palmatine and sanguinarine are toxic to insects and vertebrates and inhibit the multiplication of bacteria, fungi and viruses. Biochemical properties which may contribute to these allelochemical activities were analysed. Acetylcholine esterase, butyrylcholinesterase, choline acetyl transferase, alpha 1- and alpha 2-adrenergic, nicotinergic, muscarinergic and serotonin2 receptors were substantially affected. Sanguinarine appears to be the most effective inhibitor of choline acetyl-transferase (IC50 284 nM), while the protoberberines were inactive at this target. Berberine and palmatine were most active at the alpha 2-receptor (binding with IC50 476 and 956 nM, respectively). Furthermore, berberine and sanguinarine intercalate DNA, inhibit DNA synthesis and reverse transcriptase. In addition, sanguinarine (but not berberine) affects membrane permeability and berberine protein biosynthesis. In consequence, these biochemical activities may mediate chemical defence against microorganisms, viruses and herbivores in the plants producing these alkaloids.

To be complete this library is including information on the safety of Sanguinaria, which is excerpted from a 125 page monograph published by the FDA.  Although the focus of the monograph is on products used for dental purposes, the extensive testing performed upon the active ingredients and the results clearly apply to the complete safety profile of an ingredient as if it were ingested or consumed systemically.  The results posted in this monograph on the safety of Sanguinaria are that it is completely safe.

PROPOSED RULES

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration

21 CFR Part 356

[Docket No. 81N-033P]

RIN 0910-AA01

Oral Health Care Drug Products for Over-the-Counter Human Use;
Antigingivitis/Antiplaque Drug Products; Establishment of a Monograph

Thursday, May 29, 2003

*32232 AGENCY: Food and Drug Administration, HHS.

ACTION: Advance notice of proposed rulemaking.

SUMMARY: The Food and Drug Administration (FDA) is issuing an advance notice of proposed rulemaking that would establish conditions under which over-the- counter (OTC) drug products for the reduction or prevention of dental plaque and gingivitis are generally recognized as safe and effective and not misbranded.  This notice is based on the recommendations of the Dental Plaque Subcommittee of the Nonprescription Drugs Advisory Committee (NDAC) and is part of FDA's ongoing review of OTC drug products.

A. Submissions by Firms

 
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                    Table 1.--Firms and Submitted Products                     
                 Firm                              Submitted Products          
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American Xyrofin (Morgan, Lewis &        Xylitol All Natural Toothpaste, Xytol 
  Bockius) Washington, DC 20036            32 Dental Cream.                    
Amer Co., Montecito, CA 93150            Insadol Toothpaste, Pyoralene         
                                           Toothpaste.                         
Angus Chemical Co., Northbrook, IL       Hexetidine solution.                  
  60062                                                                         
Chesebrough Pond's USA Co., Greenwich,   CloseUp Antiplaque Toothpaste,        
  CT 06836                                 Mentadent P Toothpaste.             
Church & Dwight Co., Inc., Princeton,    Arm & Hammer Dental Tooth Powder,     
  NJ 08543                                 Dentifrice, and Gel.                
CIBA-GEIGY Corp., Greensboro, NC 27419   Irgasan DP, Irgacare MP.              
Clinical Product Research, Inc.,         Prozyme Toothpaste, Anti-Plaquer Oral 
  Shreveport, LA 71109                     Rinse, Anti-Plaquer Toothpaste.     
Colgate-Palmolive Co., Piscataway, NJ    Colgate Tartar Control Toothpaste,    
  08855                                    Gelkam Oral Care Rinse, Dentaguard  
                                           Toothpaste.                         
E. Merck, Frankfurter, Germany           Thera-Med, Cholordont M.              
E. B. Michaels Research Associates,      Therasol Brush & Rinse Antiplaque Oral
  Inc., Milford, CT 06460                  Hygiene Solution, Therasol Brush &  
                                           Rinse Liquid Dentifrice Oral        
                                           Irrigant.                           
Leaf, Inc., (Hyman, Phelps & McNamara)   Xylitol.                               
  Washington DC 20005                                                          
Lion Corp. (America), Memphis, TN 38138  Check-Up Gingival Toothpaste.         
Madaus Medtech, Inc., (ACC Consulting    Parodontax Toothpaste.                 
  Group, Inc.) Washington DC 20036                                             
Pfizer Inc, New York, NY 10017           Plax Pre-Brushing Dental Rinse.       
Pierre Fabre, S.A., 81106 Castres        Eligydium Toothpaste, Eludil          
  Cedex, France                            Mouthwash.                          
Prevention Laboratories (formerly 7-L    Prevention Mouth Rinse.               
  Corp.), Harrisburg, IL 62947                                                 
Procter & Gamble Co., Cincinnati, OH     Crest Gum Care Toothpaste.            
  45242                                                                        
SmithKline Beecham Consumer Brands       Cepacol Gold and Mint Mouthwashes,    
  (Marion Merrell Dow, Inc.),              Gly-oxide Liquid.                   
  Parsippany, NJ 07054                                                         
Vipont Pharmaceuticals, Fort Collins,    Viadent Toothpaste and Oral Rinses.   
  CO 80522                                                                      
Warner-Lambert Co., Morris Plains, NJ    Listerine Antiseptic Mouthwash.       
  07950                                                                        
WhiteHill Oral Technologies, Inc.,       Omni-Med Brush-On Tooth Medication,   
  Hazlet, NJ 07730                         Perio-Med Spray, Take-5 Plaque      
                                           Fighter Brushless Dentifrice,       
                                           Smokers Take-5 Plaque and Stain     
                                           Fighter.                            
Witkins, Roy T., Westport, CT 06880      Perimed Oral Hygiene Rinse.           

