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Elevation of serum high density lipoprotein cholesterol by Rowachol, a proprietary mixture of six pure monoterpenes
47
Atherosclerosis, 36 (1980) 47-54 Scientific 0 Elsevier/North-Holland
Publishers,
Ltd.
ELEVATION OF SERUM HIGH DENSITY LIPOPROTEIN CHOLESTEROL BY ROWACHOL, A PROPRIETARY MIXTURE OF SIX PURE MONOTERPENES
G.D. BELL l, J.P. BRADSHAW 3, A. BURGESS ‘, W. ELLIS I, J. HATTON 3, A. MIDDLETON 3, B. MIDDLETON 3, T. ORCHARD 4 and D.A. WHITE 3 Departments of ’ Therapeutics and 2 Clinical Pharmacology, City Hospital, Nottingham, and Departments of 3 Biochemistry and 4 Medicine, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham NG7 2UH (Great Britain) (Received 24 August, 1979) (Revised, received 5 November, (Accepted 2 January, 1980)
1979)
Summary Rowachol, a proprietary choleretic containing 6 pure monoterpenes markedly elevates serum HDL cholesterol (SHDL-C) concentrations in man. The concentration of SHDL-C showed a progressive increase in 16 patients treated with 6-9 capsules of Rowachol daily for periods of 2-28 weeks. There was no accompanying significant change in the concentrations of serum total cholesterol or triglyceride. In view of the significant inverse relationship between SHDL-C concentration and the risk of developing ischaemic heart disease, it is suggested that Rowachol and possibly other terpenes merit further investigation as possible therapeutic agents in the prevention and treatment of atheroma. Key words:
High density lipoprotein cholesterol - Hypercholesterolaemia
- Monoterpenes
Introduction A strongly significant ischaemic heart disease
inverse relationship between the development of and serum high density lipoprotein cholesterol
Financial support (A.M. and W.E.) and supplies of Rowachol from Rowa Limited, Bantry, Co. Cork. Eire, are gratefully acknowledged, as is the SUPPCA of the British Heart Foundation to J.P.B. Correspondence to Dr. D.A. White, Department of Biochemistry, University of Nottingham Medical School. Queen’s Medical Centre. Nottingham NG7 2UH. Great Britain.
48
(SHDL-C) concentration has been shown in many cross-sectional [1,2] and prospective studies [3]. Patients developing other atherosclerotic conditions such as cerebrovascular [4] or peripheral vascular disease [ 51 also tend to have lower concentrations of SHDL-C than matched control groups. Conversely, raised concentrations of SHDL-C appear to predispose against atherosclerosis [6]. If the association between SHDL-C and ischaemic heart disease is shown to be causal, then agents which raise the concentration of SHDL-C merit careful evaluation. Alcohol consumption, which in moderate quantities may lower the risk of coronary heart diseases [7] or its severity [8] or both, elevates the concentration of SHDL-C [9] ; the greater the weekly intake of alcohol, the higher the resulting concentration of SHDL-C [lo]. It has been reported that mixtures of pure monoterpenes lower serum cholesterol concentrations [ 111. Rowachol, a proprietary choleretic containing the following pure mono- and bicyclic monoterpenes: methanol (32% w/w), cineole (2% w/w), borne01 (5% w/w), menthone (6% w/w), camphene (5% w/w) and pinene (17% w/w) in olive oil, reduces the lithogenicity of bile [12] and causes the dissolution of cholesterol gallstones [ 131 in man. Since Rowachol has been reported to prevent atheroma formation in a cholesterol-fed rabbit model [ 141, it is possible that the anti-atherogenic properties of the terpene mixture may be explained by an effect on SHDL-C metabolism. It was decided to. investigate this in an unselected group of gallstone and hypercholesterolaemic patients currently undergoing Rowachol therapy. Patients
and Methods
Fasting serum lipids (total cholesterol, HDL cholesterol (HDL-C) and triglycerides) were measured in 3 treated groups of subjects: (i) hypercholesterolaemic patients; (ii) gallstone patients; (iii) healthy volunteers; both before and at approximately 2-6 week intervals while on Rowachol capsules (Rowa Ltd., Bantry, Co. Cork, Eire) (each capsule contains 32 mg menthol, 17 mg pinene, 6 mg menthone, 5 mg bomeol, 5 mg camphene and 2 mg cineole). The majority of the subjects were studied between September, 1978 and January, 1979. A group of 8 medical students aged betwetn 21 and 22 years (5 male; 3 female) acted as an untreated control group from whom blood samples were taken in September, 1978 and again approximately 3 months later. Hypercholesterolaemic patients Details of the seven patients in this group are given in Table 1. All except one (A.R. who also had gallstones) had consumed a diet low in cholesterol and saturated animal fat for at least 6 months before the study. B.B. and K.B., who have type IIa hyperlipoproteinaemia, have a strong family history of premature ischaemic heart disease [15]. None of the patients had clinical evidence of ischaemic heart disease. Gallstone patients and treated controls The details of the 5 gallstone patients and the 4 healthy volunteers are given in Table 1. All took 6 Rowachol capsules per day except P.G. and A.B. who
49
TABLE
1
DETAILS
OF
SUBJECTS
Subject
AND
THERAPY SIX
Age (Yr)
Con
Weight
Daily
(kg)
dose
Rowachol (capsule
no.)
trozs A.M.
