Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
YACUANG LIU
PHARMACEU'rl CAL COM POS I T I ON
Ct)NTAINING PURI~ SAN- MAI-SE:N
B<~ckground o~ the Invention
~ield of the Inv~ntion
This invention relates to a new pharmaceutical com-
positlon for treatment and prevention o~ cardiovascular
disease, and increasing the immune function.
Specifically, this invention provides a new composi-
tion o~ four major active ingredients: Cinsenoside,
Ophiopogonin, Sesquicarene and Chamigrene.
~escrlption of Prior Art
Clnseng roots have long ~een used in Asi~ ~o prepare
drugs and medicines. Many methods o~ extractin~ the ac~ive
ingredients of ginsen~ roots have been used over the years
including sCewing, or extract-lon with solvents such as
ethanol.
Summ~ry of th~ In_~.rltion
There is a nee~ to provLde a composi~ion compresing
severa] ~ctive ingre~ients which, in combination, are
useul in treating and prevenCing cardiovascular disease
in human
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, ~ody and in increasing resistance to infection (immune
response.) this inventlon provides a pharmaceutical com-
position referred to as Pure San-Mai-Sen (YSMS) and a
process for preparing the same. PSMS comprises Ginsenoside
(I), Ophiopogonin (II), Sesquicarene (III) and Chamigrene
(IV), all of which are derived from plant materials. The
approximate composition of PSMS and the sources of $ts
components are listed below:
TABLE 1
Component source Approximate
Concentration in
Weight Precent in
PSMS
Cinsenoside (I) 25-60
Roots of Panax quinquefolium L,
or Panax ginseng C. A. Mey,
generally referred to as ginseng
l, Ophiopogonin (II) 25-60
i Ophiopogon .
! japonica Ker-Cawl
Sesquicarene (III)
and Chamigrene (IV) 5-50
Schisandra chinensis
Baill
The process ior producing PSMS comprises extracting
! ground of the above natural materials with appropriate
I solvents such as alcohol or water, removing lipids by
,,
~ Z3~
extraction with ether where necessary, vacuum-distilling
the extracts with and without addition of butanol, and
preparing PSMS by mixing its components in the desired
proportion.
Brief descrlp~l9~L~ YL~
The chemical structures of tlhe components of PSMS
are shown in Figures 1-5.
Cinsenoside (I) is a mixture of Ginsenoside b, and
g, as illustrated in Figures 1 and ~.
Ophiopogonin (II) is a mixture of Ophiopogonin B
and D ~s illustrated in Figure 3.
Sesqulcarene (III) and Chamigrene (IV) are isomers
occurring in a mixture. Thelr structures are illustrated
in Figures 4 and 5.
PSMS is produced by mixing the above components in
the proportions given in Table 1 above.
.1
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`:
9~
C~13 ~3 R3
When
R1 ~ Cluco~e 6 ~ Gluco~e
~2 ~ Gluco~e 2_ _ 1 Gluco~e
R3 ~ H
the compound ia Gln~enc~ide bl ,Melting Point ,198-202l'.
Wllen
R1 ~ Clucoae
R2 ~ H
R3 ~ -0- Gluco~e
the coMpound 1~ Ginsenoside gl,Meltlng Po~nt, 192-194C.
.
~i~, 1 Che~llc~ truc~ure of Gin~enoslde ~I)
~2~ 6
~ zO~lf
0~
~1 ~U I Glucose 6 1 Glucose
~f
o~
t~ o~
~2~`
0~ ~ Glucose 2 1 loco~e
~ ~ I
~ . -O- Glucose 2 _1 Rhan~ose~ I
~
o~ 011 , . " i
', , .i' . I
Fig, 2 Chemical Structure of Substituen~cs
of G:Lnserloside
_5_
f f
~'. . `.'
~ i2~
,'
. e~3 _ CH ~ ~3
C~
~ ,
t~O~;~O~ 1~0
H~/ ~
~ o~ .
When R = H the component is Ophiopogonin B,
which has a melting point of 269-271C.
When R = as following chemical structure
. ' .
~ O
the component is Ophiopogonin D, which has a
melting point of 263-265C.
