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Notes on Salvia divinorum Options
 
Entropymancer
#1 Posted : 10/5/2011 4:43:36 AM

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This thread ended up meandering far enough that I think it needs a table of contents Laughing. Initially I just posted some notes about Aaron Jenks' Ph.D. dissertation that deals with the evolutionary history of S. divinorum. Then the thread got stickied so I added some notes on the pre- and early- recorded history of the plant. Then one of our members was kind enough to provide me with a wide variety of theses and dissertations dealing with S. divinorum, and this thread seems like as good a place as any to share my notes on those sources. I've added a post for each paper as I finished reading it. There are a few other things peppered in here and there.


Table of Contents
  • Post #1: Notes on Jenks' "Systematics and ethnobotany of Salvia subgenus Calosphace and origins of the hallucinogenic sage, Salvia divinorum"
  • Post #8: Notes on the prehistory and early recorded history of S. divinorum
  • Post #9: Notes on Willard's "Forensic analysis of Salvia divinorum and related Salvia species using chemometric procedures"
  • Post #14: Notes on Addy's "That deep internal voice: controlled administration of Salvia divinorum"
  • Post #15: Notes on Gruber's "Quantification of salvinorin A from tissues of Salvia divinorum (Epling & Játiva-M.)"
  • Post #16: 1994 Palenque Norte talk by Rob Montgomery with Jonathan Ott and Terence McKenna
  • Post #18: Guide to the literature on models for salvinorin A binding to opioid receptors
  • Post #19: Notes on Kane's "Molecular recognition of G-protein coupled receptor ligands: insights into salvinorin A and xanomeline"
  • Post #20: Notes on Yan's "Molecular mechanisms by which salvinorin A binds to and activates the kappa-opioid receptor"
  • Post #24: Notes on Kutrzeba's "Biosynthesis of salvinorin A, a potent hallucinogen from Salvia divinorum Epling & Jativa"
  • Post #27: Notes on Lawrence's "Total synthesis of salvinorin A"
  • Post #28: Notes on Lovell's "Synthesis and pharmacological evaluation of salvinorin A analogues as opioid receptor probes"
  • Post #30: Notes on Lozama's "Preparation of neoclerodane diterpenes as probes for the opioid receptor system”
  • Post #31: Notes on McGovern's "Salvinorin A: fragment synthesis and modeling studies"
  • Post #32: Notes on Nygård's "Listening to the sage: the experience of learning from the Salvia divinorum altered state"
  • Post #33: Notes on Pandit's "3D-QSAR studies of salvinorin A analogs as kappa opioid agonists"
  • Post #34: Notes on Phipps' "The neuropharmacological assesment [sic] of Salvia divinorum Epling and Jativa-M"
  • Post #35: Notes on Stevens' "Extraction and photodegradation of the plant species Salvia divinorum"
  • Post #36: Notes on Stewart's "Phytochemical investigation of Salvia divinorum"
  • Post #37: Notes on Tidgewell's "Development of novel analgesics from the neoclerodane diterpene natural product salvinorin A"
  • Post #38: Notes on Valdés' "The pharmacognosy of Salvia divinorum (Epling & Jativa-M)"
  • Post #39: Notes on Rasakham's "Kappa opioid receptor regulation of ERK1/2 MAP kinase signaling cascade: molecular mechanisms modulating cocaine reward"
  • Post #40: Relevant patents and patent applications





In terms of the evolutionary history of Salvia divinorum, there are a couple of particularly contentious points that have been discussed or speculated on over the years.

One is the question of why the plant seems to have such low reproductive viability. While some cultivators have been able to induce the plant to set seed, this has never been observed in the wild in its native region; there the plant seems to exclusively propagate vegetatively (whether though humans taking cuttings or tall plants breaking off at the stem and setting new roots). Some have speculated that this is because the plant is a hybrid with low reproductive viability, while others suggest that it's a result of inbreeding depression, a consequence of having been subject to human cultivation for a long period of time (particularly if the plants in cultivation had limited genetic diversity to begin with). The other issue is whether S. divinorum is monoclonal (that is, if all the plants are genetically identical). This speculation arose from the fact that the plant propagates vegetatively, and there are no truly wild populations of the species known. All populations are either under human cultivation, or appear to be feral populations that were once under human cultivation, so it would not be inconceivable that they all represent a single genotype.

In 2007, Daniel Siebert mentioned that he had a colleague at UC Riverside doing research on the plant for a PhD dissertation, including genetic analysis of S. divinorum to resolve the issue of whether the plant is monoclonal. Then just last month, the Journal of Plant Research published a paper by Aaron Jenks discussing the evolution and origins of S. divinorum. The paper did resolve the question of hybridity: long story short, S. divinorum is not a hybrid species. If you're really into botany, the paper also established that S. divinorum does not belong in section Dusenostachys of subgenus Calosphace, where Epling had originally placed it, but it does clearly belong within Calosphace. Aside from those two points, the paper left me with more unanswered questions than I'd have liked.

However, in reading the paper I noticed that Jenks was affiliated with UC Riverside, and that the paper was originally part of a PhD dissertation. So I hopped over to UC Riverside's archive of dissertations, and sure enough he'd submitted the dissertation in 2009, and the full text of it was freely available. And luckily, the dissertation contained a lot more juicy bits of information than the recently-published paper. I guess this information has been out there for a while, I just have never seen any mention of it (in the published literature or elsewhere). I've summarized the more interesting bits:

  • As previously mentioned, S. divinorum does not appear to be a hybrid based on the low incidence of polymorphisms in genetic sequences that are inherited biparentally.

  • In the sequences analyzed to assess hybridity, two of the six plants analyzed each displayed a single polymorphism (both at the same locus). This means that there is at least some genetic variability in the species, so it can safely be said that it's not monoclonal. It's still possible that genetic variations are extremely limited; more thorough examination of a larger number of plants collected from wild populations would be necessary to assess the species' genetic diversity.

  • S. divinorum is closely related to a Columbian species, S. venulosa. The data indicates that Central America was the primary region of diversification of the subgenus Calosphace, with dispersal events carrying some species to South America. The simplest explanation would be that the common ancestor of these two species arose in Mexico, and a dispersal event carried the ancestor of S. venulosa to the Northern Andes. But it can't be ruled out that the common ancestor of both species arose in South America (after being carried there by a dispersal event), and the ancestor of modern S. divinorum was returned to Mexico in a back-dispersal event. To resolve this question, phylogenetic studies would have to be conducted on a variety of related South American species.

  • It's not certain whether S. divinorum once occurred over a wider geographic range, but if this were the case it would help explain the plant's apparently limited reproductive viability in its current natural habitat. Nahua trade routes pssed though the eastern Mazatec region, possible providing an explanation of S. divinorum's presence there (assuming it previously occurred over a wider geographic range). “Regardless, of whether or not S. divinorum is native to the Sierra Mazateca, the conditions there do not seem fully adequate for sexual reproduction, suggesting that the origins of this plant are elsewhere.”

  • Jenks frequently mentions the possibility pipiltzintzintli being S. divinorum as possible evidence for a more broad distribution in the past... I find the evidence for that identification problematic, as detailed in my Notes on Pipiltzintzintli. But the fact that a deity's headdress appears to be decorated with the flower of S. divinorum in the Dresden Codex of the pre-Columbian Maya would certainly tend to support the notion that S. divinorum occurred outside the Mazatec region some centuries ago.

  • The close association of the plant with humans does not, in itself, mean that the species is a cultigen (requiring human intervention to prosper). While S. divinorum is not reproductively robust (about 50% of pollen grains aborted), it does seem to be reproductively viable. Nutlet maturation in self-pollinated flowers has been reported to be fairly low (11 nutlets from 108 flowers), seed set in some species of Calosphace appears to be naturally low. The reported 30% germination rate is not abnormally low for species in this subgenus.