58
F
50.8
A.B.
20
F
64.2
P.G.
21
M
71.0
W.E.
31
M
81 .O
Hypercholesterolaemic
patients
R.T.
32
M
92.5
G.W.
65
M
68.0
B.B.
16
M
K.B.
19
M
A.R.
54
F
54.4
R.J.
28
M
57.6
F.S.
74
F
52.3
M.N.
46
F
72.2
J.B.
70
M
95.2
V.B.
76
F
71.2
Gallstone
patients
took 9 capsules daily. No patient in either ischaemic heart disease.
group had clinical evidence of
Methods Serum total cholesterol and triglycerides were measured enzymatically by standard automated laboratory methods [16,17]. HDL-C was measilred after precipitation of low density and very low density lipoproteins by heparin (1.4 mg/ml) and manganous chloride (92 mM) as recommended by Wamick and Albers (1978) [ 181. HDL-C in the supernatant was determined enzymatically by the method of Rijschlau et al. (1974) [19]. The precipitate of manganous phosphate was removed by centrifugation before making optical measurements at 405 nm in the Gilford 240. Samples of freezedried control serum (Boehringer Precilip, Boehringer Corp. (London) Ltd.) were assayed for total and HDL cholesterol concentrations with each batch of sera analysed. Statistical analysis Since the period of drug treatment varied, serum lipid samples taken during treatment have been arranged in 4 temporal groups to enable a comparison between initial and subsequent serum lipid concentrations to be made for each patient. A Wilcoxon matched-pairs signed-ranks test was used to evaluate the data. Result8
Untreated control group The mean serum HDL-C and total cholesterol concentrations
for the 8
50
medical period.
students
were
not
significantly
changed
over the
3-months
study
Patients treated with Rowachol Effect on serum HDL-cholesterol concentration In contrast to the 8 untreated control subjects, the mean SHDL-C concentrations in the Rowachol-treated group rose significantly between 2 and 6 weeks from the start of treatment (Table 2). The smallest change occurred in the 4 treated controls who received Rowachol for the shortest period. The most significant elevation of HDL-cholesterol occurred in patients who were treated for the longest period. It can be seen from Fig. 1 that the concentration of SHDL-C rose progressively throughout the period of study in 4 patients typical of this group. In 8 patients who were treated for more than 140 days the rate of increase of SHDL-C showed a statistically significant progression with time (r = 0.63, P < 0.001). In contrast to this, adult members of a family with type IIa hyperlipidaemia (K.B. and B.B.) showed no apparent change in concentration of SHDL-C on Rowachol treatment. Effect of Rowachol treatment on serum total cholesterol concentrations No consistently significant changes in serum total cholesterol concentration occurred during therapy (Table 3), and though the concentration appeared to fall slightly in the hypercholesterolaemic individuals, the difference was not statistically significant (Table 3).
TABLE SHDL-C
2 CONCENTRATIONS
(mM) Duration
Subject
0 Con
DURING of therapy 2-6
THERAPY (weeks) 7-12
13-20
.20
tro1s A.M.
0.57
A.B.
0.88
P.G.
0.82
1.03
W.E.
0.86
1.12
Hypercholesterolaemic
1.09
1.28 1.14
patients
R.T.
0.33
G.W.
0.66
B.B.
0.30
0.60
0.60
K.B.
0.60
0.60
0.60
A.R.
0.59
0.62
R.J.
0.96
1.05
F.S.
0.34
M.N.
0.83
1.07
1.10
1.47
J.B.
0.78
1.40
1.49
1.72
Gallstone
increase
0.59
0.78
0.97
0.62
1.46
1.38
1.06
1.29
0.52
0.86
patients
V.B. Significance
0.49
of in
concentration
1.07
1.57
0.87
0.68
1.43
-
P < 0.05
P < 0.02
P < 0.05
P < 0.01
(n = 9)
(n = 9)
(n=
(n = 8)
6)
51
1.6-
1
I 20
0 Fig.
1.
excess
The of
I 40 Days on effect
140
of
days.
TABLE
3
TOTAL
SERUM
I 60
Rowachol
A G.W..
on
a A.R.,
CHOLESTEROL
Subject
Duration
0 Con
I ‘00
I 60
treatment
the
I lx)
I 140
concentration
0 R.J.,
I 180
I 160
of
SHDL-C
I 200
in 4 individuals
studied
over
0 R.T.
CONCENTRATIONS
of therapy 2-6
(mM)
DURING
THERAPY
(weeks) 7-12
13-20
>20
tr01s A.M.
