Composition for oral application having HMG-COA reductase inhibitory activity

Abstract

Disclosed is a composition comprising an olive extract and a Scutellaria baicalensis extract for use as an HMG-CoA reductase inhibitor in the treatment of dyslipidemia.

Description

[0001] The present invention relates to a composition comprising an Olea europaea extract and a Scutellaria baicalensis extract for use in the treatment of dyslipidemia.

[0002] State of the art

[0003] It is estimated that 2.6 million deaths worldwide are attributed to hypercholesterolemia. HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase is the enzyme responsible for the liver cholesterol biosynthesis. Statins are the most widely used drugs in the treatment of hypercholesterolemia. Their mechanism of action is the competitive inhibition of the enzyme. The decrease in intracellular cholesterol content leads to an increase in LDL receptors, thus leading to greater absorption and internalization of cholesterol from the bloodstream, thus eliminating it. Statins have a dose-dependent effect. The treatment with statins is not always well tolerated, especially due to their known possible side effects (hepatotoxicity and myopathy). 10-13% of patients develop myopathy. The main reason for non-compliance with statin therapy is the muscle symptoms associated with statins, including the nocebo / drucebo effect. In addition, in a significant percentage of patients (10-15% of low responders), statin therapy does not achieve adequate therapeutic goals. In this case, in borderline cases and / or in primary prevention, in addition to ezetimibe, complementary agents (nutraceuticals) are prescribed.

[0004] In particular in the last decade, various clinical trials have been carried out which confirm the activity of the Olea europaea leaf and / or fruit extract on blood cholesterol levels, but olive oil is not included in this class, since it is composed almost entirely of fatty acids, with a much lower proportion of polyphenols with respect to the extract, making their composition almost completely different from that of the extract. These studies do not always lead to the conclusion that there is a significant beneficial effect, i.e. a significant reduction in "bad" (LDL) cholesterol.

[0005] The study by Lockyer et al. (2017) concludes that the daily administration of a polyphenol-rich leaf extract, for a total of 136 mg of oleuropein + 6 mg of hydroxytyrosol per day, for 6 weeks, reduces LDL by 6.37%.

[0006] The study by Stevens et al. (2021) concludes that the daily administration of a polyphenol-rich leaf extract, for a total of 83.5 mg of oleuropein per day, for 8 weeks, has no effect on LDL.

[0007] The study by De Bock et al. (2013) concludes that the daily administration of a polyphenol-rich leaf extract, for a total of 51.1 mg of oleuropein + 9.7 mg of hydroxytyrosol per day, for 12 weeks, has no significant effect on LDL.

[0008] The study by Filip et al. (2015) concluded that the daily administration of a leaf extract rich in polyphenols, totaling 100 mg of oleuropein per day, for 12 weeks, reduced LDL by 20.73%.

[0009] The study by Araki et al. (2019) concluded that the daily administration of tea leaves for 12 weeks significantly reduced LDL in prediabetic patients.

[0010] The study by Georgakouli et al. (2016) concluded that the daily administration of a yogurt rich in 50 mg of polyphenols from the fruits of the olive tree for 2 weeks reduced LDL by 6.45%.

[0011] The study by Pais et al. (2016) concluded that the daily administration of a fruit extract, totaling 50 mg or 100 mg of hydroxytyrosol per day, for 11 days, did not reduce LDL.

[0012] In other clinical trials, olive tree extracts were combined with other herbal extracts, and we will cite some examples of them; however, we have excluded those related to the combination with statins or similar substances, such as monacolin K and monacolins usually present in fermented liquids using Monascus purpureus (such as red yeast), since the latter are compounds with known anticholesterolemic activity, very similar to pharmacological statins.

[0013] The study by Sánchez Macarro et al. (2020) concluded that the daily administration of a combination of grape and bitter orange extracts, totaling 250-300 mg of flavonoid glycosides and 175-200 mg of flavones per day, leaf extracts rich in polyphenols, totaling 85-90 mg of polyphenols (mainly oleuropein), for 8 weeks, reduced LDL by 6.10%.

[0014] The study by Wong et al. (2014) concluded that the daily administration of an olive leaf extract, totaling 160-240 mg of oleuropein, at least 150 mg of other polyphenols, a green coffee bean extract, totaling 90-100 mg of chlorogenic acids, 20-30 mg of coffee tannins, and 100-110 mg of other polyphenols, and 300 mg of beet extract, for 12 weeks, did not reduce LDL.