Further Reading
  • Jenks, A.A. 2009. "Systematics and ethnobotany of Salvia subgenus Calosphace and origins of the hallucinogenic sage, Salvia divinorum." PhD dissertation, Plant Biology: University of California Riverside. (fulltext pdf).
  • Jenks, A.A., J.B. Walker, and S.-C. Kim. 2011. "Evolution and origins of the Mazatec hallucinogenic sage, Salvia divinorum (Lamiaceae): a molecular phylogenetic approach." Journal of Plant Research 124(5): 592-600.
  • Reisfield, A.S. "The botany of Salvia divinorum (Labiatae)." SIDA 15(3): 349-366. (fulltext pdf)
 

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jamie
#2 Posted : 10/5/2011 5:11:08 AM

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hmm..interesting..thanks for the info!

I have been growing salvia for 4-5 years now..it is a beautiful plant to grow and next fall I hope that I can get at least one plant to flower and eventaully get some to seed..isn't continual propegation via cuttings problematic in that in time the plant looses genetics? I have wondered about the effects this will have somewhere down the line on the plant..it is good that people have gotten then plant to produce viable seeds that are being grown out.
 
corpus callosum
#3 Posted : 10/5/2011 6:15:02 AM

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Good stuff Entropymancer;thanks for sharing this.

As a side-note, I love looking at PhD theses as they often analyse minutiae in great detail.

Gibran2 is a bit of an expert in growing and working with salvia so I look forward to any thoughts he may have on this.

Once again, good stuff!Smile
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Metanoia
#4 Posted : 10/5/2011 4:31:39 PM

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Very interesting, even for a layman such as myself. I've been growing it for more than a year now myself, and I was also wondering the same thing as fractal. I'd love to be able to get my plants to seed, but alas, I don't really have much of a greenthumb. I can grow things well enough, but I wouldn't say I have a gift for it like some people do.

Thanks for sharing!
 
Entropymancer
#5 Posted : 10/5/2011 5:00:06 PM

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If you can get your plants to flower, it's been reported that manual pollination can produce about one seed per ten flowers. And the seeds themselves have about a 30% germination rate.

Fractal is right that continuous clonal propagation does reduce genetic diversity and can impair reproductive fitness... this is probably a factor in why S. divinorum doesn't set seed very easily. But since the species isn't monoclonal and people are producing seeds these days, these cultivators are helping to boost the genetic diversity of the species.


This paper made me realize that I've been missing a potentially robust source of information in theses and dissertations. Last night I searched a database and turned up thirteen other papers that would be worth getting my hands on. Only one is available for free download, but it's on that's actually of interest to laypeople. It's a double-blind placebo-controlled study where 30 participants smoked either a placebo or a S. divinorum extract containing 1 mg salvinorin A on four different occasions. I haven't finished reading the paper, but it sounds interesting. I'm not sure why it didn't receive as much attention as the more widely-publicized study by Johnson et. al. that involved only four subjects.

The paper:
  • Addy, P.H. 2010. "That deep internal voice: Controlled administration of Salvia divinorum." PhD dissertation, Clinical Psychology: Institute of Transpersonal Psychology. (fulltext pdf)


And the other controlled study of salvinorin A in humans:
  • Johnson, M.W., K.A. MacLean, C.J. Reissig, T.E. Prisinzano, and R.R. Griffiths. 2010. "Human psychopharmacology and dose-effects of salvinorin A, a kappa opioid agonist hallucinogen present in the plant Salvia divinorum." Drug and Alcohol Dependence (Article in press), doi:10.1016/j.drugalcdep.2010.11.005. (fulltext pdf)
 
gibran2
#6 Posted : 10/5/2011 5:05:59 PM

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Interesting information. Thanks for putting it all together for us!

Because of the way I harvest my plants, they rarely reach the flowering stage – I generally harvest an entire plant after it’s been growing for 3-4 months. I once had a plant that produced flowers after about 6 months (it produced flowers around this time of year), but it never produced seed.

Salvia grows in such a way that when a stem reaches a certain height, it takes very little for it to snap off. I imagine that in a moist environment, broken branches can easily root and can propagate quite effectively this way.
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tigerstrike92
#7 Posted : 10/5/2011 11:20:40 PM

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Wow! This is all very interesting, and I hope to keep reading more.

Salvia has such an interesting history, with a lot of mystery surrounding her. It is truly awesome when fellow salvianauts can work at unraveling some of these questions.

I can't wait until I'm at a place where I can grow my own!
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Entropymancer
#8 Posted : 10/10/2011 3:45:48 PM

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Well shoot, since this thread ended up stickied, I feel obligated to give it a little more oomph. My apologies for the somewhat clunky flow in the following history. Much of it is simply extracted from my notes on the subject. More care is currently being put into constructing a complete and coherent history.





The earliest possible indication of the use of S. divinorum comes from the Dresden Codex, a pre-Columbian book from the Mayan culture dating to the 11th or 12th century. Botanist William Emboden noted that the flower on the front of the Death god’s headdress (on p. 13a and p. 13b of the Dresden Codex; see below) is suggestive of S. divinorum based on the bilabiate flower in a conspicuous calyx, though he admits the identification is tentative. The god on p. 13a is holding a bowl containing what appear to be cacao pods; on 13b he is holding a glyph representing maize. The depictions are thought to relate to divinatory practices. (Emboden 1983)


The image on the left is from p. 13a, the image on the right from p. 13b. Both are from the Kingsborough edition of the Codex


Aside from that, there is essentially no evidence for the use of the plant prior to the 20th century. Ott argues that the plant was probably introduced to the Mazatecs relatively recent, and speculates that it might have been used by other indigenous cultures of Mexico in the past (Ott 1995):
Jonathan Ott wrote:
Before summarizing the human pharmacology of Salvia divinorum and salvinorin A, I would like to list my reasons for regarding the shamanic use of this drug to be a post-Conquest innovation in the Sierra Mazateca. I had previously mentioned the lack of a truly indigenous name for Salvia divinorum among the Mazatecs. It is suspicious that the Mazatecs associate the plant with the Biblical Mary, and with sheep, both post-Conquest introductions to the Sierra Mazateca, and Valdés documented remedial use of infusions of 4-5 pairs of Salvia divinorum leaves to treat a disease called panzón de barrego (sic), ‘big lamb’s belly’ (Valdés et al. 1983). We also have the precedent of the mushroom Psilocybe cubensis (Earle) Singer, introduced to Mexico by Europeans along with the cattle in whose dung it grows. Some Mazatec curanderos have come to ultilize this mushroom as a shamanic inebriant, others eschew it (and, tellingly, those who do use it hold it to be the ‘least esteemed’ species). This is exactly what we find with Salvia divinorum — we have seen that María Sabina held it in low esteem. Like the leaves of Mary Shepherdess, P. cubensis lacks a truly indigenous name, being known prosaically in Mazatec as the ‘sacred mushroom of the bull’s dung’; or in Spanish as honguillo de San Isidro Labrador, the ‘mushroom of St. Isidore the Plowman,’ patron saint of Madrid! (Wasson & Wasson 1957). The fact that the Mazatecs put Salvia divinorum in the same ‘family’ as two species of Coleus known to be post-Conquest introductions to Mexico is further evidence for this hypothesis. What clinches the argument for me, however, is how little the Mazatecs seem to know about using the drug. They believe the leaves to be inactive when dried, but this is not true—the dried leaves preserve their activity indefinitely and salvinorin A is highly stable. Valdés suggested the dried leaves were unsuitable for preparing the aqueous infusion, but Pendell has shown they can be successfully rehydrated for oral ingestion, one way the Mazatecs have been documented using the fresh leaves. Valdés saw in the strange method of preparing an infusion of the fresh leaves: “a pharmaceutically elegant way of preparing a microsuspension or emulsion of salvinorin A,” while Wasson dismissed this as “certainly an inefficient method.” Siebert’s studies showed it to be indeed an inefficient method — a marginal, low dose which provoked no effects in an imitation of the Mazatec technique (and the same dose which was all but inactive for Albert Hofmann, even when prepared under the supervision of María Sabina) was “consistently effective” at evoking “definite psychoactive effects” utilizing the simple quid method, readily discovered by American ‘basement shamans,’ but not divined by the Mazatecs. Far from being an ‘elegant way’ of ingesting the leaves of Salvia divinorum, this seems rather a crude adaptation of the standard Mazatec (and other Mesoamerican Indian) technique for preparing the psilocybian mushrooms and the entheogenic morning glory seeds, which are traditionally crushed on a metate and infused in water (Wasson 1963). It is as ‘though the Mazatecs had adapted this standard technique for processing entheogenic plants for ingestion, which is indicated in the case of the mushrooms and seeds, but barely effective in the case of the leaves ... as ‘though they had learned comparatively lately of this drug, which was given a name inspired by the religion and economy of their conquerors, and to process which they simply adapted their existing technique for processing entheogens, despite the fact that it hardly works in this novel case. So ineffective is this adapted processing, that the leaves of Mary Shepherdess have the reputation among the Mazatecs of being much less powerful than the psilocybian mushrooms. Even Valdés’ informants regarded Salvia divinorum to be weaker than the morning glory seeds or the mushrooms (Valdés et al. 1983). Hofmann found 0.2% psilocybine (dry weight) in cultivated Psilocybe caerulescens Murrill from a strain collected in July 1956 in Huautla de Jiménez (Heim & Hofmann 1958), while Valdés isolated 0.18 % salvinorin A from dried leaves of Salvia divinorum — making the leaves, gram per gram, nearly 10 times as potent as the mushrooms (since salvinorin A is roughly 10 times the potency of psilocybine)! If the Mazatecs have a long familiarity with the leaves, if in reality they have developed a ‘pharmaceutically elegant’ way of processing them for ingestion, then why do they fail to perceive them as being far and away the most potent entheogen available to them?

