Purification of benzophenanthridine alkaloids extracts from alkaloid extracts

A method for separating and purifying individual benzophenanthridine alkaloids from alkaloid extracts by using the pH differences in formation of their respective alkanolamine or base forms and iminium ions. The insoluble base form of the alkaloid is collected as a precipitate and dissolved in an organic solvent and converted to the insoluble iminium ion form with a mineral or organic acid.

BACKGROUND OF THE INVENTION 
Sources of benzophenanthridine alkaloids include five plant families: 
Papaveraceae, Fumariaceae, Rutaceae, Capifoliaceace, and Meliaceae. Two of 
the most important sources of benzophenanthridine alkaloids are found in 
the Papaveraceae family. These plant species are Sanguinaria canadensis L. 
(bloodroot) and Macleaya spp. Sanguinaria canadensis L. is commonly known 
as bloodroot puccoon, teterwort and is a perennial plant native to North 
America. The plant and its extracts have been used as a folk remedy for 
treating asthma, bronchitis, dysentery, ringworm and other ailments. The 
rhizome of the plant has been used as an expectorant in cough syrups and 
in homeopathic medicine. 
Recently Sanguinaria extract derived from the rhizome has been used as an 
antiplaque and gingivitis agent as shown in U.S. Pat. No. 4,145,412 with 
zinc chloride, and as an antimicrobial agent in U.S. Pat. No. 4,406,881 
and USSR Patent No. 230,387. The following patents describe the use of 
benzophenanthridine alkaloid extracts in medical ailments: U.S. Pat. No. 
4,376,115; U.K. Patent No. 2,042,336; German Patent No. 2,907,406; and 
Belgian Patent No. 888,843. 
As a result of their activity in treating medical conditions it has become 
increasingly important to develop methods for extraction of these 
benzophenanthridine alkaloids from plant biomass. It has also become 
important to develop commercial methods for purifying and separating these 
benzophenanthridine alkaloids from each other. The benzophenanthridine 
alkaloids of interest are sanguinarine, chelerythrine, sanguilutine, 
chelilutine, chelirubine and sanguirubine. In the past, methods for the 
separation of these alkaloids from each other included column 
chromatography and thin layer chromatography. However, these methods do 
not lend themselves to production of commercial quantities of these 
alkaloids. 
The prior art for extracting the benzophenanthridine alkaloids from plant 
biomass include the extraction of cut or ground bloodroot with methanol at 
elevated temperatures, filtering the liquid extract, evaporating the 
extract to dryness, dissolving the dried extract in chloroform, adjusting 
the pH of the chloroform solution with acid such as hydrochloric acid, 
collecting the filtered extract, and drying of the extract. This procedure 
is disclosed in U.S. Pat. No. 4, 145,412, incorporated herein by 
reference. Other patents for the extraction of benzophenanthridine 
alkaloids from plant biomass include USSR Patent No. 495,331 for 
extracting Chelidonium majus from benzophenanthridine alkaloids. 
German Patent No. 2,856,577 discloses a method of benzophenanthridine 
alkaloid extraction by treating chopped plant materials with an ammonia 
solution and subsequent extraction with trichloromethane solution. 
Sulfuric acid is added and the solvent is distilled off. The residue is 
basified with ammonia to precipitate the alkaloid free bases. The bases 
are collected. 
U.S. Pat. No. 4,818,533 describes the extract of plant material for 
benzophenanthridine alkaloids using a pH 8.5 solution of water and 
co-solvent such as methylene chloride to dissolve the alkaloid. The 
methylene chloride solution is washed with acidified water and the 
benzophenanthridine alkaloids are precipitated as the acid salt. 
Other U.S. patents describing the extraction of benzophenanthridine 
alkaloids from a plant biomass include: 4,767,861 and 4,769,452. 