B. Active Ingredients Submitted For Review

 Labeled Ingredients Contained in Marketed Products Submitted to the Subcommittee:

Alkyl dimethyl amine oxide

Alkyl dimethyl glycine

Aloe vera

Bromchlorophene

Carbamide peroxide

Cetylpyridinium chloride

Chlorhexidine digluconate

Dicalcium phosphate dihydrate

Eucalyptol

Hexetidine

Hydrogen peroxide

Menthol

Methyl salicylate

Peppermint oil

Polydimethylsiloxane

Poloxamer

Povidone iodine

Sage oil

Sanguinaria extract

Sodium bicarbonate

Sodium citrate

Sodium lauryl sulfate

Soluble pyrophosphate

Stannous fluoride

Stannous pyrophosphate

Thymol

Triclosan

Unsaponifiable fraction of corn oil

Xylitol

Zinc chloride

Zinc citrate

H. General Guidelines on Safety and Effectiveness

1. General Statement

 The Subcommittee arrived at its conclusions and recommendations regarding the safety and effectiveness of all active ingredients after considering all pertinent data and information submitted.  The Subcommittee adopted the following general "points to consider." These are not intended to restrict investigators, but are recommendations for studies recognized as desirable approaches to determine the safety and effectiveness of OTC antigingivitis/antiplaque active ingredients.  In some cases, other methods may be equally applicable, or newer methods may be preferable.  Also, these recommended studies may not produce all information necessary to determine that an ingredient is generally recognized as safe and effective.

2. Guidelines

 An OTC drug included in a monograph is described in §  330.10 as generally recognized among qualified experts as safe and effective for use and as not misbranded.  Proof of the safety of an OTC drug ingredient consists of adequate tests by methods reasonably applicable to show the drug is safe under the prescribed, recommended, or suggested conditions of use.  This proof shall include results of significant human experience during marketing.  General recognition of safety shall ordinarily be based upon published studies which may be corroborated by unpublished studies and other data.  Proof of effectiveness of an OTC drug ingredient consists of controlled clinical investigations as defined in §  314.126(b) (21 CFR 314.126b)) by qualified experts to show that the drug provides clinically significant relief of the type claimed in its labeling.  The latter requirement may be waived if it is not reasonably applicable to the drug in question or *32244  essential to the validity of the investigation and an alternative method of investigation is adequate to substantiate effectiveness.  Effectiveness may be corroborated by partially controlled or uncontrolled studies, and reports of significant human experience during marketing.  General recognition of effectiveness shall ordinarily be based upon published studies that may be corroborated by unpublished studies and other data.