... I doubt Salvia divinorum was (inadvertently) introduced to Mesoamerica by Europeans, although it is ineluctably associated by the Mazatecs with sheep, which of course were. It seems more likely that the plant was used since pre-Columbian times by another group of Mesoamerican Indians. We have seen that Emboden suggested the ancient Maya knew of Salvia divinorum, and Wasson proposed that the Nahua peoples of central Mexico were familiar with the plant, and used it for its entheogenic properties, under the name pipiltzintzintli. Although of course we cannot prove Wasson’s assertion beyond any doubt, we have seen that Salvia divinorum fits the available, albeit scanty, evidence, and that none of this evidence would preclude the identity of Salvia divinorum and pipiltzintzintli...


Ott's reasons for suspecting that the plant is a post-Conquest introduction to Mazatec culture seem quite well-founded. But we see that much of his argument for its prior use by other Mesoamerican cultures relies perhaps too heavily on the proposed identification of S. divinorum as pipiltzintzintli. I really do not enjoy having to disagree with Ott, but on this point I find that I must disagree with him strongly. I've surveyed the data on pipiltzintzintli very extensively, and it seems that there are very few reasons to accept the identification, and a great many reasons to reject it. A brief summary of my thoughts on the pipiltzintzintli issue can be found in my thread "Notes on the Identity of Pipiltzintzintli."

So prior to the 20th century, that's all we have to go on: a somewhat ambiguous image in an ancient Codex, a contentious argument over the identity of an obscure healing plant, the botanical evidence noted above, and some linguistic and anthropological arguments.

The first recorded account that seems to refer unambiguously to S. divinorum comes from the American anthropologist Jean Bassett. In 1938, he visited the town of Huatla de Jimenéz to observe Mazatec culture. The next year, he published a couple of papers where he notes that there is a leaf, know as Hierba Maria (Johnson 1939):
Jean Bassett Johnson wrote:
In addition to the mushrooms, some people use a seed called "Semilla de la Virgen", others use "Hierba Maria"... The leaf is beaten well, and a tea is made thereof.


We get a similarly sparse comment from the Austrian expatriate and consummate ethnobotanist Blas Pablo Reko (Reko 1945, translated by Valdés 2001):
Blas Pablo Reko wrote:
I cannot leave unmentioned here another magical plant whose leaves produce visions and which the Cuicatecs and Mazatecs (of the districts of Cuicatlán and Teotitlán) call "leaf of prophecy." The loose leaves I have obtained do not allow its scientific identification at the present time.


In 1952, we finally receive a more rigorous account of the visionary sage from the Austrian-born Mexican anthropologist Roberto Weitlaner. He describes its preparation and use:
Quote:
Asking Don S. about the mushroom Teonanacatl, which is found in Huautla de Jiménez, he said it wasn’t used in Jalapa, but he mentioned another plant that was called Yerba de María.

The plant somewhat resembles yerba mora [Solanum nigrum] but its leaves are a little wider; only the leaves are used, and they are put in water. First they are rubbed (crushed) in the hands, the water is not boiled, and they are used for very specific means...

For example, if someone is ill, and the doctors don’t know the disease, then with this herb they can divine the illness. The curandero who brings the leaves first asks the sick person if they are addicted to alcohol, for a person who doesn’t drink is prescribed fifty leaves, but one who does is prescribed one hundred. The ill one drinks the water in which the leaves have been squeezed; at midnight the curandero goes with them and another person to a place where there is no noise, as for example, a house where the sick person drinks the potion. They wait a quarter of an hour for the effects of the drug, and the sick person begins to describe the type of illness they are suffering from. The sick one finds themselves in a semi-delirious state... At daybreak the curandero bathes the sick person with the same water that they took, and with this they are cured. (Weitlander 1952, Valdés 2001)


A few years later, a botanist named Arturo Gómez Pompa also learned of the plant while visiting the Sierra Mazateca in 1957. He noted that the Mazatecs called it xka Pastora and reported it to be hallucinogenic. Unfortunately he found no flowering specimens during his visit, and so was unable to classify the species botanically, aside from identifying it as a member of the genus Salvia, the sages. It would be five more years before material sufficient for botanical identification would be collected.

The next major player in the introduction of Salvia divinorum to Western consciousness is R. Gordon Wasson, the American banker-cum-ethnomycologist who introduced psilocybin-containing mushrooms to popular awareness. After reading R.E. Schultes' early papers on teonanácatl, he immediately set about finding guidance in traveling to Mexico to observe the Mazatec use of the mushrooms. Schultes referred Wasson to Reko, who in turn introduced him to Weitlaner, and in the town of Huatla de Jimenéz, the same town where Johnson first observed the use of "Hierba Maria," Wasson first ingests the mushrooms under the guidance of Doña Maria Sabina. Here he also learned of the psychoactive sage, though he had little time to seek further information on it, being consumed with his mycological efforts. (Wasson 1962)

By 1960, he had reached a point in his mushroom research where he had time to turn some of his attention to the psychoactive sage that he'd learned of several years prior. He began making collections of the plant, which he sent to Schultes and to Carl Epling, the preeminent authority on New World sages for identification. Unfortunately he was unable to find any of the hidden gardens of the plant, and so was at the mercy of the Mazatecs to bring him specimens. In all cases, the plants were either not flowering, or were otherwise unsuitable for definitive botanical identification.

In 1962, Wasson invites Albert Hofmann and his wife Anita to join the annual expedition to Mexico. Hofmann has identified psilocybin from the mushrooms, and has prepared a large number of capsules of synthetic psilocybin to present as a gift to María Sabina. In the end, this expedition turns out to be largely successful; they purchase some whole flowering plant material which is pressed and sent off to Carl Epling, as well as obtaining a sample of the leaf juice, preserved in alcohol, for Hofmann to experiment with (Hofmann 1990). By the end of the year, Wasson and Epling have published papers side-by-side in Harvard University's Botanical Museum Leaflets announcing the discovery of a new psychoactive plant (Wasson 1962) and describing it botanically as Salvia divinorum (Epling & Játiva-M 1962).