[0015] Regarding the mechanism of action, some in vitro studies have demonstrated that oleuropein (a polyphenol present in olive extract) reduces the activity of hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase), resulting in decreased cholesterol synthesis in rat hepatocytes (Priore et al. 2014). When the mechanism of action of olive polyphenols was studied in an in vitro model with glial cells, the same research group found that, at a pharmacological concentration (25 μM), hydroxytyrosol had a down-regulating effect on the synthesis of fatty acids and cholesterol, reducing HMG-CoA reductase activity by 16% (Priore et al. 2017). More recently, a phenolic complex derived from olive oil processing waste (containing hydroxytyrosol, tyrosol, and verbascoside as the main components) was studied in an in vitro model to assess its HMG-CoA reductase inhibitory activity (Bartolomei et al. 2022): the results showed that the extract reduced enzyme activity by 10 ± 5.2%, 34.5 ± 3.2%, 48.5 ± 0.5%, 80.2 ± 1.9%, and 90 ± 0.3% at 10.0, 50.0, 75.0, 100.0, and 500.0 μg / mL, respectively.

[0016] Other compounds of plant origin have been studied and used for treatment, including Psidium guajava leaf and fruit extracts, resveratrol, Acacia catechu wood extract, Scutellaria root extract, and policosanol extracted from sugar cane.

[0017] Psidium guajava is a plant belonging to the Myrtaceae family that grows in tropical regions, such as India, Indonesia, Pakistan, Bangladesh, and South America. Although the leaves of these plants are classified as agricultural waste, they are a rich source of bioactive compounds. These leaves are used in traditional medicine in Asian countries, mainly because they have an antihyperglycemic effect (Kumar et al. 2021). A recent study in rats showed that supplementation of powdered P. guajava leaves (2.5%) in a high-fat diet improved dyslipidemia (Mamun et al. 2019). Another preclinical study on a diabetes model showed that daily administration of 400 mg / kg body weight of leaf extract reduced serum triglycerides, total cholesterol, and LDL cholesterol and increased HDL cholesterol (Tella et al. 2019).

[0018] Resveratrol is a polyphenol produced by a variety of plants as a defense mechanism against the presence of parasites and other stress situations such as climate reactions. In humans, oral treatment with resveratrol seems to modulate metabolism in different tissues; however, there is no evidence of the existence of specific receptors, particularly those related to its absorption and pharmacokinetics. Most of the studies on resveratrol report cardioprotective effects and beneficial effects in type 2 diabetes and Alzheimer’s disease, as well as antithrombotic, antiosteoporotic, and antimicrobial effects. A recent study showed that a high dose of resveratrol (250 mg) daily for 3 months reduced total cholesterol levels in patients with metabolic syndrome (Batista-Jorge et al. 2020). A meta-analysis of randomized controlled trials in 2022 concluded that resveratrol can be used to reduce total and LDL cholesterol levels, and that the dose of resveratrol is an important factor in affecting LDL-C levels (Cao et al. 2022).

[0019] Childhood hardwood flavonoids are widely used in traditional medicine, particularly in Asia, for various purposes: such as anti-inflammatory, antiviral, antibacterial, and cardiovascular health care (Srivastava et al. 2010). Water-alcohol leaf extract was administered to diabetic rats at concentrations of 200 mg / kg and 400 mg / kg for 30 days. The extract reduced total cholesterol, triglyceride, LDL, and VLDL cholesterol levels and increased HDL cholesterol levels (Jayabhara et al. 2018). An animal model study showed that supplementation of acacia nilotica polyphenols (2.5% and 5%) to mice with a high-fat diet reduced the increase in liver weight and accumulation of triglycerides and cholesterol (Ikarashi et al. 2011).