SUMMARY OF THE INVENTION 
The invention is directed to the separation and purification of the 
individual benzophenanthridine alkaloids from each other on a commercial 
scale in an economical manner. It is an object of this invention to 
provide a high-purity benzophenanthridine alkaloid with only traces of 
other alkaloids present. In particular this invention provides processes 
for purifying sanguinarine from the other benzophenanthridine alkaloids, 
chelerythrine, sanguilutine, chelilutine, sanguirubine, chelirubine, 
dihydrosanguinarine and norsanguinarine. The invention relates to a method 
for separating and purifying benzophenanthridine alkaloids from an 
alkaloid extract derived from plants. It is based upon the formation of 
the water and alcohol insoluble base forms of the alkaloids at selective 
pH values. Collection of the precipitate is followed by dissolution of the 
base form of alkaloid in an organic solvent. The addition of a mineral or 
organic acid results in crystallization of the selected 
benzophenanthridine alkaloid in high purity with only traces of the other 
benzophenanthridine alkaloids present.

DETAILED DESCRIPTION OF THE INVENTION 
The aqueous pH values for the complete conversion of these alkaloids to the 
base form is shown in Table 1. 
TABLE 1 
______________________________________ 
pH of Base Form 
______________________________________ 
Sanguinarine 8.0 
Chelirubine 7.7 
Sanguirubine 7.9 
Chelerythrine 9.0 
Chelilutine 8.7 
Sanguilutine 8.8 
______________________________________ 
The alkaloids, sanguinarine, sanguirubine and chelirubine, can be 
selectively converted to the base form with only traces (&lt;1.0%) of the 
other three benzophenanthridine alkaloids. This base formation for 
sanguinarine begins at pH 5.5. (R. Jones Journal Natural Products 49, 
1986) 
The present invention process requires the dissolution of the isolated 
alkaloid extract into a solvent such as methanol, ethanol or water. The pH 
is adjusted to pH 5.5-7.0 with any available base such as sodium 
carbonate, sodium bicarbonate, ammonium hydroxide, sodium hydroxide, 
potassium hydroxide, sodium hydride and the like. This pH adjustment 
selectively converts sanguinarine to the free base or pseudobase which is 
insoluble in alcohol and water. Chelerythrine, chelilutine and 
sanguilutine remain soluble. The free base is collected by filtration and 
washed with alcohol or water to remove any trapped soluble alkaloid. The 
alkaloid precipitate is dried. The precipitate is suspended in an alcohol 
such as methanol, ethanol, or the like and acidified with a mineral acid 
such as hydrochloric acid, sulfuric acid, or nitric acid and allowed to 
recrystallize as the mineral acid salt. 
Alternatively, the free base form can be resuspended in an alcohol and 
crystallized in the iminium ion form as the organic acid salt using 
gluconic acid, lactic acid, benzoic acid, palmitic acid and the like. The 
acid precipitated benzophenanthridine alkaloid is collected and dried. 
This procedure results in recovery of sanguinarine acid salt in a purity 
of 95 to 100% and containing 0.0 to 5% chelerythrine and/or other 
benzophenanthridine alkaloids. 
In the present invention the alkaloid extract is dissolved into an alcohol 
or water solvent. The pH is then adjusted to pH 8-10 using any available 
base. The base form extract is then allowed to stand and precipitate. The 
precipitate is collected and dried. The precipitate is resuspended in 
methanol, ethanol or the like. The suspension is heated to selectively 
improve the alcohol solubility of some of the base forms of the 
benzophenanthridine alkaloids such as sanguinarine. The suspension is hot 
filtered. The remaining precipitate is then resuspended in an alcohol and 
acidified with an mineral or organic acid such as hydrochloric acid, 
sulfuric acid, nitric acid, gluconic acid, lactic acid, benzoic acid, 
palmitic acid and the like. The alkaloid is allowed to precipitate and is 
then collected. This base precipitate procedure results in the recovery of 
chelerythrine acid salts in a purity of 95 to 100% and contains 0.0 to 5% 
sanguinarine and/or other benzophenanthridine alkaloids. 