 The characteristics of adequate and well-controlled studies have been developed over a period of years and are described in §  314.126.  Studies supporting the safety and effectiveness of OTC drug ingredients should provide sufficient details of study design, conduct, and analysis to allow a critical evaluation of the data in relationship to the above characteristics.

 In several proposed and final monographs, the agency has stated that, in order for an active ingredient to be included in an OTC drug monograph, it is necessary that the ingredient be adequately characterized and that these standards be published in an official compendium such as the United States Pharmacopeia (USP) or the National Formulary (NF) (58 FR 28194 at 28284). Such specifications are necessary to assure the identity, strength, quality, and purity of the active ingredient.  Therefore, the Subcommittee recommends that a full description of the ingredient, including its physical and chemical characteristics and stability, be provided, and that manufacturers contact and work with the USP to develop monographs for ingredients that are not currently included in that compendium.  For ingredients that are currently included in an official compendium, reference to the current edition of the USP or the NF may satisfy this requirement.

 a.  Safety.  The Subcommittee's determination of the safety of single ingredients and ingredient combinations is based on the following criteria: (1) The incidence and risk of adverse reactions and significant side effects when the ingredient was used according to adequate directions in the labeling, (2) the margin of safety under conditions of normal use and the potential for harm that might result from abuse or misuse under conditions of widespread OTC availability, (3) the potential for inducing untoward effects on the oral tissues, including irritation, ulceration, inflammation, erosion, and minor effects such as discoloration of the teeth, restorations, and prostheses, etc., and (4) assessment of the benefit-to-risk ratio.  The Panel further states that microbial safety should be determined through clinical evaluation of changes in representative oral microbial populations (e.g., the possible emergence of opportunistic organisms or potential pathogens), in order to assure that there is no adverse change in the balance of the oral microflora under conditions of expected OTC use.

 i.  Toxicological studies.  A variety of toxicological data can be obtained to demonstrate that an active ingredient is safe.  The Subcommittee recommends that manufacturers conduct the applicable studies discussed below and emphasizes that these recommendations do not preclude the use of alternative comparable methods that are currently available or better methods that may be developed in the future.  The Subcommittee recommends that the following data be available for the active ingredient(s) intended for use on the mucous membranes of the mouth and throat.

 Testing the effects of various ingredients on animal subpopulations that can reflect human subpopulations should be considered (e.g., hyposalivation studies in nonsalivating animals).  Adequate, acceptable, controlled in vivo studies of acute and chronic toxicity in several species of animals should be available.  Such studies may include single-dose gavage studies, repeat-dose gavage studies, oral irritation studies, pharmacokinetic/biodistribution studies, and dermal sensitization studies. Information regarding the genetic, reproductive toxicologic, and carcinogenic potential should be considered for ingredients that are going to be used daily on a long-term basis.  It is not necessary to determine the LD50 (lethal dose for 50 percent of the test animals) of the ingredient.  However, information about the minimal lethal dose would be useful.

 All or some of the recommended toxicological studies may not be necessary for all active ingredients.  Some circumstances that might preclude an ingredient from the above testing are: (1) It is already generally recognized as safe, (2) it is a direct food additive, (3) it has been used previously in approved dental drug products, or (4) it is the subject of an OTC drug monograph with a different but similar or related use at a similar concentration and for a similar time period.  Published articles may be considered in lieu of the testing recommended above.

 One of the Subcommittee's primary concerns regarding antigingivitis/antiplaque ingredients is whether or not swallowing the active ingredient presents a threat to the user.  The Subcommittee recommends that gavage studies be used to address concerns about potential systemic toxicity unless applicable published or unpublished studies have been conducted using a dietary admixture mode of administration and comparable toxicokinetics can be shown between gavage and dietary modes of administration.  Single administration gavage studies are typically performed using a limit-value test in the rat at a specified high dose to evaluate acute toxicity potential (Refs.  46, 47, and 48).  In the absence of adequate dietary admixture studies, repeat dose gavage studies may be employed to evaluate systemic toxicity from multiple exposures.  The test article is administered to rats on a number of consecutive days.