Fi 3. Chem1cal 9Iructure oF Ophiopogonin (II)
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C~CIt3
Fi~. 4. Chemi~al Structure of
Se~quic~rene (III)
~ C~ ~ ~
.. . . ~ ~ '
.
Fig. 5. Chemical Structure of
Chamigrene ( IY)
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Detail~d De~cription
The processes for producir~g the above-mentioned actlve
ln~redient~ of PSMS ~re d~crib0d by thg following
example~;
Example 1: Extrac~lon ~nd Purification of Gi~enoalde
000 gm3. of dried glnseng powder 1~ extracted wi~h
l 2000 ml of 957~ ethanol at room ~emper~ure ~or 24 hour~. The
; powd~r i~ recovered by filtration. Filt~ate A i8 ~aved
and the powder filtercake i~ refluxed with an ~ddi~ion~l
2000 ~1 of 95% ethansl on a s~eam ba~h. The mixture 1
. filtered again. Fi-Ltra~e B i8 saved flnd ~he powder
ilterca~e i~ reflùxed ~wo ~ore times for 6 hours with
additlonal 2000 ml batche~ o 95% ethanol and filtered,
providing Eiltrates C a~d D. ~iltra~es A, B, C, and
D nr~ combined and di~tllled at 17 ~m H~ Mbsolu~e,
whereby ethanol i~ recoveredla~nd a ~till re~idue i9
obtained,
; Thi~ ~tlll residue 1~ diss~lYed in 500 ml sf
t di~tllled w~ter. Thi3 WateF BolUtion i9 ex~r~cted flv~
. ` time~ with 500 ml of a lipophil~c ~ol~ent~ e, g, diethyl
I ether or petroleum e~her, whereby lipids are remo~ed
from the ~olutlon.
. To th~ acqueous raff~nate iB added 500 ~1 of
water-~a~urated n-butanol arld the mixture i8 di~tLlled
at 17 n~ Hg ab~olute ~o dryness, whareby a powder
reYidUe i8 obtained, Thl~ powder iB di~s~lved ln 500
ml o anhydro~l~ eth~ol, and ~ooa ml of acetone ar~.
added with agit~ion while a preci p~ t~te fo~n~
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preclpi~ate is recovere~ by filtration and w~hed 'cwice 7
with acetone and ~wice with die~hyl ether and drled.
Abou~ 60 gm~. of a white ~o light yellow powder are
recover~d, Thl~ ie Ginsenoside (I).
Example 2: Ex~cract~ on arld Purifiration o~ Ophiopogonin
1000 gm~. o~ Ophiopogon ~aponic~ Ker~G~wl powder are
soaked in 2000 ml of 75% ethanol a~ room tempe~ature or
2b, hour~. The mixture i8 filtered and ~he fil~ercake
powder i~ refluxed twice or ~wo hours wi~h 2000 ml -
o 75% ethanol an~ filte~ed. ,,
me filtrates ar~ combined and di~tilled on a steQm
bath at 17 mm Ng absolute, where~y wet etharlol i~
vaporated and an ~queous ~till re~idue l~ obtained,
This stil~ residue i~ extracted wi~h 500 ml o~ diethyl
ether ~our times to remove lipid~. Other ~ol~rents such
a8 petrole~n ether may be u~ed.
To this ~queouE) rafinat~ are added SOO ml of .
n-butanol and the mixtl~re ~ evRpor~ed to dryne~s at .
17 mm H~ ab~olute, wh~reby abou~ 30 gm9. of powder .
residue are ob~calned. Thi8 1EI Ophiopogonin (II), .~
~xample 3: RecoYery of Sequ~cQrene/Chamigrene .
1000 gm~. of Schi~andra chinen~i~ Baill i8 ground i~to c
powder and added to 2000 ml o~ distilled water. ~he
mixture iB vacuum~di~tilled and the di~tillate ~ract~on
recovered at 10 m~ Hg absolute between 80 and 130C i~ i
~eparated. Abou~ 14 ~n~, of this dist~lla~e sre obtained.
It contalns ~he lsomerE~, Seaquicarene (III) and Ch~migr2ne
(IV).