FIG. 1 illustrates the process of the invention for purifying sanguinarine 
from the alkaloid extract. Alkaloid extract is dissolved in methanol in an 
agitated tank at a temperature of about 40.degree. to 80.degree. C. 
(preferably 50.degree.-70.degree. C.) The solution is filtered to remove 
insolubles. The filtered solution is treated with a known amount of 
caustic solution to precipitate the sanguinarine as a base form. The 
precipitate is allowed to form for 16-24 hours. It is collected and dried. 
The filtrate is then moved to another tank and additional caustic is added 
to precipitate the remaining alkaloid from the methanol solution. The 
dried sanguinarine precipitate is suspended in methanol. The appropriate 
mineral or organic acid is added to reform the sanguinarine iminium ion. 
The iminium ion will dissolve in the methanol. It is filtered at about 
40.degree. to 80.degree. C. (preferably 50.degree.-70.degree. C.) to 
remove insolubles. Additional acid is added to the hot filtrate and it is 
allowed to cool for crystallization. The precipitate is collected and 
dried to obtain the purified sanguinarine chloride. 
FIG. 2 illustrates the process of the invention for purifying chelerythrine 
from the alkaloid extract. The alkaloid extract is converted to a base 
form. The base form is collected by filtration. The base form extract is 
suspended in methanol in an agitated tank. The suspension is then stirred 
and can be heated to 40.degree. to 80.degree. C. (preferably 
50.degree.-70.degree. C.). The suspension is filtered and the remaining 
precipitate is resuspended in methanol. A mineral or organic acid is added 
to the suspension. The solution is heated to 40.degree. to 80.degree. C. 
(preferably 50.degree.-70.degree. C.) and filtered. Additional acid is 
added to the filtrate. The solution is allowed to cool and crystallize. 
The precipitate is collected and dried to obtain the purified 
chelerythrine chloride or redissolved in hot solvent for 
recrystallization. The filtrate from the first methanol suspension and 
filtration contains 50% sanguinarine and 50% chelerythrine. This base form 
product can be acidified and collected as a precipitate. This precipitate 
can be used to produce sanguinarine by selective base precipitation as 
described in FIG. 1. 
EXAMPLE 1 
A 9.7 gram sample of Macleaya Extract containing 5.7 grams of sanguinarine 
and 2.9 grams of chelerythrine (total alkaloid 8.6 grams) was dissolved in 
388 ml of methanol. The solution was heated to 50.degree. C. and filtered 
through a CUNO 50S Zeta filter. 2.3 ml of a 50/50 ammonium 
hydroxide/methanol solution (v/v) was added to the hot filtrate to raise 
the pH to 6.3. 
The filtrate was allowed to cool overnight and precipitate. The precipitate 
was collected on a Buchner funnel washed with methanol and then dried at 
40.degree. C. overnight. The precipitate (6.0 grams) was suspended in 240 
ml of methanol after which 1.2 ml of concentrated hydrochloric acid was 
added. The solution was stirred and heated to 50.degree. C. for fifteen 
minutes. The solution was filtered through Whatman No. 1 filter paper. The 
hot filtrate was stirred and an additional 1.2 ml of hydrochloric acid was 
added. The solution was stirred for an additional fifteen minutes. The 
heat was removed and the filtrate was allowed to stand overnight. The 
precipitate formed was collected, dried and analyzed. The yield was 4.8 
gram of 97% sanguinarine chloride and 2.7% chelerythrine chloride. 
EXAMPLE 2 
A 10 gram sample of Macleaya Extract was dissolved in 400 ml of methanol. 