5.0
A.B.
5.4
6.7
P.G.
4.5
4.6
W.E.
6.0
5.8
Hypercholesterolaemic R.T.
11.7
9.9
14.1
10.8
12.0
10.7
B.B.
10.3
10.5
10.9
K.B.
13.7
10.4
12.3
A.R.
5.1
patients 13.2
G.W.
5.8
8.9
8.7
R.J.
5.1
5.3
F.S.
6.6
11.2 8.7
9.8
9.4
7.1
7.6
7.0
6.2
N.S.
N.S.
Gallstone patients
M.N.
7.0
J.B.
6.5
V.B.
7.6
Significance of change
in
concentration
5.2
5.3
7.5 7.2 7.2
7.5
N.S.
N.S.
periods
in
52
TABLE SERUM
4 TRIGLYCERIDE
Subject
CONCENTRATIONS Duration
0 Con
(mM)
of therapy 2-6
DURING
THERAPY
(weeks) 7-12
13-20
>20
tro1.s 1.4
A.M.
1.4
A.B.
0.7
1 .o
P.G.
0.6
0.4
W.E.
1 .a
1.6
Hypercholesterolaemic
patients
R.T.
1.8
G.W.
2.8
B.B.
1 .o
1.1
1.8
1.6
1.3
2.7
2.7
1 .l
2.7
3.3
1.6
1.5 1.9
K.B.
1.5
1.3
A.R.
3.0
3.3
R.J.
1.2
0.7
F.S.
2.1
M.N.
1 .l
J.B.
0.8
V.B.
1.5
2.3
1.6
-
N.S.
N.S.
Gallstone
1.2
patients
Significance of change
1.2
1.1 2.6
2.6
0.9
1 .o
N.S.
N.S.
1.5 1.7
in
concentration
Effect of Rowachol treatment on serum triglycerides Rowachol treatment did not significantly alter the fasting serum triglyceride concentrations (Table 4). Side effects of Rowachol Rowachol was well tolerated. Two of the 16 subjects receiving the drug noticed a slight camphor or minty taste after eructation. The symptom was mild and infrequent and no adjustment of dose was required. Discussion Administration of Rowachol, a mixture of pure terpenes, to man increased the concentration of SHDL-C but had no significant effect on the concentration of total serum cholesterol. In 8 patients treated for more than 20 weeks, there was an approximately constant progression with time and this implies that the percentage increase per week is inversely related to the initial SHDL-C concentration. Hordinsky and Hordinsky [20] similarly reported that daily administration of Rowachol(9 capsules/day) for 6 weeks increased the concentration of SHDL-C by approximately 2-fold in 10 patients with various forms of hyperlipidaemia. In contrast to the present study these patients also achieved a significant fall in total serum cholesterol concentration, and this has also been observed by others [ll]. This fall may reflect the differing dietary regimes used in these studies, where all patients were restricted to low lipid diets from the start of treatment with Rowachol. In the present study patients’
53
dietary regimes were not changed on starting treatment. The impressive rise in the concentration of SHDL-C in patients treated with Rowachol is unlikely to be related to such factors as seasonal changes or “laboratory drift”, since no similar changes or trend was noted in the untreated control group and analysis of the control serum gave reproducible results over the test period. The apoprotein components of serum high density lipoproteins are synthesized in the liver and intestine and the final particle may be derived from the [ 211. Rowachol catabolism of chylomicrons and very low density lipoproteins [22] and some of the individual terpene components [23] affect hepatic cholesterol synthesis in vivo by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase. These hepatic effects may be relevant to the altered serum cholesterol distribution seen in patients dosed with Rowachol. However, it is not yet known whether the increase in SHDL-C seen in man after Rowachol treatment is due to an increased number of HDL particles (as would be indicated by an increase in the serum concentration of apoprotein A,), or to an increase in the amount of cholesterol carried by each particle. This is currently under investigation. Rowachol is sold on the continent of Europe mainly because of its choleretic properties. It has been marketed for over 25 years, and is freely available in the U.K. on prescription. Experience to date would suggest that it has a remarkably low incidence of side effects [24] and, furthermore, is relatively inexpensive. In the light of the present findings and the association between SHDL-C concentration and atheroma formation [25], Rowachol merits further investigation, especially since the preparation has already been shown to prevent atheroma in experimental animals [ 141. Acknowledgements The authors are especially grateful to Professor M.J.S. Langman for much helpful criticism during the preparation of the manuscript. We also thank Dr. J.R. Hampton for permission to study 4 patients under his care. References 1 Miller, G.J. and Miller, N.E., Plasma high density lipoprotein concentration and development of ischaemic heart disease, Lancet. 