By a strange bit of coincidence, an American psychiatrist named Sterling Bunnell was visiting Mexico at virtually the same time as Wasson and Hofmann. While his main purpose was to collect cultures of psilocybian mushrooms, he also happened to obtain a live S. divinorum plant which he brought home and grew in his garden in California. When he read Epling and Wasson's articles the following year, he donated a cutting to the botanical gardens where Epling worked, and also began disseminating the plant among the "psychedelic" subculture. One notable person to receive one of his plants was none other than Dr. Alexander Shulgin. (Valdés 2001)

Ultimately Hofmann's efforts to characterize the active principle of the leaves were fruitless, and he was unable to achieve any discernible psychoactive effects from the leaf juice that he brought back (Hofmann 1964; Hofmann 1990). Shulgin's investigations were similarly fruitless. He noted only possible transitory effects from eating the leaves, and from an extract of the leaves which he named "salvose" he experienced no effects at all (Shulgin 2007; a full transcription of Shulgin's notes on his experiments with S. divinorum can be found in the thread Shulgin and Salvia). We may assume that both of these intrepid chemists failed on account of two significant factors. First, they presumably expected that the active constituent would be an alkaloid, but the plant is in fact apparently devoid of alkaloids. Second, they presumably expected it to be active by oral ingestion, while in reality it appears that the compound is primarily absorbed through the oral mucosa, with very little effect coming from absorption in the stomach.

From here, the history of the plant begins to meander. It stays low-key for many years, and many people come to doubt that the plant is even active at all. While some investigations continue to occur, and salvinorin A is in fact isolated in 1982, a significant wave of interest in the plant doesn't emerge until the mid-1990s. A full three decades after the plant is botanically identified, Siebert publishes his landmark paper establishing salvinorin A as the primary active compound in the leaves, and demonstrating that the plant is vastly more active when taken buccally rather than orally (Siebert 1994).




Citations
  • Emboden, W.A. 1983. "The ethnobotany of the Dresden Codex with especial reference to the narcotic Nymphaea ampla. Botanical Museum Leaflets Harvard University 29(2): 87-132. (fulltext of this journal volume from botanicus.org)
  • Epling, C., and C.D. Játiva-M. "A new species of Salvia from Mexico." Botanical Museum Leaflets Harvard University 20(3): 75-76. (fulltext of this journal volume from botanicus.org)
  • Hofmann, A. "Mexicanische Zauberdrogen und ihre Wirkstoffe." Planta Medica, 12(3): 341-352.
  • Hofmann, A. "Ride through the Sierra Mazateca in search of the magic plant 'Ska María Pastora'." In: Reidlinger, T.J. (Ed.) The Sacred Mushroom Seeker: Essays for R. Gordon Wasson. Dioscorides Press, Portland, OR. pp. 115-127. Originally published in: Hofmann, A. LSD: My Problem Child. Translated by Jonathan Ott (1980). McGraw-Hill, New York, NY. pp. 127-144.
  • Johnson, J.B. 1939. "The elements of Mazatec witchcraft." Góteborgs Etnografiska Museum Etnologiska Studier 9: 119-149.
  • Reko, B.P. 1945. "Mitobotánica Zapoteca." Privately published, Tacubaya, México, D.F.
  • Ott, J. 1995. "Ethnopharmacognosy and human pharmacology of Salvia divinorum and salvinorin A." Curare 18(1): 103-129.(fulltext from sagewisdom.org)
  • Shulgin, A. 2007. "Pharmacology Notes I (April 1960 – December 1976)." Unpublished notebook. Last updated 15 Dec 2007. Accessed 4 Mar 2008 from http://www.erowid.org/li...logynotes01shulgin.pdf. See also Shulgin and Salvia.
  • Siebert, D.J. 1994. "Salvia divinorum and salvinorin A: new pharmacologic findings." Journal of Ethnopharmacology 43(1): 53-56. (fulltext from sagewisdom.org)
  • Valdés, L.J., III. "The early history of Salvia divinorum." Entheogen Review 10(2): 73-75.
  • Wasson, R.G. "A new Mexican psychotropic drug from the mint family." Botanical Museum Leaflets Harvard University 20(3): 77-84. (fulltext of this journal volume from botanicus.org)


Threads referenced
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Entropymancer
#9 Posted : 10/11/2011 8:08:41 PM

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As one of our members was kind enough to provide me with a number of theses and dissertations dealing with S. divinorum, I'll be posting my notes on them in thread. Just finished the first one Smile


Willard, M.A.B. 2011. "Forensic analysis of Salvia divinorum and related Salvia species using chemometric procedures." Masters thesis, Criminal Justice: Michigan State University.

Willard developed a means of differentiating between S. divinorum and other Salvia species by applying principal component analysis (a multivariate statistical procedure, eliminating the subjectivity of simple visual analysis) to GC-MS data. In addition to differentiating for the presence of salvinorin A, this technique is able to differentiate S. divinorum from other plant materials spiked with salvinorin A.

The Salvia genus is in fact polyphyletic [that is, not arising from a single common ancestor] (one clade being predominantly Old World, another being exclusively New World, and a third of Asiatic origin). S. divinorum belongs to the second clade (exclusively New World). [This information is credited to Walker et al. 2004]

“As of 2011, 26 individual states have regulated either the plant or salvinorin A (shown in Table 1.1) and several others have pending legislation… in South Dakota, possession of less than two ounces of either S. divinorum or salvinorin A is a misdemeanor and possession of greater than two ounces is a felony. In Tennessee, possession of salvinorin A is a misdemeanor, whereas in Wisconsin possession is not regulated, but manufacturing and sale are illegal.” [I’ve only quoted specific information in regards to states where the table does not adequately represent the situation]



“States in which only S. divinorum is regulated, such as Delaware and Mississippi, may have problems in the forensic identification of the controlled substance. Identification may be challenging if salvinorin A was extracted from S. divinorum and then spiked onto a non-regulated plant material.” [A significant loophole for vendors to slip through]

Methods previously reported for extraction of salvinorin A are summarized in Table 1.2:


[19]=Valdés et al. 1984 ; [20]=Siebert 2004 ; [21]=Medana et al. 2006 ; [22]=Giroud et al. 2000


Pigments are indicated as potential factors in the degradation of salvinorin A in solution (vide Tsujikawa et al. 2008; specifically in solution in acetonitrile).

Methanol extracts are undesirable for forensic analysis as methanol could potentially cleave ester groups from salvinorin A or siloxane groups from the stationary phase of GC-MS columns.

Soxhlet extraction of S. divinorum with acetone, methanol, DCM, and hexane (at their respective boiling points) for 24 hours resulted in no salvinorin A detected in any sample. This was presumed to be attributable to salvinorin A degrading under the application of prolonged heat.

Extracted by agitating for 24 hours at room temperature in acetone, methanol, DCM, and hexane. DCM pulled the greatest quantity of salvinorin A, followed by methanol and acetone (hexane pulled negligible quantities). Methanol pulled by far the largest quantity of other (undesired) phytochemicals.

Simple one minute extractions at room temperature were performed with the abovementioned solvents, as well as chloroform and acetonitrile. DCM pulled the greatest quantity of salvinorin A, followed by acetone and chloroform. Acetone was the most selective (that is, contained the greatest quantity of salvinorin A per gram of total extract). Acetone and DCM extracted the fewest number of compounds, primarily salvinorins A–D. When stored in solution at 4°C for 24 hours, salvinorin A was most stable in DCM, and least stable in acetone. DCM was concluded to be the best solvent for forensic analysis purposes. Acetone appears to be the next most suitable, though analysis of acetone extracts should be done immediately on account of the apparent instability of salvinorin A stored in this solvent.

[Takeaway notes for people extracting salvinorin A at home: Acetone is likely still the best solvent choice since DCM is carcinogenic. Short extraction times and rapid removal of solvent are likely crucial to optimize yield. Excess heat is probably undesirable, so rapid evaporation would be better encouraged by large surface area and use of a fan with a spark-free motor; care must still be taken to avoid igniting the flammable fumes of acetone.]