[0020] Scutellaria baicalensis Georgi root is used in traditional Chinese medicine for the treatment of inflammation, fever, cough, dysentery, and hypertension. Baicalein is the most abundant flavonoid found in S. baicalensis. Pharmacokinetic studies using baicalein in the range of 100 mg-2800 mg doses have not shown absorption to be directly proportional to the oral dose (Li et al. 2014; Dong et al. 2021). Recent pharmacological studies have confirmed some of the indications in traditional medicine, such as anti-tumor, anti-inflammatory and anti-infective activities, and liver protection and neuroprotective effects. Existing literature indicates that most of the biological activities are attributed to neocloudane-type flavonoids and diterpenoid compounds. Despite the use of Scutellaria in clinical practice, the research remains limited (Shen et al. 2021), which is attributed to the limited expertise in its HMG-CoA reductase inhibition potential. In an animal model, HMG-CoA reductase expression was significantly lower in the liver homogenate of mice in the group supplemented with S. baicalensis compared to the unsupplemented group (Lee et al. 2011). Screening of 98 different polyphenolic compounds demonstrated activity of baicalein and baicalin at a concentration of 10 pg / mL, and both were found to have lower inhibitory activity than pravastatin (Son et al. 2018). Anti-hypercholesterolemic activity of straight-chain aliphatic alcohols, known as policosanols, especially those with at least 60% content of octacosanol, has been evaluated in numerous clinical trials. There is no consensus on the effects of policosanols. A recent meta-analysis concluded that policosanols have no effect on the lipid profile (Osadnik et al. 2022), while a meta-analysis from 2005 concluded that policosanols are more effective than phytosterols and phytostanols in lowering LDL cholesterol levels (Chen et al. 2005). A meta-analysis examining the combined data on the combination of Hongqu Mi, berberine, and policosanols demonstrated that the combination effectively improved the lipid profile, including cholesterol (Millan et al. 2016). Regarding the mechanism of action and the involvement of HMG-CoA reductase in endogenous cholesterol synthesis, a 2019 study on rats showed that policosanols did not affect the mRNA expression of HMG-CoA reductase, while they decreased the activity of HMG-CoA reductase during the development of hypercholesterolemia induced by a high-fat, high-cholesterol diet. In addition, the addition of policosanols stimulated the phosphorylation of AMPK, which can effectively inhibit cholesterol levels by inactivating HMG-CoA reductase (Nam et al. 2019).

[0021] WO 20212094948 describes compositions comprising policosanols in combination with Annurca apple and olive extract for the treatment of dyslipidemia. The application does not report any synergistic effect between policosanols and Olea extract.

[0022] Based on the existing literature, it is evident that the effect of the reduction of the enzymatic activity of HMG-CoA reductase is dose-dependent. Given the low bioavailability of polyphenols and their low content in natural extracts, it is desirable to find a solution that produces a beneficial effect on patients suffering from hypercholesterolemia without the need to use high doses of polyphenols, given the economic and safety drawbacks involved (for example due to side effects, interference with ongoing drug treatments and overload of the excretory organs required to dispose of the catabolic residues). Given the need for long-term treatment, it is even more important to find a solution that allows the use of small doses of biologically active compounds, since it is necessary to reduce cardiovascular risk factors, such as LDL cholesterol, every day and compliance with treatment is essential to ensure lipid targets and thus cardiovascular risk control; such a solution can improve compliance, safety of use and economic sustainability.

[0023] Description of the application

[0024] It has now been found that the combination of Olea fruit and leaf extract and Scutellaria root extract and possibly policosanol inhibits HMG-CoA reductase in a synergistic manner.

[0025] One aspect of the application therefore relates to a composition comprising or consisting of Olea fruit and leaf extract, Scutellaria root extract and possibly policosanol as synergistic components, for use as an inhibitor of HMG-CoA reductase in the treatment of dyslipidaemia.

[0026] The Olea extract obtained by extraction of the leaves and fruits using a mixture of ethanol and water preferably has a polyphenol content ranging from 5% to 95%, more preferably from 10% to 70% and even more preferably from 15% to 50% by weight of the extract. For example, the polyphenol content can be about 16% by weight. The Scutellaria extract is a hydro-alcoholic root extract.

[0027] The Scutellaria root extract has a baicalin content ranging from 5% to 95%, preferably from 10% to 70% and more preferably from 15% to 50% by weight of the extract.

[0028] Olea and Scutellaria extracts are well known and commercially available on the market.

[0029] According to one preferred aspect, the composition of the application comprises or consists of:

[0030] - Olea fruit and leaf extract, policosanol and Scutellaria root extract;

[0031] - Olea fruit and leaf extract and policosanol, preferably free of other active ingredients, such as plant extracts;

[0032] - Olea fruit and leaf extract and Scutellaria root extract.

[0033] The weight ratio of Olea fruit and leaf extract to propolis ranges from 1 : 1 to 50: 1, preferably from 2: 1 to 25: 1 ; and more preferably from 3: 1 to 10: 1.

[0034] The weight ratio of Olea fruit and leaf extract to Scutellaria root extract ranges from 0.1 : 1 to 5: 1, preferably from 0.2: 1 to 2: 1, and more preferably from 1 : 1 to 0.5: 1.