1 (1975) 16. 2 Castelli, W.P., Doyle, J.T., Gordon, T., Hames. C.G., Hjortland. M.C.. Hulley, S.B.. Kagan, A. and Zukel, W.J., HDL cholesterol and other lipids in coronary heart disease -The cooperative lipoprotein phenotyping study, Circulation, 55 (1977) 767. 3 Miller, N.E., Fdrde, O.H., Thelle, D.S. and Mjds. O.D., The Tromsd Heart Study - High density lipoprotein and coronary heart disease: a prospective case-control study, Lancet, 1 (1977) 965. 4 Riissner. S.. Mettinger, K.L., Kjellin. K.G.. Siden, A. and Soderstrom. C.E., Normal serum cholesterol but low HDL cholesterol concentration in young patients with ischaemic cerebrovascular disease, Lancet, 1 (1978) 577. 5 Zilcher, H., Kaliman, J. and M6ller. M., HDL cholesterol in peripheral vascular disease, Lance& 1 (1979) 558. 6 Glueck, C.J.. Fallat. R.W., Mfflett. F. Gartside, P.. Elston, R.C. and Go, R.C.P., Familial hyper-alphalipoproteinaemia -Studies in 18 kindreds, Metabolism, 24 (1975) 1243. heart disease among 7 Yano, K., Rhoads. G.G. and Kagan, A., Coffee. alcohol and risk of coronary Japanese men living in Hawaii, N. Engl. J. Med., 297 (1977) 405. 8 Bsrboriak, J.J., Rimm, A.A., Anderson, A.J., Schmidhoffer. M. and Tristani. F.E., Coronary artery occlusion and alcohol intake, Brit. Heart J., 39 (1977) 289.
54 9
10
11 12 13 14 15 16 17 18 19 20 21 22 23
24 25
Johansson. B.G. and Medhus, A., Increase in plasma o-lipoproteins in chronic alcoholics after acute abuse. Acta Med. Stand.. 195 (1974) 273. Castelli. W.P., Doyle. J.T.. Gordon, T.. harries, C.G., Hjortland, M.C., Hulley, S.B., Kagan, A. and Zukel, W.J., Alcohol and blood lipids - The co-operative lipoprotein phenotyping study, Lancet. 2 (1977) 153. Hankiewicz, J. and Szeruda-Rudzka, E., The effects of terpichol treatment on cholesterol concentration in serum, Pol. Arch. Med. Wewn., 56 (1976) 401. Doran, J., Keighley, M.R.B. and Bell, G.D.. Rowachol - a possible treatment for cholesterol gallstones, Gut, 20 (1979) 312. Bell, G.D. and Doran, J., Gallstone dissolution in man using an essential oil preparation, Brit. Med. J., l(1979) 24. Benko. S., Macher. A.. Szarvas, F. and Tiboldi, T.. Effect of essential oils on atherosclerosis of cholesterol-fed rabbits, Nature (Lond.), 190 (1961) 731. Dunn, K. and Orchard, T.J., Families with hyperlipoproteinaemia - Do they want to know? Practitioner, 221 (1978) 401. Boehringer, On Technicon Autoanalyser 11, Cholesterol CHOD/PAP, Method Cat. No. 148393. Boehrlnger, Fully enzymatic method for triglycerides, Cat. No. 16448. Warnick, G.R. and Albers, J.J.. A comprehensive evaluation of the heparin--manganese precipitation procedure for estimating high density lipoprotein cholesterol. J. Lipid Res., 19 (1978) 65. Bestimmung des Gesamt-Cholesterins im Roschlau, P., Bernt, E. and Gruber. W., Enzymatische Serum, Z. Klin. Chem. Klin. Biochem., 12 (1974) 403. - Seine Verwendung bei Hyperliplmie, Arch. ArzHordinsky. B.Z. and Hordinsky, W.. Rowachol neither.. 1 (1979) 45. Tall, A.R. and Small, D.M., Plasma high density lipoproteins, N. Engl. J. Med., 299 (1978) 1232. Middleton, A., Middleton, B.. White, D.A. and Bell, G.D., The effects of monocyclic terpenes on hepatic S-3hydroxy-3-methylglutaryl-CoA reductase in viva, Biochem. SOC. Trans.. 7 (19791 111. Clegg. R.J., Middleton, B., Bell, G.D. and White, D.A., Inhibition of hepatic cholesterol synthesis and and S3-hydroxy-3-methylglutaryl CoA reductase by mono- and bicyclic monoterpenes administered in vlvo. Biochem. Biophys. Res. Commun., In press. Bell, G.D.. Drugs used in the management of gallstones. In: M.N.G. Dukes (Ed.), Side Effects of Drugs, Annual 3, Excerpta Medica, Amsterdam, Oxford, 1979, p. 294. Miller, G.J. and Miller N.E., Do high density lipoproteins protect against coronary atherosclerosis? In: A.M. Gotto. N.E. Miller and M.F. Oliver (Eds.), High Density Lipoproteins and Atherosclerosis, Elsevler/North-Holland Biomedical Press. Amsterdam, 1978, p. 103.
