CA2425912A1 - Use of buchu extracts for hypertension - Google Patents

Abstract

A method is described for obtaining a composition for the treatment of inflammatory and/or hypertensive conditions. The plant (Barosma betuliona, agosthoma betuliona or agosthoma crenulata) is vacuum steam distilled and fractions rich in one or more of diphenol, diosmin, quercetin and rutin are separated and used in dilutions of from 1:400 to 1:3200.

Description

USE OF BUCHUEXTRACTS FOR HYPERTENSION
TECHNICAL FIELD OF THE INVENTION
This invention relates to a composition of matter which is useful in the treatment of hypertension and inflamznatozy conditions BACKGROUND ART
Inflammation and hypez-tension are two well known conditions and it is not considered necessary to explain, in a patent specification, the medical reactions responsible for the hypertensive and inflammatory conditions. , It is known that oil of buchu (Barosma betuliona or agosthoma betulina or 1 p agosthoma crenulata) has been used as an anti-inflammatory agent, usually in the form of a tea prepared from the leaves of the plant.
As a result of extensive experimentation, it has surprisingly been found that certain fractions of the distillate of the buchu oil have unexpectedly beneficial properties.
DISCLOSURE OF THE INVENTION
According to the invention a composition for the treatment of inflammatory and/or hypertensive conditions includes one or more fi~actions of the vacuum steam distillation of buchu oil, whether as the raw product obtained directly from the plant or as a residue from a process aimed at the separation of fractions for othex purposes , the fractions comprising those rich in one or more of diphenol, diosmin, quercetin and rutin.
Hesperidin and Vitamin B and E may also be present in substantial quantities.
Such fractions are determined by experimentation and it has surprisingly been shown that such fractions exhibit an action far in excess of other fractions.

- _2_ As an anti-inflammatory agent, the fractions exhibit unexpected action on neutrophil and monocyte functions; whereas as an anti-hypertensive agent the administration leads to unexpectedly good normalisation of blood pressure without any need for additional medication. This may be due to the increased secretion of renin by the kidneys.
The invention also extends to a method of manufacturing a composition useful in the treatment of inflammatory and/or anti-hypertensive conditions, comprising the steps of selecting one or more fractions of the vacuum stream distillation of buchu oil, the fractions being rich in diphenol, diosmin, quercetin and rutin.
A. ANTI-INFLAMMATORY ACTION .
~~"l'~I~MEltITAL PRC?CEDURES:
t Neui;rophil Tunctinns:
.. e) Adhesfuri rn~5lecules:
Upon activation by complement peptides, the neutrophils up-regulate the expression of the adhesion molecules on their surfaces in order to adhEre more efficiently to the cell.
' wall of the bacteria and endothelial cells. 'these adhesion molecules axe called the ~3 Integrins. There axe 2 that are present on the cell surfaces of neutropIuls and both are expressed constitutionally but up re~.tlated (expressed more) upon activation:
the so-called LFA-1 (CDIIa/CD18 heterodimer) and the Mac-1 (CDlIblCDl8 heterodimer).
Both of these surface molecules can be measured and quantified by means of flow cytotraetry (a specialized tecluuque that measures the amount of binding which takes place between the antibody specific far these n~olecuIes and the cell in question).
Hepaxinised blood from healthy vplunteers was obtained in vacuutainer tubes.
The donating volunteers were not on any anti-ulflammafiory medication. An aliquot of wlxole blood was pre-incubated {20 ax~inutes at room temperature) with various dilutions of the $uchu essc,ntial oil distillate prior to being stimulated using PMA (phorbol tnyristate acetate, art activator used in vilrvr). The experitxa.ental outline was as follows:
I. I3lood incubated with Illediun~
II. Mood with stimulant (nMA) III_ Blood with stimulant (I'MA) E1,ND Buchu oil dilution in a increasing doses Measurement of Mac-I was conducted using antibodies that bind to these stmctures card analysed by flow cytometry. Briefly, the blood aliquot pre-incubated artd activated witl the activator was treated with a solution to lyre the red blood cells. The ncm-lysed cell:;
were washed rr~ith an isotonic solution and analysed on a flow cytometry.
Fifteen thousand events were accumulated and the % positive events as wc;ll as the degre:c of positivity (mem channel fluorescent: MCF) rvas measured.
Respiratory burst:
When the ncutrophils arrive at the site of inl:ection, they eat the organisms invading the host and once inside the cytoplasts of the cells, activate a series of en~yrnes on the inside of the membrane to generate toxic oxygen radicals. This is referred to as the oxygen-dependent killing naechaniszn. This respiratory burst can be measured by using--Iluorochromes which change colour when in the presence of the oxygen radicals generated. This change can be quantified by flow cytoanetry.
A connnercial kit (BurstTest) was used to measure the ability of neutrophils to znouzlt a respiratory burst. For this, blood was obtained from h~aItlzy volunteers as above- The aliquots were pro-incubated with diluttons of the Buchu ails prior to being activated with .- whole bacteria (included in the commercial kit) and processed further. The experizxiental outline was as follows-I. Blood with medium II. Blood with activator (B coli) III. 'Blood with activator (as ahave) AND Buchu oil in. increasing doses Following the pre-incubation and aetivati.on,, the red blood cells were lysed and the non-lysed cells washed and analysed on a flow cytometry. 1'lze % positive calls as well as the MCJ,Y measured. Fifteen thousand evatits were accumulated for the analysis.
' 25 ~ IVIt~nocyte Functions:
~t} )ExpreSSion of atlhesiori molecules:
Similarly to the measurement of adhesion molecules on neutrophils, antlbodte5 binding to the Mac-I will he used to measLZre the expression of these molecules when monocytes are activated in fhe presencelabsence of Buehu oil extract (sea abcwe}. The Same reagents are used for this procedure.

