PiHKAL #133 (http://www.erowid.org/library/books_online/pihkal/pihkal133.shtml)
(http://www.erowid.org/library/books_online/pihkal/pihkal133.shtml)) are the end products I had in mind.10.1016/0031-9422(79)83072-1 (http://dx.doi.org/10.1016/0031%2D9422%2879%2983072%2D1)
Rh's site (https://www.thevespiary.org/rhodium/Rhodium/chemistry/benzaldehydes.kmno4.html)
(https://www.thevespiary.org/rhodium/Rhodium/chemistry/benzaldehydes.kmno4.html), or you can take a look at the original article: S Lai et al. Synthesis (2001) 1644, DOI:10.1055/s-2001-16760 (http://dx.doi.org/10.1055/s%2D2001%2D16760)
.http://www.herbmed.org/viewherb.asp?varHerb_ID=14 (http://www.herbmed.org/viewherb.asp?varHerb_ID=14)
http://public.srce.hr/ccacaa/CCA-PDF/cca1999/v72-n4/CCA_72_1999_827-834_STANIC.pdf (http://public.srce.hr/ccacaa/CCA-PDF/cca1999/v72-n4/CCA_72_1999_827-834_STANIC.pdf)
It is obvious that the highest content of esculin and fraxin was found in the oldest bark samples (branch diameter of 5 cm) during the whole growing period. The values differed from 3.61% in autumn to 5.96% in summer for
esculin and from 1.53% to 2.62% for fraxin. In the same period, the coumarin content in the youngest bark (branch diameter of 1 cm) varied in a range from 1.06% to 3.01% for esculin and from 0.45% to 1.04% for fraxin.
As regards the quantities of esculin and fraxin throughout the seasons, it was found that all summer samples were the richest plant material (Figure 4). In contrast, the samples from autumn or spring contained the lowest amount of these substances. It is also evident that the ratio of fraxin and esculin varied from about 1:2 to 1:3 in all samples analyzed.
Also, no severe damage to the trees is done, as the article states that the external bark layer (the cork) contains much more aesculin than the internal one. ;)
One could perhaps extract the dried, ground bark with alcohol, evaporate this to dryness, wash residue with petroleum ether, add acidified water, remove the coloring with a little chloroform, and extract the glucoside fraction with ether.
This is how the glucoside hydrangin is extracted from the shrub Hydrangea arborescens L., as described in the American Journal of Pharmacy, Vol. 59 (1887):
http://www.ibiblio.org/herbmed/eclectic/journals/ajp1887/03-hydrangea.html (http://www.ibiblio.org/herbmed/eclectic/journals/ajp1887/03-hydrangea.html)
http://www.swsbm.com/SayreMM/Sayre%27s_Materia_Medica-3.pdf (http://www.swsbm.com/SayreMM/Sayre%27s_Materia_Medica-3.pdf)
A Manual of Organic Materia Medica and Pharmacognosy by Lucius E. Sayre, B.S., Ph. M (4th edition, 1917)
331. ÆSCULUS HIPPOCASTANUM Linné.—HORSE-CHESTNUT. (Bark and Fruit.)
Habitat: Asia; cultivated as an ornamental tree in Europe and North America.
The bark contains a bitter glucosid, esculin, isomeric with quinovin in cinchona bark, for which it is used as a substitute in Europe.
[...]
Preparation of Esculin.
—Precipitate a decoction of the bark with lead acetate, treat the filtrate with H2S, evaporate and recrystallize.
Yuck! ;D This is just posted as an historical example and it is not recommended to perform it the crazy old chemist's way.
Patent WO02053552 (http://l2.espacenet.com/dips/viewer?PN=WO02053552&CY=gb&LG=en&DB=EPD)
Patent SU595320 (http://l2.espacenet.com/dips/viewer?PN=SU595320&CY=gb&LG=en&DB=EPD)
Patent SU595320 (http://l2.espacenet.com/dips/viewer?PN=SU595320&CY=gb&LG=en&DB=EPD)
, as requested by Vitus_Verdegast above as well as in Post 460563 (not existing).Patent US6337095 (http://l2.espacenet.com/dips/viewer?PN=US6337095&CY=gb&LG=en&DB=EPD)
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a process for the isolation of compound scopoletin which is used as nitric oxide synthesis inhibitor from Artemisia annua and other plant families, said process comprising:
a) extraction of dried powdered material of different plant parts with an aqueous acetonitrile solvent in the ratio of 1:5 for 6 to 8 hrs.,
b) concentration of the extracted solvent up to 30% of its original extract under vacuum,
c) partitioning the concentrated extract with halogenated solvent to transfer scopoletin in the non-polar halogenated solvent,
d) drying halogenated solvent over anhydrous sodium sulphate and evaporating the solvent,
e) crystallizing the residues in methanol and filtering the crystals,
f) concentrating the filtrate and chromatographed on silica gel,
g) eluting scopoletin in chloroform methanol mixture; and crystallization of the fractions containing the scopoletin to get the pure scopoletin compound.
In one embodiment of the invention, Artemisia annua plant was selected for the isolation of scopoletin. Artemisia annua ariel parts from Spain have reported 0.02% scopoletin and England (cult) 0.034%. China has also reported the presence of scopoletin from the whole plant (Brown, G. D.: Two new compounds from Artemisia annua: J.Nat. Prod. 58, 300 (1992); Marco, J. A.; Sawz, J. F.; Bea, J. F.; Barber, O.: Phenolic constituents from Artemisia annua: Pharmazie 45, 382-383 (1990); Liu, H. M.; Li, G. L., Wu, H. Z.: Studies on the constituents of Quinghao:Yao Hsuch Hsuch Pao, 37, 129-143 (1979)).