The methanol solution was heated to 60.degree. C. and filtered through a 
CUNO 50s filter. 1.6 ml of a 50/50 ammonium hydroxide/methanol solution 
(v/v) was added to the hot filtrate to raise the pH to 6.5. The filtrate 
was allowed to cool overnight and precipitate. The precipitate (3.3 grams) 
was collected, washed with methanol and dried. The precipitate was 
suspended in 130 ml of methanol and 0.65 ml of concentrated hydrochloric 
acid was added. The solution was heated to 63.degree. C. and filtered. An 
additional 0.65 ml of concentrated acid was added to the filtrate. The 
solution was allowed to stand overnight and crystallize. The precipitate 
was collected and dried at 40.degree. C. The yield was 2.6 g of 99.4% 
sanguinarine chloride and 0.6% chelerythrine chloride. 
EXAMPLE 3 
A 10 gram sample of Macleaya Extract was dissolved in 400 ml of methanol at 
57.degree. C. The methanol solution was filtered and 55 ml of a 1% sodium 
hydroxide in methanol solution (w/v) was added to the hot filtrate to 
raise the pH to 6.7. The filtrate was allowed to cool for 3 hours to 
25.degree. C. The precipitate was collected, washed with methanol and 
dried overnight at 40.degree. C. The precipitate was suspended in 175 ml 
of methanol and heated to 55.degree. C. 1.75 ml of concentrated 
hydrochloric acid was added to the suspension. The solution was filtered 
hot and allowed to cool overnight. The crystals were collected and dried 
at 40.degree. C. The yield was 3.5 g of 98.9% sanguinarine chloride and 
1.1% of chelerythrine chloride. 
EXAMPLE 4 
5000 grams of Macleaya Extract containing 51% by weight sanguinarine and 
29% by weight chelerythrine was dissolved in 156 liters of methanol. The 
methanol solution was heated to 59.degree. C. and filtered through a CUNO 
50S filter. The hot filtrate was stirred and 27.5 liters of 1% sodium 
hydroxide was added to the filtrate. The filtrate was allowed to stand 
overnight and precipitate. The precipitate was collected, washed with 
methanol and dried under a vacuum of 20 inches mercury at 35.degree. C. 
overnight. The filtrate was saved for precipitation of alkaloid by raising 
the pH to 10 with a 20% sodium hydroxide solution. The precipitate (1,914 
grams) was suspended in 64 liters of methanol and heated to 55.degree. C. 
320 ml of concentrated hydrochloric acid (HCl) was added to the 
suspension. The solution was filtered through a Whatman No. 1 filter 
paper. An additional 320 ml of concentrated HCl was added to the filtrate. 
The filtrate was stirred and allowed to stand overnight for 
crystallization. The precipitate was collected and dried for 48 hours 
under a vacuum of 20 inches of mercury at 35.degree. C. The yield was 
1,510 grams of 98.8% sanguinarine chloride 1.2% chelerythrine chloride and 
0.1% norsanguinarine. 
EXAMPLE 5 
A 10 gram sample of Sanguinaria Extract containing 40% by weight 
sanguinarine and 40% other benzophenanthridine alkaloids (chelerythrine, 
sanguirubine, chelirubine, sanguilutine and chelilutine) was dissolved in 
400 ml of methanol and heated to 57.degree. C. The solution was filtered 
and 11 ml of a 5% sodium hydroxide/methanol (w/v) solution was added to 
the hot filtrate to raise the pH to 6.6. The filtrate was allowed to cool 
and precipitate overnight. The precipitate was collected, washed with 
alcohol and dried at 50.degree. C. The precipitate was suspended in 130 ml 
of methanol and 0.65 ml of concentrated HCl was added to the suspension. 
The solution was heated to 60.degree. C. and filtered. An additional 0.65 
ml of HCl was added to the hot filtrate. The filtrate was allowed to cool 
overnight and crystallize. The precipitate was collected and dried at 
40.degree. C. The yield was 3.3 g of 96% sanguinarine chloride, 1.1% 
chelirubine chloride, 0.8% sanguirubine chloride and 1.2% chelerythrine 
chloride. 