b) Release of pro-inflarrnumtiory monolcincs by monocytes:
When activated, monoc3~tes release IL6 and TNF-a that are responsible far the recruitnzent of other in~znune cells and initiate a self=pepetuating chronic infh~imnzatory process. These factors caz, be quantified by activating monocytes with bacterial products (endotoxin) and measuring the soluble fiactors by immunological assays (1;LISA) using specific antibodies. The aims of these e:cpcrimcnts rvere to measure the release of 1L6 in the followity way. Whole blood was collected izzto vacuutainer tubes and the mononuc;lcar cells isolatrci by density centrifuga lion. The ntonocyte rich fraction was obatined by adherence to the wells of a mufti-rvell cultuxe plate. The adlzerez~t cells were stimulated to release the naonok.ine 1LG by being activated by a pre-determined concentration of bacterial endotoxin (Lipopolysaccharide). The experimental outline Was..
as follows:
1. Cells with medium lI. Cells with endotoxin III. Cells with endotoxin vvith Buchu oil in increasing doses The cells were incubated for 17 hours at 37°C and S% CO2, The .following day, the supernatant was collected and assayed for their contents in IL6 by an in-house ELISA
method using extez~nal sta~~dards as calibratprs. The absolute concentrations ol' ILC~ in the respective supernatmts were calculated ~'rozu a st~n~lard curve derived from the standards.
'l:'he results were expressed as Units of Ih6 (pglml) released by the respective culture wells.
~S~(TLTS:
l;xpression of Adhesion molecxtles (MAC'-1) on Neutrophits and Mcznocytes:
As spawn in laigure 1, the neutrophils pra-incubated with the various dilutions of Huchu oil distillate showed a statistically significant decrease in their ability to express the MAC-1 adhesion molecules as measured lay flow cytometiy. Indeed, when the cells are activated by PIvIA, they increase their expression of MAC-l (compare negative control versus positi~~c conizol). When the cells are activated in the prcsc;ncc of increasing dilutions of the Buchu oil, the expression is significantly inhibited (p <
0.05) by diltions of 1:400; 1:8x0 and 1:1600. TherealLer, the inhibitory effects are diluted out. Tlus effect was not paralleled by the monocytes raider identical conditions of culture.
Figure 1: Expressia~ of MA,~-1 (~C1711b/CD18) by IVeutrophiles and monocytes post activation in the pr~sencel~t~sen~crv nil' Buchu c~iI
distillate nt different tiilutions: -,~ aooo U
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m OD

a -._-_._ 0 ~ O N~utrophns lsoo l ~ Monocytes c ..