In India CIMAP, Lucknow, has developed a new variety of Artemisia annua "Jeevan raksha" producing high content of artemisinin and biomass (stems & leaves). Artemisinin and its derivative are reported as potent against chloroquine resistant multi drug resistant and severe complicated malaria.(!) Artemisia annua plant is the only source of artemisinin.
Screening of all the three major parts of the A. annua plant of the new variety for scopoletin was carried out by HPLC. The content of scopoletin obtained in different plant parts are as follows; leaves 0.2%, Stems 0.3% and roots 0.004%. The yield of the scopoletin is very high as compared to other reported plants. The stem part of the plant Artemisia annua is a waste material as no artemisinin is present in the stems. The biomass of the stem portion of the plant is five times more than of the leaves. Therefore, we have selected the stem part of the plant Artemisia annua for the isolation of scopoletin. Also the stem portion of the stems was found to contain less colouring and fatty material which eases the isolation and purification of scopoletin.
In the prior art, it has been observed that non polar solvents for extraction of the plant materials were employed, resulting in less recovery of coumarins. Still, polar solvents (methanol and ethanol) used for the extraction of coumarins resulted in a higher amount of total extract having more colouring and fatty material. The separation of fatty and colouring matter is a difficult task. In the present invention, we have selected aqueous acetonitrile solvent for the extraction which yielded a higher amount of scopoletin with less amount of colouring and fatty material. Also, the separation of water from acetonitrile for recovery of the pure solvent for reuse is much easier.
In the prior art the scopoletin was purified from the crude extract through acid base treatment or by sublimation method which reduces the amount of scopoletin due to rearrangements and thermal decomposition. In the present invention of the improved process, selective transfer of the coumarins from the aqueous extract into the non polar phase was carried out by partitioning the aqueous phase with chlorinated solvent (Carbon tetrachloride, dichloromethane, chloroform). By employing this step, most of the colouring and fatty material is left in the polar phase thereby enriching the scopoletin in the non polar phase which is easily crystallisable (50-60%) in the crude extract itself The scopoletin left in the mother liquor after crystallization is subjected to column chromatography over silica gel in ratio of only 1:10 for complete isolation of the pure scopoletin. The partition of scopoletin from aqueous extract to non polar solvent reduces the bulkiness of the crude extract by 60-70% which in terms requires less amount of silica gel and solvent in the process.
The process consists of the following operations:
1. Shade drying and grinding of the stems of Artemisia annua.
2. Extracting the powdered Artemisia annua stems with aqueous acetonitrile solvent by cold percolation.
3. Concentrating the total extract under vacuum.
4. Partitioning of the aqueous acetonitrile phase with halogenated solvent.
5. Removal of moisture from the total halogenated extract.
6. Distillation of the halogenated solvent for obtaining the residual extract.
7. Crystallisation of the scopoletin from the residual extract.
8. Filtration and concentration of mother liquor.
9. Column Chromatography of mother liquor over silica gel for recovery of pure scopoletin.
The present invention is to provide a process for the extraction and isolation of scopoletin from the plant Artemisia annua to overcome the drawbacks of the hitherto known process. The invention more particularly provides a process which gives a cheaper and higher yield of nitric oxide synthesis inhibitor compound scopoletin from the natural source.
Accordingly, the present invention provides a process for the extraction and isolation of scopoletin from Artemisia annua which comprises extraction of a plant part, preferably dried stem powder of Artemisia annua with aqueous acetonitrile solvent in the ratio of 1:5, concentration of the extract under a vacuum, partitioning of the concentrated extract with a non polar halogenated solvent, distillation of the halogenated solvent, crystallization of the residue in methanol, filtration of scopoletin, concentration of the mother liquor and performing chromatography over silica gel for obtaining pure scopoletin.
In an embodiment, of the invention the solvent used for the extraction is selected in different ratios of acetonitrile:water from 1:9 to 9:1.
In another embodiment, of the invention the halogenated solvent used for partitioning is to be selected from dichloromethane, carbon tetrachloride, chloroform etc. In another embodiment of the invention the plant part for extraction of scopoletin is selected from stems, leaves, roots etc.
In another embodiment, of the invention the separation of scopoletin over silica gel whereas the ratio of crude extract to silica gel is selected from 1:5 to 1:20 preferably either from 1:5, 1:10 or 1:20.
The details of the invention provided in the following examples are given by way of illustration only should not be construed to limit the scope of the present invention
Yet another embodiment of the invention, extraction and isolation of scopoletin is from plant families such as Umbelliferae, Rutaceae, Compositae, Leguminosae, Moraceae, Caryophyllacae, etc.
Still another embodiment of the invention, the scopoletin is crystallized in the solvent which is selected from chloroform, acetone, methanol and mixtures thereof.
One another embodiment of the invention, scopoletin isolated from different parts of Artemisia annua is in the range of 0.25-0.30% in stem, 0.16-0.20% in leaves, and 0.003-0.004% in roots.
Post 460298 (https://www.thevespiary.org/talk/index.php?topic=9811.msg46029800#msg46029800)
(ClearLight: "translation coming", Methods Discourse)