[0035] The dosage unit generally falls within the following ranges:

[0036] - 5 mg - 300 mg of Olea fruit and leaf extract; preferably 20 mg - 150 mg; more preferably 30 mg - 100 mg;

[0037] - 2 mg - 30 mg of propolis; preferably 10 mg - 25 mg; more preferably 15 mg - 20 mg;

[0038] - 5 mg - 300 mg of Scutellaria root extract; preferably 20 mg - 200 mg; more preferably 50 mg - 150 mg.

[0039] The composition of the present application can also comprise 10 mg - 400 mg of Psidium guajava leaf and / or fruit extract per dosage unit; preferably 20 mg - 200 mg; more preferably 50 mg - 150 mg as additional active ingredient.

[0040] Other herbal extracts can also be present in the form of extracts or powders, fermentates, probiotics, yeast, bacterial lysates, postbiotics, prebiotics, oils, essential oils, polyphenols, bioflavonoids, berberine, phytosterols, phytostanols, fungi or seaweed in the form of extracts or powders, lipoic acid, glutathione (GSH), melatonin, resveratrol, GABA, coenzyme Q10, choline, amino acids, vitamins and minerals.

[0041] The composition of the present application is formulated into a dietary, nutritional or pharmaceutical preparation using conventional techniques and excipients.

[0042] Examples of preparations include swallowable tablets, chewable tablets, hard capsules, soft capsules, granules or powders reconstituted with water in a bottle or sachet or taken directly orally, solutions, suspensions, liquids, dissolvable or orally dispersible bars, vials or two-phase ampoules.

[0043] To demonstrate the synergistic profile of the composition of the present application, several natural extracts were evaluated for their inhibitory effect on HMG-CoA reductase: Olea europaea leaf and fruit extract (total polyphenol content 16%; plant / extract ratio = 8:1), policosanol extracted from sugar cane (total policosanol content > 98%, octacosanol content > 60%), resveratrol extracted from Polygonum cuspidatum (trans-resveratrol content > 98%), Scutellaria baicalensis root extract (baicalin content > 30%), Butea frondosa wood extract (catechin and epicatechin content > 21%), Punica granatum peel extract (gallic acid content > 40%), Psidium guajava leaf extract (guaijaverin content > 20%; plant / extract ratio = 4:1), Psidium guajava fruit extract (guaijaverin content > 20%).

[0044] The screening was performed using an analytical kit for the evaluation of HMG-CoA reductase inhibition. The assay is based on the spectrophotometric measurement of the absorbance decrease at 340 nm, which is indicative of the oxidation of NADPH by the catalytic subunit of HMG-CoA-reductase in the presence of the HMG-CoA substrate. The reaction is as follows: HMG-CoA + 2NADPH + 2H + → Acetyl-CoA + 2NADP + + CoA-SH. The modulation of the enzymatic activity and, in particular, its inhibition, was calculated as a percentage of the variation with respect to the activity measured under control conditions, i.e. in the absence of any inhibitor. The positive control, pravastatin, was used to verify the biochemical system at doses of 212.5 mg (100 mM), 80 mg, 40 mg and 20 mg.

[0045] The first test phase was performed using pravastatin as a positive control to calculate the theoretical concentration of the administered dose in the systemic circulation (5 L, mean blood volume of an adult male weighing about 70 kg);

[0046] Table 1 presents the data of the activity of the enzyme in the presence of pravastatin

[0047] Table 1

[0048]

[0049] Each test was performed in triplicate and the compounds were tested at different concentrations.

[0050] Several phases were performed to evaluate the inhibitory activity of each ingredient, so as to extrapolate the following oral administration doses:

[0051] Policosanol 10 mg

[0052] Psidium guajava leaf extract 100 mg

[0053] Psidium guajava fruit extract 100 mg

[0054] Pomegranate extract 100 mg

[0055] Scutellaria extract 100 mg

[0056] Resveratrol 10 mg

[0057] Catechu extract 100 mg

[0058] Olea leaf and fruit extract 100 mg

[0059] The first test phase (Test 1) was performed on a 1:200 dilution of the theoretical concentration of all the ingredients, in a dose for administration in the systemic circulation (5 L, average blood volume of an adult male of about 70 kg).

[0060] Table 2 presents the data relating to the activity of the enzyme in the presence of the various ingredients under the test conditions.

[0061] Table 2

[0062]

[0063] As mentioned above, the combinations of ingredients that performed best in Test 1 at a 1:200 dilution were investigated, with the aim of finding a solution that would have a beneficial effect on patients with hypercholesterolemia, without the need to use large doses of polyphenols. We decided to include in these combinations the total extract of Olea, even though it did not have inhibitory activity at a 1:200 dilution, since it is unanimously considered a valuable component of the Mediterranean diet, with many healthy properties.