 Where there is a concern that antigingivitis/antiplaque active ingredients may induce untoward effects on the oral mucosa, the dosage to be used for these studies should be justified based on the concentration of human exposure levels.  An appropriate dosage range may extend, for example, from a low dose comparable to swallowing a single dose of mouthrinse or the amount remaining following expectoration of a mouthrinse to a high dose that either causes dose- limiting toxicity or is several orders of magnitude greater than the clinical exposure levels.  Such studies usually use four applications per day for a period of 28 consecutive days.  The oral irritation should include both a negative and a positive control group.  All test articles should be applied in an identical manner.  A negative control group may consist of animals that are treated with either water or saline, and the positive control is a group of animals that are treated with the solution that is known to cause a minimal degree of irritation without being inhumane to the animals (e.g., 5-percent solution of sodium lauryl sulfate).

 The Subcommittee recommends that the study include abraded mucosa in order to determine whether the test ingredient delays or prevents the healing of oral lesions.  The parameters to include are any gross observations of changes in the oral tissue, such as sloughing, ulceration, or bleeding. Following the sacrifice of each animal, the histopathology of oral tissues should be examined.

 ii.  Studies in older adults.  The Subcommittee is concerned that older adults might be at greater risk for potential systemic toxicity from the use of antigingivitis/antiplaque active ingredients.  This is of particular concern because of the continually *32245  increasing size of the older adult population, who are retaining more natural teeth and becoming a significant population for use of antiplaque/antigingivitis products.

 Publications have described differences in drug responses in the elderly.  Changes in pharmacokinetics have been reviewed (Ref. 49).  Absorption can theoretically be altered by noted changes in gastrointestinal function, but the majority of studies have shown no difference in rate or extent of absorption of the drug examined.  Distribution of a drug within the body is affected because fat content of body weight increases and intracellular water decreases.  For example, albumin concentration is reduced and drugs which bind to albumin are more free to distribute to the rest of the body.  Hepatic metabolism may be altered.  Reduction of blood flow to the liver will decrease clearance of some drugs.  Renal excretion is affected in some older adults by loss of renal mass and functional nephrons.

 Russell (Ref. 50) noted that despite numerous reports in the literature of impaired GI function with aging, most functions remain relatively intact because of the large reserve capacity of the intestine, pancreas, and liver. In a review critically analyzing available information on age-related changes in the digestive and absorptive GI physiology of lipids, data suggested lipid digestion and absorption are well-preserved in the aging.  However, intercurrent illness or experimental stress may produce impairment in aging animals and humans that is not seen in younger controls (Ref. 51).

 Atillasoy and Holt (Ref. 52) noted that the GI tract represents an organ system characterized by rapid proliferation.  Contrary to generally held prejudices, the authors write, a state of hyperproliferation, not hypoproliferation, occurs in the epithelial cells of the stomach, small intestine, and large intestine of stable-fed, aged rodents when compared to young adult rodents.

 In a gavage study (Ref. 53) Yamada et al. investigated renal ammoniagenesis in isolated nephron segments from control, acidotic senescent (exhibiting deteriorating teeth due to aging), and young adult rats.  No significant difference was seen in glutamine-dependent ammonia production in the segments. However, ammonia production in glomeruli from old rats was significantly greater than in young rats.

 There appear to be no available consistent findings to warrant that additional gavage studies of antigingivitis/antiplaque active ingredients in older animals will produce more meaningful findings relative to older adults than the usual gavage studies in adult animals.  This is due to the great diversity which exists in the health and fitness status of the elderly population.  The Subcommittee considers a comment by Ahronheim (Ref. 54) appropriate:

 Although much has been written about age-related alterations in drug disposition, there is disagreement as to the extent and inevitability of these changes.  Studies focusing on aged individuals suffer from several problems. Cross-sectional studies comparing young and old subjects sometimes compare young, healthy individuals with aged subjects gathered from hospitals or nursing homes.  If the aged subjects are "healthy" they may nonetheless have subclinical disease, which can alter outcomes in studies that seek to determine a drug's disposition and effects.  However, aged subjects that are truly healthy may represent an elite minority so that the study's results may not be applicable to the general elderly population.  Longitudinal studies are almost impossible to complete and data is sparse, but recent findings indicate that the geriatric population is, indeed, heterogeneous.

