CA1266236A - Cardiovascular composition - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A new pharmaceutical composition for treatment and prevention of cardiovascular disease, and increasing immune function contains Ginsenoside, Ophiopogonin, Sesquicarene and Chamigrene. Processed for producing these components and the composition, referred to as Pure San-Mai-Sen (PSMS), are provided.

Description

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 ,' .` I

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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:

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.

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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) ~, .
., ~66~
.
ll . ' ~~
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C~CIt3 Fi~. 4. Chemi~al Structure of Se~quic~rene (III) ~ C~ ~ ~
.. . . ~ ~ ' .

Fig. 5. Chemical Structure of Chamigrene ( IY) lZfi6~:36 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 ;, . .

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 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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Claims (6)

A process for producing ginsenoside comprising:

1. A new pharmaceutical composition, Pure San-Mai-sen, for cure and prevention of cardiovascular disease and increasing the immune function, comprising, in weight percent, Ginsenoside, 25-60%; Ophiopogonin, 25-60%;
and Sesquicarene/Chamigrene, 5-50%.

2. A process for producing Ophiopogonin comprising:
(a) soaking ground Ophiopogon japonica Ker-Gawl powder in aqueous ethanol, (b) separating the powder from the ethanol extract, (c) refluxing the powder from (b) with additional aqueous ethanol, and separating it therefrom, (d) again separating the powder from the ethanol extract, (e) repeating steps (c) and (d) 1-3 times, (f) distilling the ethanol extracts, thereby removing wet ethanol, and obtaining an aqueous still residue, (g) extracting said aqeuous still residue 1-5 times with a lipophilic solvent of low mutual miscibility with water, and recovering an aqueous raffinate, and (h) evaporating said aqueous raffinate to produce Ophiopotonin powder.

3. A process according to Claim 2 wherein said refluxing in step (c) is carried out on a steam bath.

4. A process according to Claim 2 wherein the lipophilic solvent is selected from a group consisting of diethyl ether and petroleum ether.

5. A process according to Claim 2 wherein step (h) is carried out upon addition of n-butanol.

6. A process for producing a mixture of Sesquicarene and Chamigrene comprising:
(a) distilling a mixture of powdered schisandra Chinensis baill and water at a pressure of 7 to 15 mm Hg absolute, and (b) recovering a distillate fraction between 75 and 150°C.