Various extraction times were explored with DCM. Quantity of salvinorin A extracted reached a maximum between three and six minutes (total extracted quantity of salvinorin A was actually smaller with the longer extraction times). With a 5 minute extraction, 97.6% of the total salvinorin A was extracted from the sample. This five minute extraction in DCM at ambient temperature is recommended as standard practice in forensic analysis of materials suspected to contain salvinorin A.



There are a few errors in the paper: She claims (without supporting evidence) that the plant has been used by the Mazatecs for centuries, citing a source (Valdés et al. 1983) that makes no such claim. Valdés has generally argued against assigning a long history of use to the plant. She also states that the leaves were traditionally brewed into a tea or infusion, when in fact infusions would be entirely ineffective; emulsifying the material is crucial to aqueous preparations.

Those points notwithstanding, I have to give credit where credit is due. In discussing the dubious reports of psychoactivity from S. splendens, she cites a thread on the Corroboree. Also, to simulate inert plant material spiked with S. divinorum extract, she followed an extraction procedure from erowid for producing fortified leaf.



References cited
  • Giroud, C., F. Felber, M. Augsburger, B. Horisberger, L. Rivier, and P. Mangin. 2000. "Salvia divinorum: an hallucinogenic mint which might become a new recreational drug in Switzerland." Forensic Science International 112: 143-150.
  • Medana, C., C. Massolino, M. Pazzi, and C. Baiocchi. 2006. "Determination of salvinorins and divinatorins in Salvia divinorum leaves by liquid chromatography/multistage mass spectrometry." Rapid Communications in Mass Spectrometry 20(2): 131-136.
  • Siebert, D.J. 2004. "Localization of salvinorin A and related compounds in glandular trichomes of the psychoactive sage, Salvia divinorum." Annals of Botany 93(6): 763-771.
  • Tsujikawa, K., K. Kuwayama, H. Miyaguchi, T. Kanamori, Y.T. Iwata, T. Yoshida, and H. Inoue. 2008. "Determination of salvinorin A and salvinorin B in Salvia divinorum-related products circulated in Japan." Forensic Science International 180: 105-109.
  • Valdés III, L.J., J.L. Díaz, and A.G. Paul. 1983. "Ethnopharmacology of Ska María Pastora (Salvia divinorum, Epling and Játiva-M.)." Journal of Ethnopharmacology 7(3): 287-312.
  • Valdes III, L.J., W.M. Butler, G.M. Hatfield, A.G. Paul, and M. Koreeda. 1984. "Divinorin A, a psychotropic terpenoid, and divinorin B from the hallucinogenic Mexican mint, Salvia divinorum." Journal of Organic Chemistry 49(24): 4716-4720.
  • Walker, J., K. Sytsma, J. Treutlein, and M. Wink. 2004. "Salvia (Lamiaceae) is not monophyletic: implications for the systematics, radiation, and ecological specializations of Salvia and tribe Mentheae." American Journal of Botany 91(7): 1115-1125.
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gibran2
#10 Posted : 10/11/2011 10:12:12 PM

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Thanks for all the information.

I wonder if experimenters are taking into account degradation due to UV light? I think Siebert pointed out that salvinorin A when in solution is very sensitive to UV light. I wonder if the acetone degradation results were due to UV?

Also, looking at the lists of legal status, under Wisconsin it says misdemeanor/felony. This is only for manufacturing/selling. It is not illegal to possess (even for minors!). One of the strangest laws that a State has passed.
gibran2 is a fictional character. Any resemblance to anyone living or dead is purely coincidental.
 
Entropymancer
#11 Posted : 10/11/2011 10:55:24 PM

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gibran2 wrote:
I wonder if experimenters are taking into account degradation due to UV light? I think Siebert pointed out that salvinorin A when in solution is very sensitive to UV light. I wonder if the acetone degradation results were due to UV?


Hmmm... it's possible, but only if the UV-degradation is just specific to particular solvents. In Tsujikawa et al. 2008 (referenced in post #8), they left two extracts in acetonitrile for 24 hours at 4°C without any light shielding. One was a crude extract, the other had been cleaned of pigments (by treating with graphite carbon powder). After 24 hours, there was significant degradation of salvinorin A in the crude extract, but virtually no degradation in the cleaned extract. This leads me to suspect that the particular pigments extracted by the acetone are the primary culprit.
 
Rising Spirit
#12 Posted : 10/12/2011 6:03:21 AM

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I am curious if Salvia Venulosa contains the Salvinorin A molecule. Does anyone know if it has psychedelic properties? Wikipedia neglected to say or not... but apparently there is some debate if there are terpenoids present in this plant.
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Entropymancer
#13 Posted : 10/12/2011 6:10:56 AM

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Nope, S. venulosa doesn't contain salvinorin A (Jenks cites a personal communication with Siebert on this point). Chemical studies are apparently underway to characterize any terpenoids that may be present in the species. And Snu doesn't mention S. venulosa in his Garden of Eden, which probably means there isn't any recorded tradition of it being used for psychoactive effects. Still, it might be worth a bioassay if one has this plant handy.
 
Entropymancer
#14 Posted : 10/17/2011 10:35:53 PM

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Time for another installment!

Addy, P.H. 2010. That deep internal voice: controlled administration of Salvia divinorum. Ph.D. Dissertation, Clinical Psychology: Institute of Transpersonal Psychology.


Fast summary: “Pulse rate dropped 5.3 bpm between time measurements. Participants talked, laughed, and moved more often on an active dose. All 6 HRS subscales were significantly elevated on an active dose. No sex differences were noted. Participant experiences displayed 10 common themes presented in depth through qualitative analysis, profiling the phenomenology of this state, including cognitive alteration, synesthesia, and immersion in another reality.”

After eight weeks, researchers followed up to assess whether any participants met the DSM criteria for abuse of or dependence on S. divinorum. [I understand they did this to establish that data point in the literature, but it still makes me chuckle]

“Curiously, salvinorin A may cause “depressive-like symptoms” in rodents (Carlezon et al., 2006), yet several humans have reported antidepressant effects (Hanes, 2001, 2003).” [Not curious at all. Carlezon et al. used a large dose; when Braida et al. (2009) administered smaller doses, they observed antidepressant and anxiolytic effects. It would not be surprising if the depressant/antidepressant effects are dose-dependent, much like the sedative/stimulant effects are dose-dependent (as in Braida et al. 2007)]

Study was not subject to FDA regulation, as it was intended to investigate possible effects of salvinorin A on “general wellbeing” (classifying the material as a dietary supplement) rather than to prevent, diagnose, cure, mitigate, or treat a disease (intentions which would classify the substance as a “drug” subject to FDA regulation). The DEA was similarly uninvolved as there are no federal laws prohibiting salvinorin A or S. divinorum. The study was instead guided only by ethical and safety standards.

Study involved 14 women and 16 men, aged 25–49 (mean=39). “One male participant refused to give his birth date, saying he did not believe in age.” Well-educated: 23% (=7) had some college but no degree, 43% (=13) had a bachelors degree, 30% (=9) had a masters degree, 3% (=1) had a doctoral degree.

“This means that systolic BP measured after the session was lower with a placebo dose, diastolic BP was lower after a session, and diastolic BP measured after the session was lower in women.” [Though in each case, the data fell shy of statistical significance] Pulse rate was lower after a session (by a statistically significant margin with placebo sessions, and just shy of statistically significant decrease with an active dose).