Atherosclerosis, 36 (1980) 47-54 Scientific 0 Elsevier/North-Holland
Publishers,
Ltd.
ELEVATION OF SERUM HIGH DENSITY LIPOPROTEIN CHOLESTEROL BY ROWACHOL, A PROPRIETARY MIXTURE OF SIX PURE MONOTERPENES
G.D. BELL l, J.P. BRADSHAW 3, A. BURGESS ‘, W. ELLIS I, J. HATTON 3, A. MIDDLETON 3, B. MIDDLETON 3, T. ORCHARD 4 and D.A. WHITE 3 Departments of ’ Therapeutics and 2 Clinical Pharmacology, City Hospital, Nottingham, and Departments of 3 Biochemistry and 4 Medicine, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham NG7 2UH (Great Britain) (Received 24 August, 1979) (Revised, received 5 November, (Accepted 2 January, 1980)
1979)
Summary Rowachol, a proprietary choleretic containing 6 pure monoterpenes markedly elevates serum HDL cholesterol (SHDL-C) concentrations in man. The concentration of SHDL-C showed a progressive increase in 16 patients treated with 6-9 capsules of Rowachol daily for periods of 2-28 weeks. There was no accompanying significant change in the concentrations of serum total cholesterol or triglyceride. In view of the significant inverse relationship between SHDL-C concentration and the risk of developing ischaemic heart disease, it is suggested that Rowachol and possibly other terpenes merit further investigation as possible therapeutic agents in the prevention and treatment of atheroma. Key words:
High density lipoprotein cholesterol - Hypercholesterolaemia
- Monoterpenes
Introduction A strongly significant ischaemic heart disease
inverse relationship between the development of and serum high density lipoprotein cholesterol
Financial support (A.M. and W.E.) and supplies of Rowachol from Rowa Limited, Bantry, Co. Cork. Eire, are gratefully acknowledged, as is the SUPPCA of the British Heart Foundation to J.P.B. Correspondence to Dr. D.A. White, Department of Biochemistry, University of Nottingham Medical School. Queen’s Medical Centre. Nottingham NG7 2UH. Great Britain.
48
(SHDL-C) concentration has been shown in many cross-sectional [1,2] and prospective studies [3]. Patients developing other atherosclerotic conditions such as cerebrovascular [4] or peripheral vascular disease [ 51 also tend to have lower concentrations of SHDL-C than matched control groups. Conversely, raised concentrations of SHDL-C appear to predispose against atherosclerosis [6]. If the association between SHDL-C and ischaemic heart disease is shown to be causal, then agents which raise the concentration of SHDL-C merit careful evaluation. Alcohol consumption, which in moderate quantities may lower the risk of coronary heart diseases [7] or its severity [8] or both, elevates the concentration of SHDL-C [9] ; the greater the weekly intake of alcohol, the higher the resulting concentration of SHDL-C [lo]. It has been reported that mixtures of pure monoterpenes lower serum cholesterol concentrations [ 111. Rowachol, a proprietary choleretic containing the following pure mono- and bicyclic monoterpenes: methanol (32% w/w), cineole (2% w/w), borne01 (5% w/w), menthone (6% w/w), camphene (5% w/w) and pinene (17% w/w) in olive oil, reduces the lithogenicity of bile [12] and causes the dissolution of cholesterol gallstones [ 131 in man. Since Rowachol has been reported to prevent atheroma formation in a cholesterol-fed rabbit model [ 141, it is possible that the anti-atherogenic properties of the terpene mixture may be explained by an effect on SHDL-C metabolism. It was decided to. investigate this in an unselected group of gallstone and hypercholesterolaemic patients currently undergoing Rowachol therapy. Patients
and Methods
Fasting serum lipids (total cholesterol, HDL cholesterol (HDL-C) and triglycerides) were measured in 3 treated groups of subjects: (i) hypercholesterolaemic patients; (ii) gallstone patients; (iii) healthy volunteers; both before and at approximately 2-6 week intervals while on Rowachol capsules (Rowa Ltd., Bantry, Co. Cork, Eire) (each capsule contains 32 mg menthol, 17 mg pinene, 6 mg menthone, 5 mg bomeol, 5 mg camphene and 2 mg cineole). The majority of the subjects were studied between September, 1978 and January, 1979. A group of 8 medical students aged betwetn 21 and 22 years (5 male; 3 female) acted as an untreated control group from whom blood samples were taken in September, 1978 and again approximately 3 months later. Hypercholesterolaemic patients Details of the seven patients in this group are given in Table 1. All except one (A.R. who also had gallstones) had consumed a diet low in cholesterol and saturated animal fat for at least 6 months before the study. B.B. and K.B., who have type IIa hyperlipoproteinaemia, have a strong family history of premature ischaemic heart disease [15]. None of the patients had clinical evidence of ischaemic heart disease. Gallstone patients and treated controls The details of the 5 gallstone patients and the 4 healthy volunteers are given in Table 1. All took 6 Rowachol capsules per day except P.G. and A.B. who
49
TABLE
1
DETAILS
OF
SUBJECTS
Subject
AND
THERAPY SIX
Age (Yr)
Con
Weight
Daily
(kg)
dose
Rowachol (capsule
no.)
trozs A.M.