 In addition to these pitfalls, it is not known how generalizations about aging physiology, even if they are true, can be applied to drug disposition, since most drugs have not been subjected to exhaustive age-specific testing and few conclusions can be reached based on pharmacokinetic data.  Even less is known about pharmacodynamic changes because the study of age-related tissue receptor density, activity, and sensitivity is in its infancy.  We must therefore rely on clinical observations to a large extent when drawing conclusions about efficacy and potential toxicity of various agents in use.  The Subcommittee concludes that the results of the usual gavage studies are adequate.

 iii.  Irritation and delayed contact sensitization studies in humans.  Observations during adequate clinical studies are sufficient to demonstrate the irritation and sensitization potential of an ingredient or ingredient combination.  However, if necessary, a number of methods embodying the use of patch testing have proven of value in determining skin irritancy and systemic sensitization.  The Subcommittee recommends one of the following three methods of patch testing to address concerns of irritancy and sensitivity:

 • Draize testing.  In the Draize human skin irritancy and sensitization tests or one of its various modifications (Ref. 55), the testing should be performed on the skin of the subject's back or arm.

 • Method of Shelanski and Shelanski.  In this method (Ref. 56), the active ingredients or the formulation under study are applied at frequent intervals of 1 or 2 days to the test site for 3 or 4 weeks.  After a rest period of 2 weeks, a single dose of the drug is applied as a challenge.  The preliminary applications are made to detect primary skin irritants and provoke sensitization in susceptible individuals.  The challenging dose detects whether or not the drug is a skin sensitizer.

 • Maximization procedure of Kligman.  This procedure (Ref. 57) or one of its modifications uses an irritant applied over a desquamated test site. Desquamation is performed by using a rubbing technique that facilitates penetration, thereby hastening and accentuating the skin-sensitizing potential of the substance.  Other validated human models may be used.

 iv.  Microbiologic evaluation.  The Subcommittee is concerned about the potential of antigingivitis/antiplaque ingredients with antimicrobial effects to allow emergence of opportunistic pathogens, induce resistance in oral microorganisms, or allow an oral overgrowth of inherently resistant potential pathogens.  Representative microbial species and their relative proportion to the total cultivable microflora in supragingival plaque and saliva should be monitored over at least a 6-month period of continuous use of the antiplaque product to determine if a shift in the oral flora has occurred that might result in the proliferation of pathogenic microorganisms, which may include Candida species and other yeast, Staphylococcus aureus and other Staphylococcus species, beta-hemolytic Streptococci, and enteric gram-negative rods. Additionally, for those antigingivitis/antiplaque ingredients where the mechanism of action is suspected to be antimicrobial, an assessment of changes in microorganisms associated with gingival disease should be carried out.  One determination should be made prior to the start of use, one at the conclusion of the study, and one at an intermediate time.  In vitro minimum inhibitory concentrations should be assessed for representative species to determine the development of increased resistance after prolonged antimicrobial therapy.

d.  Sanguinaria extract.  The Subcommittee concludes that sanguinaria extract at 0.03 to 0.075 percent concentration is safe, but there are insufficient data available to permit final classification of its effectiveness in an oral rinse or dentifrice dosage form as an OTC antigingivitis/antiplaque active ingredient.

 Sanguinaria extract is prepared by warm acidulated alcoholic extraction of the rhizome of Sanguinaria canadensis (more commonly known as blood root or puccoon), followed by precipitation with a metal salt.  Six principal benzophenanthridine alkaloids are present in the extract with sanguinarine (50 percent) and chelerythrine (25 percent) being the major ones.  Sanguinaria extract is a bright orange, free-flowing, amorphous powder that is hygroscopic and electrostatic.  It is soluble at 25[deg] C in methanol to 1 percent weight per weight (w/w), in chloroform to 0.75 percent w/w, in water or water buffered with one percent citric acid to 2 percent w/w.  Sanguinaria extract exhibits a pH dependent lipophilicity and partitions to a significant extent into the lipid phase of a lipid/water mixture above pH 6.5.  Sanguinaria extract has been described in several pharmacopeia (Refs.  209 and 210) and textbooks (Ref. 211).  Uses include relief of spongy and red gums and in OTC cough syrups as an expectorant.  Sanguinaria extract was introduced into homeopathic practice in 1837.

 i.  Safety.  Safety studies addressing acute toxicity, irritation potential, sensitization potential, reproductive toxicity, birth defect potential, chronic organ toxicity, and carcinogenic potential were conducted in animals using sanguinaria extract and sanguinarine chloride.