Addy examined the subjective effects described in post-session interviews for common themes (pp 57–91). In the text, he has liberally interspersed quotations from the interviews to illustrate the phenomena in question. Below is a summary of the noted phenomena and frequency with which they were experienced.
  • 87% described feeling a “rush” (defined as a rapid shift from normal to altered). 40% experienced physical sensations accompanying the rush. Only 20% described an extreme sudden onset (like flipping a switch).
  • 27% described awareness of a threshold between consensus reality and another reality. 7% described feeling this threshold in the body.
  • 90% were at times aware of their own cognitive processes (“awareness of their awareness”). 27% described “resisting the altered state” and “holding onto their ego,” while 17% described surrendering to it. 20% felt “out of control” during the experience.
  • 37% described spending time dwelling on their perception of time. This included the perceptions that time had ceased to exist, or that it was being borrowed into a limited existence. Multiple descriptions of subjective timelessness and uncertainty about duration of the experience.
  • 13% described feeling dead or dying.
  • 87% experienced physical sensations. For 27%, this included altered tactile sense or the perception of being touched. 20% described their body being pulled by a force. 20% described their bodies twisting or spinning. 13% described tingling sensations. 10% felt paralyzed for part of the experience.
  • 23% described becoming cold during the experience; 23% described becoming hot during the experience. There was at least some overlap in these two groups (the extent of overlap was not commented on, but one subject described “cycling in waves: hot sweaty to cold”).
  • 13% talked about sweating during the experience, and 10% talked about having clammy hands.
  • 83% experienced closed-eye visual imagery. 77% described movement within the visions. 20% described the visions as kaleidoscopic. 13% described the images as swirling. 10% described the imagery as beautiful.
  • 80% were immersed in another reality. 30% were fully immersed and had no awareness of the external consensus reality. 37% sensed other people or beings in the experience. 17% described carnival scenes. 9% described rainforest scenes. 13% mentioned water in this other reality. 9% felt the other reality to be “more real” than consensus reality. 9% described the other reality as familiar. 9% described the other reality as ineffable.
  • 60% felt disoriented during the experience. 30% attributed this to the abruptness and brief duration of the experience. 20% felt disoriented due to feeling disconnected from their bodies.
  • 57% described some degree of synaesthesia, usually between visual imagery and kinesthetic, proprioceptive, or tactile senses.
  • 10% described merging with inanimate objects.
  • 50% talked about exploring the altered state, including commenting on the potential for insight or learning. 17% felt they’d gained something beneficial from the experience.
  • 50% described aspects of the experience as unique, not comparable to other psychedelics they had used in the past. 77% compared aspects of the experience to other altered states: 43% compared it to dreaming, 13% compared it to LSD, 10% compared it to psilocybian mushrooms, 10% compared it to cannabis, and 10% compared it to MDMA.
  • 87% experienced after-effects lasting less than 24 hours. 22% felt reflective, 22% felt emotionally sensitive, 13% felt fatigued, 13% had difficulty concentrating, 9% felt more intuitive, 9% had feelings of “floating or lightness,” 9% felt more keenly aware of beauty. 17% felt the after-effects as positive. 13% experienced a headache (although the researcher told the subjects this might happen, so there could be bias in that data).
  • 70% reported after-effects lasting more than 24 hours. 17% reported positive changes in themselves, 22% reported positive changes in family relationships, 9% reported deepening relationships with others beyond family, 13% reported increased empathy, 22% described positively integrating the experience. 13% described longer-lasting negative effects (headache persisting for three days, sense of dislocation lasting a few days)
  • 87% expressed interest in using S. divinorum again; the remainder were uncertain. 26% liked the effects of the plant, noting “an increase in creativity, self-awareness, and positive transformation.” Only 9% described the experience as fun and enjoyable.
  • The data collected in this study using Strassman’s Hallucinogen Rating Scale (HRS) indicates that 1000 μg salvinorin A vaporized was the second most intense material examined so far examined with the HRS (the most intense being 0.4 mg/kg i.v. DMT). It also had among the highest volition scores (most of those higher involved administration of ketamine). The scores on the other subscales fell roughly in the middle of the field of previously reported data. The scores were generally consistent with those obtained by Johnson et al. 2010 with a comparable dose, with the only substantial discrepancy being on the Perception subscale (1.71 from Addy, 0.95 from Johnson et al.).
  • Notably absent from the perceived effects was the visitation of places from one’s past, particularly from childhood (reported by Siebert to be a common trope).


Addy notes interesting similarities between the subjective effects of salvinorin A and other KOR agonists in humans, quoting Pfeiffer et al. 1986: “The high dose [3.8 mg/kg i.v.] resulted in somesthetic changes and disturbances in the perception of space and time. Abnormal visual experiences reported by most subjects consisted of moving lines or walls or color phenomena. There were symptoms of depersonalization, derealization, and loss of self-control. For example, one subject had frequent episodes of unmotivated and uncontrolled laughter during a 90-minute period. Two subjects became unaware of the experimental situation for periods of 20 to 30 minutes and later described their experiences as dreamlike. Although pseudohallucinations were reported, true hallucinations did not seem to occur. (Pfeiffer et al., 1986, p. 774)”



If anyone wants full citations on the sources referenced here, just let me know and I'll add them.
 
Entropymancer
#15 Posted : 10/21/2011 6:36:43 AM

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Gruber, John W. 1997. Quantification of salvinorin A from tissues of Salvia divinorum (Epling & Játiva-M.). Thesis, Master of Science: Philadelphia College of Pharmacy and Science.

Gruber developed a reversed-phase, isocratic HPLC method to detect and quantify salvinorin A from plant tissues. Salvinorin A is separated from other plant components on a C-18 column using 45% acetonitrile / 55% water as the mobile phase. The compound is detected and quantified based on UV absorbance at 208 nm.

In tested samples (quantity=20), salvinorin A content ranged from 0.89–3.70 mg/g, mean 2.45 mg/g.

He notes an interesting compound with a structure very similar to salvinorin A from a totally unrelated species:

(This compound is 3,4-didehydro-1-deoxo-salvinorin B)

In the subgenus Calosphace, 80% of studied species [as of 1995] contain neoclerodane diterpenoids.

Tried to establish callus tissue cultures without success. Living root cultures were established, but showed very little growth under any tested conditions. Also attempted to produce hairy root cultures by introducing Agrobacterium rhizogenes, also without much success.

Gruber described three types of trichomes occurring on S. divinorum, including the glandular trichome in which the neoclerodane diterpenoids are excreted (though he did not recognize them as having that function). More complete description of the trichomes can be found in Siebert 2004.

Salvinorin spots can be visualized on TLC plates by spraying the plates with 10% phosphomolybdic acid in 2-propanol followed by heating (Ortega et al. 1982), or by spraying with vanillin-sulfuric acid or anisaldehyde-sulfuric acid reagents followed by heating (citing personal communication with Siebert). For Gruber’s TLC analysis, he ran samples on silica plates using 1:1 hexane:EtOAc as the eluent, and visualized the spots by heating at 100 °C for 10 minutes after treating with spray reagent (either vanillin- or anisaldehyde- sulfuric acid). Observed retention factor (Rf) for pure salvinorin A ranged from 0.45–0.53.

λmax for salvinorin A in 45% acetonitrile / 55% water was 207.6 nm with molar absorptivity ε = 4,960 L mol-1 cm-1. Melting point of 234–235 °C is consistent with previously reported values.

Experiments on optimizing extraction: Partitioning between hexane and 90% methanol (as reported by Valdés et al. 1984) is only practical with large quantities of leaf; with smaller amounts, a greater portion of salvinorin is stuck in the hexanes (presumably this is related to the quantity needed to saturate hexanes versus the total quantity being purified). 100 minute reflux in methanol resulted in much less extracted salvinorin A than other tested methods (refluxing in chloroform, steeping in chloroform, or steeping in methanol). Shorter extraction times (no more than an hour) were found to be optimal, with the quantity of salvinorin A extracted decreases the longer the duration of the extraction. A 30 minute chloroform extraction recovered at least 89% of the total salvinorin A (due to some idiosyncrasies of the method, it’s possible the extraction actually recovered as much as 97% or more).