58
F
50.8
A.B.
20
F
64.2
P.G.
21
M
71.0
W.E.
31
M
81 .O
Hypercholesterolaemic
patients
R.T.
32
M
92.5
G.W.
65
M
68.0
B.B.
16
M
K.B.
19
M
A.R.
54
F
54.4
R.J.
28
M
57.6
F.S.
74
F
52.3
M.N.
46
F
72.2
J.B.
70
M
95.2
V.B.
76
F
71.2
Gallstone
patients
took 9 capsules daily. No patient in either ischaemic heart disease.
group had clinical evidence of
Methods Serum total cholesterol and triglycerides were measured enzymatically by standard automated laboratory methods [16,17]. HDL-C was measilred after precipitation of low density and very low density lipoproteins by heparin (1.4 mg/ml) and manganous chloride (92 mM) as recommended by Wamick and Albers (1978) [ 181. HDL-C in the supernatant was determined enzymatically by the method of Rijschlau et al. (1974) [19]. The precipitate of manganous phosphate was removed by centrifugation before making optical measurements at 405 nm in the Gilford 240. Samples of freezedried control serum (Boehringer Precilip, Boehringer Corp. (London) Ltd.) were assayed for total and HDL cholesterol concentrations with each batch of sera analysed. Statistical analysis Since the period of drug treatment varied, serum lipid samples taken during treatment have been arranged in 4 temporal groups to enable a comparison between initial and subsequent serum lipid concentrations to be made for each patient. A Wilcoxon matched-pairs signed-ranks test was used to evaluate the data. Result8
Untreated control group The mean serum HDL-C and total cholesterol concentrations
for the 8
50
medical period.
students
were
not
significantly
changed
over the
3-months
study
Patients treated with Rowachol Effect on serum HDL-cholesterol concentration In contrast to the 8 untreated control subjects, the mean SHDL-C concentrations in the Rowachol-treated group rose significantly between 2 and 6 weeks from the start of treatment (Table 2). The smallest change occurred in the 4 treated controls who received Rowachol for the shortest period. The most significant elevation of HDL-cholesterol occurred in patients who were treated for the longest period. It can be seen from Fig. 1 that the concentration of SHDL-C rose progressively throughout the period of study in 4 patients typical of this group. In 8 patients who were treated for more than 140 days the rate of increase of SHDL-C showed a statistically significant progression with time (r = 0.63, P < 0.001). In contrast to this, adult members of a family with type IIa hyperlipidaemia (K.B. and B.B.) showed no apparent change in concentration of SHDL-C on Rowachol treatment. Effect of Rowachol treatment on serum total cholesterol concentrations No consistently significant changes in serum total cholesterol concentration occurred during therapy (Table 3), and though the concentration appeared to fall slightly in the hypercholesterolaemic individuals, the difference was not statistically significant (Table 3).
TABLE SHDL-C
2 CONCENTRATIONS
(mM) Duration
Subject
0 Con
DURING of therapy 2-6
THERAPY (weeks) 7-12
13-20
.20
tro1s A.M.
0.57
A.B.
0.88
P.G.
0.82
1.03
W.E.
0.86
1.12
Hypercholesterolaemic
1.09
1.28 1.14
patients
R.T.
0.33
G.W.
0.66
B.B.
0.30
0.60
0.60
K.B.
0.60
0.60
0.60
A.R.
0.59
0.62
R.J.
0.96
1.05
F.S.
0.34
M.N.
0.83
1.07
1.10
1.47
J.B.
0.78
1.40
1.49
1.72
Gallstone
increase
0.59
0.78
0.97
0.62
1.46
1.38
1.06
1.29
0.52
0.86
patients
V.B. Significance
0.49
of in
concentration
1.07
1.57
0.87
0.68
1.43
-
P < 0.05
P < 0.02
P < 0.05
P < 0.01
(n = 9)
(n = 9)
(n=
(n = 8)
6)
51
1.6-
1
I 20
0 Fig.
1.
excess
The of
I 40 Days on effect
140
of
days.
TABLE
3
TOTAL
SERUM
I 60
Rowachol
A G.W..
on
a A.R.,
CHOLESTEROL
Subject
Duration
0 Con
I ‘00
I 60
treatment
the
I lx)
I 140
concentration
0 R.J.,
I 180
I 160
of
SHDL-C
I 200
in 4 individuals
studied
over
0 R.T.
CONCENTRATIONS
of therapy 2-6
(mM)
DURING
THERAPY
(weeks) 7-12
13-20
>20
tr01s A.M.