 The acute toxicity of sanguinaria extract was determined by oral gavage to Sprague-Dawley rats with doses from 500 to 3,000 mg/kg.  In one study (Ref. 212), the oral LD50 of sanguinaria extract was 1,440 mg/kg.  This suggests that sanguinaria extract is probably poorly absorbed orally.  The lethal dose of sanguinaria extract in two Cynomolgus monkeys was above 50 mg/kg.  The acute dermal LD50 in a limited study using 10 adult New Zealand rabbits was greater than 200 mg/kg body weight.  Acute inhalation toxicity of sanguinaria extract (2.2 mg/liter) in 10 rats resulted in mortality in 3 of 5 males and no females.  Gross pathology examination revealed no lesions or abnormalities. The LD50 from two studies of sanguinarine chloride determined by oral gavage in rats was 1,525 and 1,663 mg/kg.  The intravenous LD50 in rats was 28.7 mg/kg, and the intraperitoneal LD50 in mice was 17.7 mg/kg.
Editor’s note: The quantities of ingested material or injected material to induce death of 50% of the animals so treated is enormous and could not be duplicated even by a conscious overdose by a human being.

 Studies concerning the multidose subchronic toxicity of sanguinaria extract (Refs.  213, 214, and 215) and sanguinarine chloride (Refs.  216, 217, and 218) were conducted in rats and monkeys at doses ranging from 5 to 405 mg/kg for 2 to 13 weeks.  In a 4-week oral gavage study in monkeys (Ref. 215), 100 mg/kg of sanguinaria extract was determined to be the appropriate high-dose for a subsequent 13-week toxicity study in monkeys.  A 13-week gavage study in monkeys (Ref. 216) with 0 to 60 mg/kg showed no treatment-related toxicity except minor GI irritation of limited duration.  The study suggested a NOAEL of 30 mg/kg per day once tolerance is achieved.  A 13-week oral gavage study in rats (50 to 400 mg/kg per day) (Ref. 214) showed evidence of dose-related toxicity, principally involving GI irritation and body weight loss at all dosage levels.  Mortality was observed at doses of 100 mg/kg per day and above, with a NOAEL of less than 50 mg/kg per day.  Administration in the diet appears to protect against GI irritation.  A 4-week dietary toxicity study in rats (5 to 405 mg/kg per day) (Ref. 213) showed a group mean body weight loss at 405 mg/kg.  Based on these studies, evidence of minor treatment-related toxicity associated with sanguinaria extract and sanguinarine chloride is limited to GI irritation.

 Pharmacokinetic studies assessing metabolism, disposition, distribution, and elimination of sanguinaria extract and sanguinarine chloride were conducted in rats and mice (Refs.  219, 220, and 221).  The metabolism of sanguinaria extract was tested in vitro in rat and rabbit liver homogenates and in vivo in 10 human subjects for at least 6 months (Ref. 219).  Results indicated that no benz[c]acridine (50 parts per billion (ppb) detection limit) was formed in the rat or rabbit liver homogenates.  Neither benz[c]acridine (1 ppb detection limit) nor sanguinarine chloride (25 ppb detection limit) was found in the urine of the human subjects.

 Studies evaluating the biological disposition of radiolabeled sanguinarine chloride in rats (Ref. 220) and mice (Ref. 221) suggested low absorption, with excretion of over 50 percent (mice) and 88 percent (rats) of the total dose in feces.  Less than 1.0 percent (rats) and 0.9 percent (mice) was excreted in the urine.