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Example 4: Preparation of PSMS Liquid
120 gm~, of ~inseno~ide, 90 glll8. of ~phiopogonin, and
~8 gm~. of Sequicarene/Chamigr~ne are disper~ed and
dl~solved 1~ 500 ml o~ di~tilled w~te~. The pH o~ thi~
~olution i~ &d~u~ed to 7 by ~ddition of dilu~ce hydro-
ohLoric acld. Thi~ i~ PSMS Liquid.
On a dry basis, the esmpo~ition o~ iSMS may vary
as follow~. -
Weigh~ percen~ Preferred
compo~ltion
wei~ht ~ro~nt
~in~en~sid~ 25~60 25
Ophiopogonin . 25-60 SD
Se~quicarene/Ch~m~grene5-50 25
The dry ingredients of PSMS, prep~red in accordance
wlth the present invention, may be incorporated in t~bleta,
cap~ules and syrup~ by conventional methods which are
not part of thi~ lnvention,
This in~en~ion will now be de~crl~ed wlth re~esence
to its bene~icial effec ~, 88 illu~rated by the following
te~t~:
~xample 5:
~ '..
Culture of chick heart cell. Hearts were removed f~om
embryo~ and were dl~oclat2d ~c 37C for 45 ~n~nute~ with
0 . 2570 tryp~in ~ ma , c~pe III), 0 . 025% collagenasg
(~igma, type I), and 0.005"/0 panc~ea~ NBCo) prepa~d ~n
calclum and ma~ne~ free saline G containing 4% chick~n
' - 10,- ,
8eru~ hen ~he ti~sue i9 disper~ed lnto a single cell
~uspen~lon in culture mediu~ cont~lning 5 ~lml DNA~e I
(~lgm~). Vlable cell count~ were dete~in~d by he~4cytsmeter
coun~:lng. Cells were dlspe~ed ln~co 60 ~ cul'cu~ di~hes
(~urface sre~ 2000 ~un2) at den~itie~ of ~on cell~/m~2. - ~
Cultures were ~aintained in Ham's F~12K, cln a~ ~uQdiu~ ¦
~or myoc~rdi~l cell~ as de~cribed in K~lghn, Ml~, "Ti~aue
culture met'nod~ and ~pplications," (Kru~ and Po~terson,
edi,) pg, 54-58, Academic Prea~. 1973, and ~upple~ented
with 5% fe~al bo~ine ~er~n, gen~amlcln (5mg~1001ul).
Tis~ue ~ulture plates were lncuba~ed under 57" C02 and
95% air flt 37C.
All cell~ were counted in 20 randomly selec~ed
~ie-ld acrosa the en~ire di~h. A Zei~s micloscope 25X
ob~ ective having a ield o~ view o~ 0. 32 mm w~ used
~r cell coun~ing. In ~ low nutritlon culture ~edium,
chick myocardial cell~ tske Longer, for exa~pl~ 7 day~,
lor ~ome o~ t~e cell~ to dle. Thereore t:he ~ulnb~r
of cell~ decrea~e~. Un~e~ r condition PS~IS at
1SU m~/~l increa~e~ the number o chick my~cardi~l cells
(table 1),
Chick m~ocardial cell~ were dl~rided in~o ~ ~roupa
a~e~ belng put into culture o~ 1 day uu~er nonn~
sondition, 100% nutrition group i~ ~he nor~l ~edlu~,
twi~e ~ ~eek ~he mediurn wag ch~nged. The lO'Z nut~ition
group u~es a 10% medium ~nd 90% physiolo,~ical ~all~e
~olu~lon, Tha medi~n and physlologic~l ~allne solution
w~ no~ chenged for ~ week. Thg PSMS group i~ 10X medlum
~L~ 23 ~ .
andt~Ol1PSMS~ ~he medium is not changed for a ~eek. On the
seven (7th) day, the cell counts were as follows:
Table 1. The influence of PSMS on chick myocardial cells
under low nutrition culCure medium.
_ _ _ _
Number of chick myocardial ~ells
_ _ _ _ _
100% nutritlon 10% nutrltion PSMS ~ 10% nutrition
~ _
25.1 + 3.6 (~-10) 54.9 + 14.4 (*9)
90.0 + 5.4 (*3) _ _ P'~ _ .
.
* indicates number of sampling
Example 6:
The inf~uence of PSMS on the Auto raclioKrams of myoc~rdial
cells in a low nutrition medium.