Salvinorin A content from several S. divinorum specimens:


While none of the other Salvia species tested contained any salvinorin A, S. melissodora had a fraction with a retention time nearly identical to salvinorin A, and this fraction appears to be a terpenoid or steroid based on its reaction with the vanillin-sulfuric acid reagent used to visualize a TLC plate. Gruber suggests it may have a similar structure, and since the species is sacred to the Tarahumara of northern Mexico, Gruber suggests further investigation into the ethnobotany of this species could prove rewarding.
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Entropymancer
#16 Posted : 10/21/2011 7:10:43 AM

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Also, while I'm at it, I'd like to share an interesting slice of history: a talk given on Salvia divinorum at Palenque Norte in 1994. Rob Montgomery does most of the talking, with Jonathan Ott and Terence McKenna interjecting periodically. The sound quality is pretty bad, but it's still an interesting talk, perched on the precipice of salvia's sudden emergence into serious entheogen status.

The talk is available in mp3 format:


Or if YouTube is more your speed:





 
jamie
#17 Posted : 10/21/2011 5:23:59 PM

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Wow..thanks for that entropy! Looks like I need to get some more plants to experiment with. I have been experimenting with salvia again a bit lately after almost not touching it for a few years..the idea of other diterpenes with similar effects is interesting..
 
Entropymancer
#18 Posted : 10/21/2011 7:38:03 PM

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My next post will have my notes on Brian Kane's Ph.D. thesis considering models for salvinorin A binding in opioid receptor. A small warning: This one might be a little dull unless you're really into theoretical opioid binding epitopes. But before I get into the notes, I think it would be worthwhile to give a quick bit of background to the subject.

Kane's dissertation is not the only work to consider the mechanisms for salvinorin A binding to opioid receptors, and a better picture can be gleaned from considering all the research on the topic. The topic was first considered by Roth et al. in the 2002 paper announcing the discovery that salvinorin A is a potent and selective KOR agonist. While Kane was conducting his research, Yan et al. published a paper on similar research they had conducted (Yan would go on to compose his own doctoral dissertation on the subject in 2008), and Munro et al. published a paper approaching the issue based primarily on salvinorin analogue studies. Much of the data from this dissertation was published (in somewhat abbreviated form) in 2006, prior to the submission of the thesis I'll be discussing below. Also in 2006, Singh et al. generated a computer model for binding that produced figures on the correct order of magnitude for a number of salvinorin analogue compounds. Vortherms et al. further considered the issue in 2007, particularly examining the role of the TM II transmembrane helix, and in 2008 Kane et al. revised the binding model they proposed in 2006. In 2009, Yan et al. used the known information about the binding site to design salvinorin analogues that bind irreversibly to the KOR. At present, that's pretty much the state of things. Of course all of the work that has been done developing and testing salvinorin analogues have implications on this subject as well, but since they are largely concerned with elucidating structure-activity relationships rather than binding epitopes per se, I'll leave that topic for another day and simply refer the interested reader to Prisinzano and Rothman's capable summary on the subject from 2008.

With the exception of Kane's thesis and Yan's dissertation, all of the other papers mentioned above are available from Daniel Siebert's Sage Wisdom site. I'll include links with each citation below.


Related Reading
  • Roth B.L., K. Baner, R. Westkaemper, D.J. Siebert, K.C. Rice, S. Steinberg, P. Ernsberger, and R.B. Rothman. 2002. "Salvinorin A: A potent naturally occurring nonnitrogenous κ opioid selective agonist." Proceedings of the National Academy of Sciences of the United States of America 99(18): 11934-11939. [pdf from sagewisdom.org]
  • Munro, T.A., M.A. Rizzacasa, B.L. Roth, B.A. Toth, and F. Yan. 2005. "Studies toward the pharmacophore of salvinorin A, a potent κ opioid receptor agonist." Journal of Medicinal Chemistry 48(2): 345-348. [pdf from sagewisdom.org]
  • Yan, F., P.D. Mosier, R.B. Westkaemper, J. Stewart, J.K. Zjawiony, T.A. Vortherms, D.J. Sheffler, and B.L. Roth. 2005. "Identification of the molecular mechanisms by which the diterpenoid salvinorin A binds to kappa-opioid receptors." Biochemistry 44(24): 8643-8651. [pdf from sagewisdom.org]
  • Kane, B.E. 2006. "Molecular recognition of G-protein coupled receptor ligands: insights into salvinorin A and xanomeline." Ph.D. Thesis: University of Minnesota.
  • Kane, B.E., M.J. Nieto, C.R. McCurdy, and D.M. Ferguson. 2006. "A unique binding epitope for salvinorin A, a non-nitrogenous kappa opioid receptor agonist." The FEBS Journal 273(9): 1966-1974. [pdf from sagewisdom.org]
  • Singh, N., G. Chevé, D.M. Ferguson, and C.R. McCurdy. 2006. "A combined ligand-based and target-based drug design approach for G-protein coupled receptors: application to salvinorin A, a selective kappa opioid receptor agonist." Journal of Computer-Aided Molecular Design 20(7-8): 471-493. [pdf from sagewisdom.org]
  • Vortherms, T.A., P.D. Mosier, R.B. Westkaemper, and B.L. Roth. 2007. "Differential helical orientations among related G protein-coupled receptors provide a novel mechanism for selectivity: Studies with salvinorin A and the κ-opiod receptor." Journal of Biological Chemistry 282(5): 3146-3156. [pdf from sagewisdom.org]
  • Kane, B.E., C.R. McCurdy, and D.M. Ferguson. 2008. "Toward a structure-based model of salvinorin A recognition of the κ-opioid receptor." Journal of Medicinal Chemistry 51(6): 1824-1830. [pdf from sagewisdom.org]
  • Prisinzano, T.E., and R.B. Rothman. 2008. "Salvinorin A analogs as probes in opioid pharmacology." Chemical Reviews 108(5): 1732-1743. [pdf from sagewisdom.org]
  • Yan, F. 2008. "Molecular mechanisms by which salvinorin A binds to and activates the kappa-opioid receptor." Ph.D. dissertation, Biochemistry: Case Western Reserve University.
  • Yan, F., R.V. Bikbulatov, V. Mocanu, N. Dicheva, C.E. Parker, W.C. Wetsel, P.D. Mosier, R.B. Westkaemper, J.A. Allen, J.K. Zjawiony, and B.L. Roth. 2009. "Structure-based design, synthesis, and biochemical and pharmacological characterization of novel salvinorin A analogues as active state probes of the κ-opioid receptor." Biochemistry 48(29): 6898-6908. [pdf from sagewisdom.org]
 
Entropymancer
#19 Posted : 10/21/2011 7:40:09 PM

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Kane, Brian Edmund. 2006. “Molecular recognition of G-protein coupled receptor ligands: insights into salvinorin A and xanomeline.” Ph.D. thesis: University of Minnesota.

Only the first half of the paper is of significance to S. divinorum; the latter half is concerned with a binding model for the muscarinic acetylcholine receptors. The first twenty pages are concerned with describing the binding models currently established for a variety of traditional opioid agonists. The portions concerned exclusively with salvinorin A and its analogues are pages 22-70.

A previous model proposed by Roth indicates the Gln115 residue is involved in recognition of the furan ring; this was supported by reduced affinity observed when Kane replaced this residue with alanine.

Tyr312 was also implicated in salvinorin A binding by a previous model. Replacing it with alanine reduced affinity, supporting that implication. Replacing it with phenylalanine did not substantially alter affinity, indicating the tyrosine does not act as an H-bond donor in binding (presumably it stabilizes a hydrophobic region instead).

Glu297 plays a large role in selectivity for typical (non-neoclerodane) κ-opioid agonist binding through ionic interaction. Since salvinorin A has no labile protons, it’s not surprising that mutating this residue had no siginificant effect on binding. The κ-selectivity of salvinorin A must come from different interactions in the receptor.

A δ-opioid receptor (DOR) with the Trp284 residue replaced with a glutamate results in dramatically increased affinity for the KOR agonist gNTI. Surprisingly, this same mutation increased the affinity of salvinorin A for the DOR. Since salvinorin A would not have any ionic interactions with glutamate, Kane suggests the bulk of the tryptophan residue ordinarily prevents salvinorin A from entering the trans-membrane helix region.