 Analysis of rat tissues collected 96 hours following oral administration of 5 mg/kg indicated a total recovery of approximately 6.1 percent of the administered radioactivity.  Excretion via urine, feces, and expired air accounted for 95.1 percent of the administered dose in the 96-hour post- administration period.  Blood levels in the rat achieved less than 1.5 percent of the net dose administered orally, peaking around 8 hours and declining to near 1 hour levels by 96 hours.

 Expired air accounted for an average of 18.3 percent (mice) and 6.0 percent (rats) of the dose administered.  The nature of the blood radioactive residues and excreted 14 C-carbon was not determined.  An overall mean recovery in mice of 97.89 percent of the 14 C-carbon during the 96 hours following oral administration of sanguinarine chloride labeled at one and/or both methylene- dioxy groups suggests that a substantial portion of the radiolabeled test product may be transformed into nonlabeled benzophenanthridine metabolites. These results suggested that sanguinarine chloride is satisfactorily recovered after oral or intravenous administration.

 A cardiovascular study in dogs treated intravenously with sanguinarine *32261  chloride (0.075 mg/kg) demonstrated no treatment- related effect on heart function or cardiovascular health (Ref. 222) at a dose 30 times the maximum daily absorbed dose expected from brushing and rinsing.

 Sanguinaria extract was tested in a fertility/reproduction study in rats (Ref. 223), in developmental toxicity studies in rats and rabbits (5 to 400 mg/kg per day) (Refs.  224, 225, and 226), and in a perinatal/postnatal study in rats (5 to 60 mg/kg per day) (Ref. 227).  The NOAEL level of sanguinaria extract was 25 mg/kg per day for development toxicity in rabbits, and 15 mg/kg per day for maternal toxicity.  Sanguinaria extract had no effect on fertility, reproduction, or fetal and neonatal development in rats and rabbits at doses below those resulting in general toxicity in the adult animals.

 Mutagenicity studies were conducted with both sanguinaria extract and sanguinarine chloride with in vitro methods using microorganisms and mammalian cells in culture and in vivo in mice.  Weak positive responses were elicited only in the bacterial assay using Salmonella typhimurium (Ames assay) in the presence of metabolic activation (Ref. 228).  Studies of sanguinaria extract were negative in the bacterial assay with Escherichia coli (Ref. 229), in an unscheduled DNA synthesis assay in rat primary hepatocytes (Ref. 230), and in a micronucleus cytogenetic assay in mice (Ref. 231).  An Ames test for metabolites of sanguinaria extract in rat urine using S. typhimurium was negative.  Studies of sanguinaria chloride were negative in other Ames assays with S. typhimurium (Ref. 232), and Saccharomyces cerevisiae (Ref. 233) with and without metabolic activation.  Two mammalian cell assays (Ref. 234) with sanguinarine chloride, including a Chinese hamster ovary (CHO)-hypoxanthine- guanine phosphoribosyltransferase (HGPRT) forward gene mutation assay and unscheduled DNA synthesis assay in rat primary hepatocytes (Ref. 235) provided results that were equivocal or uninterpretable.  Neither study, however, gave a positive mutagenic response.  The CHO assay is historically difficult to conduct and interpret.

 Long-term (90 to 98 weeks) carcinogenicity studies (Ref. 236) by gavage at dosages of 0 to 60 mg/kg per day sanguinaria extract in rats did not produce treatment-related preneoplastic or neoplastic lesions to suggest a carcinogenic effect.  Dosage at 40 mg/kg per day did not produce toxicity and is considered the NOAEL dosage.  A lifetime diet carcinogenicity study of sanguinaria extract was evaluated in rats (8 to 200 mg/kg per day) (Ref. 237).  No test related hematological, biochemical, or urological changes were observed at any dosage level.  No test article related macro- or microscopic pathology changes were observed.  A 200 mg/kg per day dosage level can be considered the NOAEL level.

 Two controlled 13-week subchronic studies done in monkeys and dogs (Ref. 238) examining ocular toxicity provided no evidence that sanguinaria extract or sanguinarine chloride affected intraocular pressure or produced any other ophthalmologic changes.