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soo a~

Oil Dilution P ~ 0.05 e) Effeot<a of different Buchtt oil distillates au t>Je expression MAC-I in vitro:
' 10 Although most of the oil distillates tested yielded inhibition of the MAC-1 expression by neutropluls, It was evident when each oil eras tested separately that some of the oils had higher activity mhen analysed separately. As indicated in Figure 2, Some oils were more potent inhibitors of the. expression o1' MAC~1 upon stimulation by PMA. The wane experimental procedure was followed as above.
N~gatlve Positive 1 in 400 'I in 800 1 in 160D 1 in 300 Cptltrpl Control Tt earl be seen. froto p'.i~ut'e ~ that certain preparations of the distillate are more effective in the inhibition of MtI.C-1 expression; 1316 and 13~,SA show higher inhibition than the other 3 batches of oils tested.
Figure 2: Tlie effects of different oil t~istillates at the same dilution (1:40d) on the expx~essiou of MAC-1 (~)1~11b/CDI$) on neuthop(lils and monocytes post-activation by PMA:
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COttt~OlCrSritYbl C7if Lot p < 0.05 e) The effects of Buclm oil distillates on the Respixwtory hurat of neutrophils:
L7uring the initial pahse of neutrophil funetians, the internalised organism or particle i.s desfit'oycd by an oxygen-dependent process which ultimately leads to the generation of a~.ygen. radicals (OZ ). These radicals arc protective 'uvhcn this is generated i.ni llte cytoplasm of the cell in that it kills the bacterium. 1-Iowcwer, when these are releasod to the external medium, it can damage healthy tissues in the vicinity of the phagacytic cell.
The ability of Buchu oils to influence the generatioy of oxygen radicals was measured by flow cytametry. As shown iii Figure 3, it is evident that at the different dilutions of the oils, the oxidative burst (respiratory burst) of neutrophils was significantly inhibited by the oil distillate dilutians (fran ~ I.400 tat : 320U dilution).

Fibure 3: Tnhibitioa o~ T~espirnfory ~3r~rst of neutrc~pitils by difi'erent I3uchu oil dilution.
P~u,as you ~
Sao ~
goo . ~ D NeutrQphil~
~~o ~ ~ ManecytesJ
a oo .