Mutants in the TM VI and TM VII transmembrane helices did not tend to significantly alter the affinity of salvinorin A for the κ-opioid receptor (KOR), indicating that it does not bind in the typical opioid binding pocket. Kane notes that the lack of effect of these mutations might be “false negatives,” since salvinorin A is fairly hydrophobic and nearby residues could compensate for any single mutation, but considering the number of residues that were mutated without significant effect Kane finds it likely that these were not false negatives.

More site-directed mutagenesis studies indicate that residues in the TM I region (adjacent to TM II and TM VII) do not appear to interact directly with salvinorin A.

Based on studies with chimeric receptors (mixing parts of the KOR sequence with parts of the DOR or MOR sequences), it appears that the KOR TM II helix is crucial for binding salvinorin A. Interestingly, the affinity increased substantially when the TM V, TM VI, and TM VII regions were replaced with the DOR sequences for those regions (while affinity for the analogous KOR/MOR chimera was entirely abolished). Kane suggests that part of the MOR sequence excludes salvinorin A from entering the binding pocket (rather than the residues within the binding pocket interacting unfavorably) since salvinorin A analogues with good affinity for the MOR have been found (e.g. herkinorin).

Chimeric data further suggests significant involvement of the EL-2 extracellular loop portion of the receptor.

Overall, Kane finds that the data suggests that salvinorin A is oriented vertically in the receptor between TM II and TM VII. This places the 2-position at the TM VII/EL-3 interface “which is highly variable in sequence across the opioid receptors.” This is consistent with the fact that modifications of salvinorin A at the 2-position can alter receptor subtype affinity (e.g. herkinorin, the benzoyl ester of salvinorin B, has much greater μ-affinity and much lower κ-affinity). This hypothesis is also supported by mutational and chimeric data examining the herkinorin binding.
 
Entropymancer
#20 Posted : 10/23/2011 6:27:14 AM

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In keeping with the theme of the previous two posts, I thought it appropriate to read and take notes on Yan's dissertation next.


Yan, Feng. 2008. “Molecular mechanisms by which salvinorin A binds to and activates the kappa-opioid receptor.” Ph.D. dissertation, Biochemistry: Case Western University.

The paper starts with a very capable summary of the current state of biochemical knowledge about G-protein coupled receptors (GPCRs), and opioid receptors in particular. Very good background reading for anyone wanting a technical introduction to the topic.

Summary of previous (and unpublished) mutagenesis work:

(2) is Yan et al. 2005; (7) is Yan et al. 2008. Ki values for some of these mutants are given in the paper as well.


Tyr313 appears to be a crucial residue for hydrophobic interactions (mutating to Ala dramatically decreased affinity while mutating to Phe did not, indicating no significant hydrogen-bonding involvement).

Tyr119 and Tyr320 appear to be important for H-bonding (significant affinity lost on mutation to Phe; not much additional affinity was lost on mutation to Ala indicating little significant hydrophobic effects).

Interestingly, mutating Tyr139 or Tyr312 to Ala had little effect on the binding of salvinorin A, but significantly decreased its efficacy for activating the KOR.

To elucidate the position of salvinorin A in its bound position, several residues were mutated to Cys (accompanied by a mutation of Cys315 to serine to eliminate interference with the data… this mutation did not significantly alter affinity for salvinorin A) and the affinity of these mutants for the 2-thio-salvinorin B was characterized (presuming that a cysteine in the vicinity of the thiol group would significantly increase affinity). Mutating Tyr313 to Cys preserved affinity for 2-thio-salvinorin B but reduced affinity for salvinorin A, as predicted based on current binding models. Interestingly mutating Ile294 or Glu297 to Cys increased affinity for both salvinorin A and 2-thio-salvinorin B. The increase in affinity for the I294C mutant is accounted for by steric considerations in the binding model presented below.

In the extracellular loop EL2, replacing Leu212 or Phe214 with Ala improved affinity for salvinorin A; since these residues appear to protrude into the binding pocket, it’s possible that the bulky groups present a steric hindrance. Replacing Gln115 with Ala dramatically decreased affinity; Yan suggests Gln115 may H-bond with the furan ring.

Based on molecular modeling, salvinorin A appears to have significant interactions with TM1-3, TM6, TM7, and EL2, with the 2-acetoxy moiety positioned between EL2 and the top of TM7 (adjacent to Tyr313). The furan ring H-bonds between Tyr119 (TM2) and Tyr320 (TM7). The 4-methyl ester is oriented toward the top of TM6. The proposed binding mode is shown below:


Yan summarizes the previous work on structure-activity relationships in salvinorin analogues:
“The results suggest that the acetoxy group on C-2 position, methyl ester on C-4 and furan ring on C-12 are required for salvinorin A binding, while the lactone (C-17) and ketone (C-1) functionalities are not… Extending the alkyl chain and adding bulky aromatic groups to C-2 were found to diminish, if not totally abolish, the affinity for KOR. The acetoxy group is therefore the optimal alkyl chain length… Replacement of the oxygen with sulfur on C-2 produced the most similar analog of salvinorin A. The C-2 thioacetate isostere indeed produced comparable activity to salvinorin A, but nitrogen substitution had a diminishing effect. Intermediates, which lack a β–carbonyl at C-2 displayed moderate affinity. The difference in affinity of sulfur and nitrogen may be attributed to the presence of two lone pairs of electrons, just like salvinorin A itself, in thioacetate which may play a role in receptor recognition. A hydrogen bond acceptor at C-2 may facilitate high affinity binding, but is not critical in receptor recognition when considering the affinities of intermediates. In fact, a C-2 methoxymethyl ether analog of salvinorin A was reported to have a 7-fold increase in potency for KOR using [3H]diprenorphine as the radioligand.”

Yan reports data on the analogues tested in the lab of Bryan Roth (the majority of these data can be found in the review paper by Prisinzano and Rothman referred to in post #18).


The next section of the dissertation (modified from Yan et al. 2008) deals with conformational changes of the KOR due to G protein coupling. It’s thought that coupling with different G proteins can lead to different conformations of GPCRs. If ligands bind preferentially to certain conformations, this could be a mechanism to explain how different ligands for a particular GCPR seem to differentially modulate signaling pathways. They found that overexpression of different G proteins does in fact lead to conformational changes (particularly in TM7 and the extracellular portions of TM6 and EL2), and that these changes affect ligand binding. The affinity for salvinorin A was affected to a much larger extent than U69593 or dynorphin A.


The final section of the paper talks about the development of salvinorin A derivatives capable of covalently binding the KOR. These were 22-isothiocyanatosalvinorin A (RB-64) and 22-chlorosalvinorin A (RB-48). These ligands have the potential to help further characterize the active state of the KOR. The results of this study are reported in somewhat more detail in Yan et al. 2009.



Papers referred to in this post
  • Yan, F., P.D. Mosier, R.B. Westkaemper, J. Stewart, J.K. Zjawiony, T.A. Vortherms, D.J. Sheffler, and B.L. Roth. 2005. "Identification of the molecular mechanisms by which the diterpenoid salvinorin A binds to kappa-opioid receptors." Biochemistry 44(24): 8643-8651. [pdf from sagewisdom.org]
  • Yan, F., P.D. Mosier, R.B. Westkaemper, B.L. Roth.. 2008. "Galpha-subunits differentially alter the conformation and agonist affinity of kappa-opioid receptors." Biochemistry 47(6): 1567-1578.
  • Yan, F., R.V. Bikbulatov, V. Mocanu, N. Dicheva, C.E. Parker, W.C. Wetsel, P.D. Mosier, R.B. Westkaemper, J.A. Allen, J.K. Zjawiony, and B.L. Roth. 2009. "Structure-based design, synthesis, and biochemical and pharmacological characterization of novel salvinorin A analogues as active state probes of the κ-opioid receptor." Biochemistry 48(29): 6898-6908. [pdf from sagewisdom.org]
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