 Human exposure to sanguinarine with twice daily use of toothpaste and oral rinse has been estimated to be 0.056 mg/kg per day (Ref. 238).  Comparison of doses tested in animal studies with human doses expected from use of toothpaste or oral rinse appears to support the use of sanguinaria extract at a significantly higher concentration than contained in currently marketed products.

 Ten animal safety studies conducted between 1982 and 1984 were submitted for dentifrice formulas containing 300 to 2,000 [mu]g/mL of sanguinaria extract. None of the studies tested the currently marketed toothpaste formula containing 750 [mu]g/mL of sanguinaria extract.  Acute oral toxicity was greater than 20 g/kg in rats for a toothpaste formula containing 300 [mu]g/mL of sanguinaria extract, and 5 g/kg in rats for a formula containing 500 [mu]g/mL of sanguinaria extract (Refs.  239, 240, and 241).  Primary skin and eye irritation studies carried out in rabbits (Refs.  242 and 243) demonstrated mild irritation reaction when a toothpaste formula containing less than 750 [mu]g/mL was tested.  Mild mucosal irritation was observed when a toothpaste formula containing 300 [mu]g/mL of sanguinaria extract was tested in cheek pouches of hamsters (Refs.  244 through 248).

 Two clinical studies (Refs.  249 and 250) demonstrated only mild mucosal irritation in test subjects.  No differences were noted in the severity of lesions between the test and control groups.

 Eleven clinical studies of animal safety conducted between 1983 and 1987 (Ref. 251) were submitted.  Because modification of the oral rinse formulation from pH 3.2 to pH 4.5 began in 1989, none of these studies provided animal safety data on the currently marketed oral rinse (pH 4.5).

 Based on data on the oral rinse formula containing 450 to 1,000 [mu]g/mL sanguinaria extract at a pH of 3.2, no mucosal irritation was noted in the hamster cheek pouch (Refs.  252 and 253) or albino guinea pig studies (Ref. 254).  No signs of toxicity or pharmacological effects were observed in test animals when a rinse formula of 450 [mu]g/mL sanguinaria extract at pH 3.2 was tested (Ref. 255).

 Four human studies conducted between 1982 and 1985 evaluated the irritation and sensitization potential of dentifrice formulas containing sanguinaria extract using a repeated insult patch test design involving a 2- percent aqueous slurry (Refs.  256 through 259).  These studies demonstrated no induction of irritation or allergic contact dermatitis.  An exaggerated use study (Ref. 260) using an earlier formula (300 [mu]g/g sanguinaria extract) demonstrated no irritation or sensitization in soft oral cavity tissues.  Two 6-month studies on a toothpaste containing sanguinaria and sodium monofluorophosphate (Refs.  261 and 262) showed no adverse effects on oral hard or soft tissues.  Soft tissue examinations included inspection of the lips, tongue, hard and soft palate, gingiva, mucobuccal fold areas, inner surface of the cheeks, and sublingual areas.  Although testing of the microbial flora was inconclusive in one study (Ref. 261), sanguinaria did not promote overgrowth through the development of resistant microbial strains.

 A 6-month, double-blind, randomized study using a dentifrice containing 0.075 percent sanguinaria extract (Ref. 263) showed no significant oral irritation or adverse reactions.  A 1-week exaggerated use study showed that 18 of the 28 subjects experienced mucosal sloughing (Ref. 264).

 Although nine human safety studies were presented, only one study (Ref. 265) tested the currently marketed oral rinse containing 300 [mu]g/mL of sanguinaria extract at pH 4.5.  However, this study tested the efficacy of the formula and was not designed to test the safety of the oral rinse.  Three of the remaining eight studies showed that repeated application of the earlier oral rinse formula at pH 3.2 under a semiocclusive patch test did not induce clinically significant irritation or evidence of induced contact dermatitis in humans (Refs.  266, 267, and 268).  This earlier rinse formula gave no evidence of localized or generalized clinical manifestations in test subjects in two of the 7-day exaggerated use studies (Refs.  269 and 270).  The Subcommittee concludes that sanguinaria extract at 0.03 to 0.075 percent concentration in an oral rinse or dentifrice dosage form is safe.