5.0
A.B.
5.4
6.7
P.G.
4.5
4.6
W.E.
6.0
5.8
Hypercholesterolaemic R.T.
11.7
9.9
14.1
10.8
12.0
10.7
B.B.
10.3
10.5
10.9
K.B.
13.7
10.4
12.3
A.R.
5.1
patients 13.2
G.W.
5.8
8.9
8.7
R.J.
5.1
5.3
F.S.
6.6
11.2 8.7
9.8
9.4
7.1
7.6
7.0
6.2
N.S.
N.S.
Gallstone patients
M.N.
7.0
J.B.
6.5
V.B.
7.6
Significance of change
in
concentration
5.2
5.3
7.5 7.2 7.2
7.5
N.S.
N.S.
periods
in
52
TABLE SERUM
4 TRIGLYCERIDE
Subject
CONCENTRATIONS Duration
0 Con
(mM)
of therapy 2-6
DURING
THERAPY
(weeks) 7-12
13-20
>20
tro1.s 1.4
A.M.
1.4
A.B.
0.7
1 .o
P.G.
0.6
0.4
W.E.
1 .a
1.6
Hypercholesterolaemic
patients
R.T.
1.8
G.W.
2.8
B.B.
1 .o
1.1
1.8
1.6
1.3
2.7
2.7
1 .l
2.7
3.3
1.6
1.5 1.9
K.B.
1.5
1.3
A.R.
3.0
3.3
R.J.
1.2
0.7
F.S.
2.1
M.N.
1 .l
J.B.
0.8
V.B.
1.5
2.3
1.6
-
N.S.
N.S.
Gallstone
1.2
patients
Significance of change
1.2
1.1 2.6
2.6
0.9
1 .o
N.S.
N.S.
1.5 1.7
in
concentration
Effect of Rowachol treatment on serum triglycerides Rowachol treatment did not significantly alter the fasting serum triglyceride concentrations (Table 4). Side effects of Rowachol Rowachol was well tolerated. Two of the 16 subjects receiving the drug noticed a slight camphor or minty taste after eructation. The symptom was mild and infrequent and no adjustment of dose was required. Discussion Administration of Rowachol, a mixture of pure terpenes, to man increased the concentration of SHDL-C but had no significant effect on the concentration of total serum cholesterol. In 8 patients treated for more than 20 weeks, there was an approximately constant progression with time and this implies that the percentage increase per week is inversely related to the initial SHDL-C concentration. Hordinsky and Hordinsky [20] similarly reported that daily administration of Rowachol(9 capsules/day) for 6 weeks increased the concentration of SHDL-C by approximately 2-fold in 10 patients with various forms of hyperlipidaemia. In contrast to the present study these patients also achieved a significant fall in total serum cholesterol concentration, and this has also been observed by others [ll]. This fall may reflect the differing dietary regimes used in these studies, where all patients were restricted to low lipid diets from the start of treatment with Rowachol. In the present study patients’
53
dietary regimes were not changed on starting treatment. The impressive rise in the concentration of SHDL-C in patients treated with Rowachol is unlikely to be related to such factors as seasonal changes or “laboratory drift”, since no similar changes or trend was noted in the untreated control group and analysis of the control serum gave reproducible results over the test period. The apoprotein components of serum high density lipoproteins are synthesized in the liver and intestine and the final particle may be derived from the [ 211. Rowachol catabolism of chylomicrons and very low density lipoproteins [22] and some of the individual terpene components [23] affect hepatic cholesterol synthesis in vivo by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase. These hepatic effects may be relevant to the altered serum cholesterol distribution seen in patients dosed with Rowachol. However, it is not yet known whether the increase in SHDL-C seen in man after Rowachol treatment is due to an increased number of HDL particles (as would be indicated by an increase in the serum concentration of apoprotein A,), or to an increase in the amount of cholesterol carried by each particle. This is currently under investigation. Rowachol is sold on the continent of Europe mainly because of its choleretic properties. It has been marketed for over 25 years, and is freely available in the U.K. on prescription. Experience to date would suggest that it has a remarkably low incidence of side effects [24] and, furthermore, is relatively inexpensive. In the light of the present findings and the association between SHDL-C concentration and atheroma formation [25], Rowachol merits further investigation, especially since the preparation has already been shown to prevent atheroma in experimental animals [ 141. Acknowledgements The authors are especially grateful to Professor M.J.S. Langman for much helpful criticism during the preparation of the manuscript. We also thank Dr. J.R. Hampton for permission to study 4 patients under his care. References 1 Miller, G.J. and Miller, N.E., Plasma high density lipoprotein concentration and development of ischaemic heart disease, Lancet. 