c 200 1n0 Negative Positive 1 in QUO 1 if? 800 '! in 16110 1 in ~20A .
Control Control ail Ditutirrn e) Effects c~f various l3uchu ail distillates c~zz the ftespiratoryr Burst of lVeutz-ophils:
It was evidezat during the measurement of this cellullr function that certain bitches were Amore hOtCIit in their inhibitory ability. 'this is shown in Figure ~ v~~hexe various oil distillate$ were compared at the saam dilution (1:h00). rt is obvious that batch 816, B25,A and B~.5I3 were the most effective is this aspect although all had inhibitory activities (p < 0.05) _g_ ~, looa 9dd d aoo goo ,i soo l~l~ Neukrophits~
a, son 4nD I ~ Monocytes~j sno c zoo ~n Inn n Fi~nre 4: Effects of different Baehtt; rail distiiIEI~es on the Respirntony ' Burst of Neutt~opliiIs: nII oils ere tesi:ed at the sane cIIIution (1:4UU).
e) The effects of I3ueliu vil distillate nn the release of ILG by activated rrtonocytcs_ During the chronic phases of ini'3atnmation, tlae cellular infiltrate made up prztxtarily of tnonocytes and other monouclear cells reh;pse IL6, a factor with variod biological activities itxcluding fever, pain, calcium molailization from b~yne, etc. This factor.
pet~etuates the on-going inflaixunatiotx atxd tlms is pivotal in the tissue damage seen in these chronic cotxditions.
Blood rnonocytcs were; pre-incut~attsd with various dilutions of Buchu ail prior to being I-0 activated by endotoxin in order to induce the synthesis and release of TL6. As shown in Figure 5, it is evidetxt thlt various dilution.s of the l3uchu oils were ~L~Ic to significantly inhilait the release of IL6 fronx monocytes.
Nspat3vc PoVttve 874 81S B16 82SA 82513 Control Control ail Lot Figure 5: I<tthik~itintt c~f LL(i relwse from monocytes in the presettce of 73uc:hu oil distillate:
2~0 '115 ,,~, 'f 50 ~f ~5 o tU0 ~~= L.Pa ~1:~1U i ,~5 ~ i~F~ 1 100 J
'"' S0 ~5 .,fi~,ti4 '1 ~4C~ ~~40 ~~~4 ~~'0 tam Oil Diiutiatt ~ =o.o~
At both stimulus concentration {LPS 1:100 and LPS 1:10}, the 1:100 dilution was the most active in its inhibition. Once the oil was diluted out, this activity rWas lost.
nxsc:ussmrr:
It has ~2GI2 SI201~V11 by the present report that vaxious Buchu oiI
distillates are able to iz~llhlt various processes involved during inilamtnatiozl. The acute phase of the inflammatory process as measured Uy the expression of adhesion molecules such as 11~1,C-1 (CDlIblCDlB) on both tnonocytes and ~.3eutraphils as well as the respiratory burst of neutrophils were significantly inhibited by low dilutioris of the oils tested. This in~bition was dose dependent since titering out of the oil Icad to this ei'fect being lost.
Some oils wexe more effective in ibis process.

One can mimic the chronic phase of inflammation by inducing and measuring the release of IL6 from activated monocytes. This was measured in the presence of Buchu oil and shown to be significantly inhibited at low dilutions of the oils.
B. ANTI-HYPERTENSIVE ACTION
Experimental Cases:
Case l:
A 70 year old woman (RH) who for 2 years suffered from high blood pressure.
She started drinking 250 ml of Buchu water (an oil extract of the Buchu plant mixed into spring.
water) each day and within a week, her blood pressure dropped from 150/90 to a more acceptable 140/80 (normal considering the age of this patient).
Case 2:
Mrs GK: suffered from high blood pressure for several years (4 years) necessitating medication which could not control this chronic condition. Several changes of her medication still did not manage to control her blood pressure which stood at 220/120. Four years later, she was introduced to the Buchu water. Within 2 months of using this product regularly, her hypertension has been controlled at 145/0 without any other medication for this serious condition.
Case 3: Mrs DG
This patient was diagnosed with a blood pressure of 160/95 and at other times p 170/100. She was prescribed various medications 'that caused side effects and she was advised to stop the medication. She discovered the Buchu water product and since using the product, her condition improved: her blood pressure now stands at 130/75. She has since never shown signs of her previously chronic high blood pressure.

Claims (6)

1. A method of preparing a composition for the treatment of inflammatory and/or hypertensive conditions characterised in that buchu oil or a product containing buchu oil is vacuum steam distilled, those fractions which are rich in one or more of diphenol, diosmin, quercetin and rutin being separated and incorporated into a pharmaceutically acceptable form.

2. The method according to claim 1 characterised in that the fractions separated also contain one or more of hesperidin, Vitamin B and vitamin E.

3. The method according to either claim 1 or claim 2 characterised in that the fractions are diluted by a factor of between 1:400 to 1:3200.

4. A pharmaceutical composition for the treatment of inflammatory and/or hypertensive conditions characterised by including one or more fractions of the vacuum steam distillation of buchu oil, whether as the raw product obtained directly from the plant or as a residue from a process aimed at the separation of fractions for other purposes, the fraction/s comprising those rich in one or more of diphenol, diosmin, quercetin and rutin.

5. The composition according to claim 4 characterised in that the fraction/s also contain one or more of hesperidin, Vitamin B, and vitamin E.

6. A method of treating a subject suffering from inflammatory and/or hypertensive conditions characterised in the administration of a pharmaceutically sufficient amount of a composition according to claim 4 or claim 5.