Autogradiograms - cellular labelling was conducted in medium
prepared without unlabelled thymidine and containing 5~e/ml
methyl- H~thymidine (New England Nuclear) at a specific
acCivi~y of 50.8 Ci/mole. Cultures were labelled in medium
containing 3H~thymidine labelling. After labelling plates
were rinsed in saline, fixed in formal: ethanolll:9)3
stained with periodic ~cid-schiEf (PAS) and coated with
Kodak NTB-3 emulsion, a nucleAr emulsion diluted 1:1 with
water. Auto~radiograms were e~posed for 7 days then developec
in DekcoL counterstained with 1% agueous fast green and ~ir
dried.
At least 1000 nuclei per culture were counted a~ random,
cells havin~ more than 50 silver grains over the nucleus
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r
were scored as positive labelling, background was usually
less than 5 grains. The cellular density of each culture
was also ~etermine by recording the number of microscopic
fields counted to assess the total area scored. A Zeiss
25X objective having a field of vlew of 0.32 mm2 was used
for cell counting.
Under similar condition d~scribed in table 1,
experiment of Autogradiograms was performed. From table 2
we can see PSMS at 150~g/ml can increase percent of
myocardial cell nuclei labelled. That is PSMS increases
DNA synthesis of chick myocardial cells.
Table 2. The influence of PSMS on percent of myocardial
cell nuclei labelled.
~n the seventh (7th) day, 3~-Td~ was added (5~Ci/ml medium)
Percent of myocardial cell nuclei pabelled
_ .
100~/~ nutrition 10% nutrition 10% nutrition -1- PSMS
11.5 ~ 1.6 (*7) 17.0 + 2.3 (*7)
19.0 + 1.2 (*3) _ P< 0.01 ~ ` _
Example 7.
_he influence of PSMS on the cardiovascular experiment in
animals.
Cardiovascular experiments are carried out in
accordance with procedures outlined in: Kirby, M.L.
JournaI of Molecular and Cellular Cardiology (1~83)
15 (10) : 6~5
3 6
The above three compounds in animal experiments
reflect the following results:
A. PSMS can slgnificantly increase myocardlal DNA
synthesis; treated group/control group = 365.7%, P~0.001.
B. PSMS can prolong significantly the survival time
of mice under hypoxia. Living minute o~ the control
group is 10 + 5 min. PSMS group is 56 + 3 mni., P~0.001
~2 about 5%)~.
C. PSMS can enhance markedly the coronary flow on
lsolated perfused hearts. drug group/control group =
183.69%. Myocardia uptake of 86Pb was raised in rats.
Example 8: ~
the influence of PSMS on the immune function in animals.
A. PSMS caused markedly increase lymphocyto -
blastogenesls. Mlce was injected with acetoprednisolone
malcing up a model of inhibitecl immunity, then establishing
the index of stimulatlon of lymphocyto blastogenesis.
control group: 16.87 PSMS group: 30.12 (P 0.001)
B. PSMS caused increase of serum complement content
in guinea plgs.
control group: 301 /ml PSMS group: 359 /ml (P~0.05)
C. PSM~ was fo~lnd to ~arkedly increase the formation
sf roset in guinea pigs.
control group: 42.7% PSMS group: 6~.3% (PcO.~l)
D. PSMS could increase the content of serum lyso~yme
in mice.
control group: 9.0 gtml PSMS group: 11.7 g/ml (P~0.01)
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, Example 9:
;~ Toxicity of PSMS
A. The toxicity of PSMS in human body and animal was
found to be very low. When PSMS was administered orally
to mice at a dose of 48g/kg neither death nor toxic effect
was observed other than a sedative appearance.
B. LD50: 1295m~/kg injection in abdominal cavity in
mice.
il C. Each dose for an adult is 20mg. Using 50 kg as
ll the average weight of an adult the dosage is 0.4mg/kg,
!I therefore it is very safe.
! The embodiment of the invention described here can
¦¦ be modified within the spirit and scope of the present
j invention. Numerous modifications and variations of the
present invention are possible in light of the above
teachings.
Having described a composition referred to as PSMS,
a process for producing PSMS, and furthermore described
its pharmacological effect on cardiovascular disease as
well as other effects studied by tests carried out by
approved procedures, I clalm:
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