The precipitate was dissolved in 110 ml methanol and 0.5 ml of concentrated 
HCl was added. The solution was heated to 60.degree. C. and filtered. The 
solution was heated to 60.degree. C. and filtered. The filtrate was cooled 
and crystallized. The precipitate was collected and dried overnight. The 
yield was 2.8 grams of 98% sanguinarine chloride, 1.0% chelirubine 
chloride, and 0.7% chelerythrine chloride. 
EXAMPLE 6 
A 10 gram sample of Macleaya extract was dissolved in 400 ml of methanol 
and filtered at 55.degree. C. 55 ml of a 1% NaOH/methanol solution was 
added to the hot filtrate to raise the pH to 6.5. The filtrate was cooled 
and precipitated. The precipitate was collected and dried. The precipitate 
was suspended in 130 ml methanol. A 4 gram sample of benzoic acid in 10 ml 
methanol was prepared. The suspended precipitate was heated to 50.degree. 
C. and the benzoic acid/methanol solution was added. The solution was 
allowed to cool and crystallize. The yield was 3 grams of 97% sanguinarine 
benzoate and 1.5% chelerythrine benzoate. 
EXAMPLE 7 
A 10 g sample of benzophenanthridine alkaloid extract containing 35% 
sanguinarine and 65% chelerythrine in the base form at pH 9 was suspended 
in 200 ml of methanol. The suspension was stirred and heated to 55.degree. 
C. The hot suspension was filtered through Whatman No. 541 filter paper. 
The filtrate was transferred for recovery of an extract containing 55% 
sanguinarine/45% chelerythrine. The remaining precipitate was resuspended 
in 200 ml methanol. The suspension was heated to 50.degree. C. and 
filtered through Whatman No. 541 filter paper. The 6.4 g of precipitate 
was collected and suspended in 100 ml methanol. 3 ml of concentrated 
hydrochloric acid was added. The solution was heated to 55.degree. C. and 
filtered. An additional 3 ml of concentrated hydrochloric and was added to 
the hot filtrate. The solution was allowed to stand overnight and 
crystallize. The precipitate was collected and dried at 40.degree. C. The 
yield was 4.8 g of 98% chelerythrine chloride and 2.0% sanguinarine 
chloride. 
EXAMPLE 8 
A 5 g sample of benzophenanthridine alkaloid extract containing 20% 
sanguinarine and 80% chelerythrine in the base form at pH 9.2 was 
suspended in 100 ml of methanol. The suspension was stirred and heated to 
50.degree. C. The hot suspension was filtered through Whatman No. 1 filter 
paper. The filtrate was transferred for later recovery of an extract 
containing 53% sanguinarine and 47% chelerythrine. The remaining 
precipitate was resuspended in 100 ml methanol and heated to 50.degree. C. 
The suspension was hot filtered through Whatman No. 2 filter paper. The 
3.0 g of precipitate was collected and suspended in 50 ml of methanol. 2 
ml of concentrated hydrochloric acid was added. The solution was heated to 
50.degree. C. and allowed to cool and crystallize. The yield was 2.7 g of 
99% chelerythrine chloride and 0.5% sanguinarine chloride. 
EXAMPLE 9 
A 10 g sample of benzophenanthridine alkaloid extract in the base form at 
pH 9.6 containing 34% sanguinarine/66% chelerythrine was suspended in 100 
ml of ethanol. The suspension was stirred at room temperature. The 
suspension was filtered through Whatman No. 541 filter paper. The 
remaining precipitate was resuspended 5 more time in 100 ml ethanol and 
stirred at room temperature. The 4.3 g of precipitate remaining was 
collected and suspended in 50 ml of methanol. 3 ml of concentrated 
hydrochloric acid was added. The solution was heated to 50.degree. C. and 
allowed to cool and crystallize. The yield was 3.9 g of 96% chelerythrine 
chloride with 1.2% sanguinarine chloride.