1 (1975) 16. 2 Castelli, W.P., Doyle, J.T., Gordon, T., Hames. C.G., Hjortland. M.C.. Hulley, S.B.. Kagan, A. and Zukel, W.J., HDL cholesterol and other lipids in coronary heart disease -The cooperative lipoprotein phenotyping study, Circulation, 55 (1977) 767. 3 Miller, N.E., Fdrde, O.H., Thelle, D.S. and Mjds. O.D., The Tromsd Heart Study - High density lipoprotein and coronary heart disease: a prospective case-control study, Lancet, 1 (1977) 965. 4 Riissner. S.. Mettinger, K.L., Kjellin. K.G.. Siden, A. and Soderstrom. C.E., Normal serum cholesterol but low HDL cholesterol concentration in young patients with ischaemic cerebrovascular disease, Lancet, 1 (1978) 577. 5 Zilcher, H., Kaliman, J. and M6ller. M., HDL cholesterol in peripheral vascular disease, Lance& 1 (1979) 558. 6 Glueck, C.J.. Fallat. R.W., Mfflett. F. Gartside, P.. Elston, R.C. and Go, R.C.P., Familial hyper-alphalipoproteinaemia -Studies in 18 kindreds, Metabolism, 24 (1975) 1243. heart disease among 7 Yano, K., Rhoads. G.G. and Kagan, A., Coffee. alcohol and risk of coronary Japanese men living in Hawaii, N. Engl. J. Med., 297 (1977) 405. 8 Bsrboriak, J.J., Rimm, A.A., Anderson, A.J., Schmidhoffer. M. and Tristani. F.E., Coronary artery occlusion and alcohol intake, Brit. Heart J., 39 (1977) 289.
54 9
10
11 12 13 14 15 16 17 18 19 20 21 22 23
24 25
Johansson. B.G. and Medhus, A., Increase in plasma o-lipoproteins in chronic alcoholics after acute abuse. Acta Med. Stand.. 195 (1974) 273. Castelli. W.P., Doyle. J.T.. Gordon, T.. harries, C.G., Hjortland, M.C., Hulley, S.B., Kagan, A. and Zukel, W.J., Alcohol and blood lipids - The co-operative lipoprotein phenotyping study, Lancet. 2 (1977) 153. Hankiewicz, J. and Szeruda-Rudzka, E., The effects of terpichol treatment on cholesterol concentration in serum, Pol. Arch. Med. Wewn., 56 (1976) 401. Doran, J., Keighley, M.R.B. and Bell, G.D.. Rowachol - a possible treatment for cholesterol gallstones, Gut, 20 (1979) 312. Bell, G.D. and Doran, J., Gallstone dissolution in man using an essential oil preparation, Brit. Med. J., l(1979) 24. Benko. S., Macher. A.. Szarvas, F. and Tiboldi, T.. Effect of essential oils on atherosclerosis of cholesterol-fed rabbits, Nature (Lond.), 190 (1961) 731. Dunn, K. and Orchard, T.J., Families with hyperlipoproteinaemia - Do they want to know? Practitioner, 221 (1978) 401. Boehringer, On Technicon Autoanalyser 11, Cholesterol CHOD/PAP, Method Cat. No. 148393. Boehrlnger, Fully enzymatic method for triglycerides, Cat. No. 16448. Warnick, G.R. and Albers, J.J.. A comprehensive evaluation of the heparin--manganese precipitation procedure for estimating high density lipoprotein cholesterol. J. Lipid Res., 19 (1978) 65. Bestimmung des Gesamt-Cholesterins im Roschlau, P., Bernt, E. and Gruber. W., Enzymatische Serum, Z. Klin. Chem. Klin. Biochem., 12 (1974) 403. - Seine Verwendung bei Hyperliplmie, Arch. ArzHordinsky. B.Z. and Hordinsky, W.. Rowachol neither.. 1 (1979) 45. Tall, A.R. and Small, D.M., Plasma high density lipoproteins, N. Engl. J. Med., 299 (1978) 1232. Middleton, A., Middleton, B.. White, D.A. and Bell, G.D., The effects of monocyclic terpenes on hepatic S-3hydroxy-3-methylglutaryl-CoA reductase in viva, Biochem. SOC. Trans.. 7 (19791 111. Clegg. R.J., Middleton, B., Bell, G.D. and White, D.A., Inhibition of hepatic cholesterol synthesis and and S3-hydroxy-3-methylglutaryl CoA reductase by mono- and bicyclic monoterpenes administered in vlvo. Biochem. Biophys. Res. Commun., In press. Bell, G.D.. Drugs used in the management of gallstones. In: M.N.G. Dukes (Ed.), Side Effects of Drugs, Annual 3, Excerpta Medica, Amsterdam, Oxford, 1979, p. 294. Miller, G.J. and Miller N.E., Do high density lipoproteins protect against coronary atherosclerosis? In: A.M. Gotto. N.E. Miller and M.F. Oliver (Eds.), High Density Lipoproteins and Atherosclerosis, Elsevler/North-Holland Biomedical Press. Amsterdam, 1978, p. 103.