[0064] Surprisingly, the Olea extract, which did not have inhibitory activity (see Test 1), showed an unexpected synergism with policosanol (+40.9% compared to the sum of the individual ingredients) and with the Scutellaria root extract (+44.1% compared to the sum of the individual ingredients).

[0065] Even more surprisingly, a synergism was found between the Olea fruit and leaf extract, policosanol and the Scutellaria root extract. In fact, the mixture of the three ingredients recorded a percentage of inhibition not only greater than the sum of the individual ingredients (+42.2%), but also greater than the sum of the combinations of only two ingredients (Olea + policosanol and Olea + Scutellaria), for which a synergism had already been found.

[0066] Not only was there no interference between the ingredients, which is the expected event using other combinations, but the result was significantly greater than the sum of the individual results for inhibiting the HMGR enzyme. See Table 3.

[0067] Table 3

[0068]

[0069] As an example, the percentage of HMGR enzyme inhibition of Olea europaea and other combinations of ingredients that performed best in Test 1 are shown below, demonstrating that other combinations were ineffective. See Table 4.

[0070] Table 4

[0071]

[0072] To establish the exact synergy between Olea europaea, Pristilane and Scutellaria baicalensis, different doses and weight ratios between them than those described above were tested, maintaining the dilution (1 :200). See Table 5.

[0073] Table 5

[0074]

[0075] The preparation of the formulations of the application using conventional techniques and excipients is shown in the following examples.

[0076] Example 1-3: Hard capsules

[0077] Example 1

[0078]

[0079] Example 2

[0080]

[0081] Example 3

[0082]

[0083] Example 4-5: Tablets / coated tablets

[0084] Tablets can be prepared by direct compression of the ingredients or by compression of a granular form thereof (dry or wet granulation). The ingredients can advantageously be distributed between two or more layers of the tablet having different release kinetics of the ingredients.

[0085] Example 4

[0086]

[0087] Example 5

[0088]

[0089] Example 6: Sachets / sticks containing water-dispersible and / or mouth- dispersible powders and / or granules

[0090]

[0091] Example 7: Soft capsules

[0092] Before the capsule filling process is started, two preparation processes are usually carried out simultaneously but separately, thus forming two parts of the soft capsule, namely the preparation of (1) the gel substance and (2) the matrix used as the filler of the capsule.

[0093]

[0094] Example 8: Solution / suspension

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[0096] List of documents

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Claims

1. A composition comprising or consisting of an Olea europaea fruit and leaf extract and a Scutellaria radix extract for use as an inhibitor of HMG-CoA reductase in the treatment of dyslipidemia.

2. The composition for use according to claim 1, wherein the Olea europaea fruit and leaf extract has a polyphenol content ranging from 5% to 95%, preferably from 10% to 70%, and more preferably from 15% to 50% by weight of the extract.

3. The composition for use according to claim 1 or 2, wherein the Scutellaria radix extract has a baicalin content ranging from 5% to 95%, preferably from 10% to 70%, and more preferably from 15% to 50% by weight of the extract.

4. The composition for use according to claim 1, 2 or 3, further comprising policosanol.

5. The composition for use according to one or more of claims 1 to 4, which is free of any other plant extract.

6. The composition for use according to one or more of claims 1 to 5, wherein the weight ratio of the Olea europaea fruit and leaf extract to policosanol ranges from 1 : 1 to 50: 1, preferably from 2: 1 to 25: 1 ; and more preferably from 3: 1 to 10:

1.

7. The composition for use according to one or more of claims 1 to 6, wherein the weight ratio of the Olea europaea fruit and leaf extract to the Scutellaria radix extract ranges from 0.1 : 1 to 5: 1, preferably from 0.2: 1 to 2: 1, and more preferably from 1 : 1 to 0.5:

1.

8. The composition for use according to one or more of claims 1 to 7, comprising from 5 mg to 300 mg of Olea europaea fruit and leaf extract per dosage unit; preferably from 20 mg to 150 mg; and more preferably from 30 mg to 100 mg.

9. The composition for use according to any one of claims 1 to 8, comprising from 2 mg to 30 mg of policosanol per dosage unit; preferably from 10 mg to 25 mg; and more preferably from 15 mg to 20 mg.

10. The composition for use according to any one of claims 1 to 9, comprising from 5 mg to 300 mg of Scutellaria radix extract per dosage unit; preferably from 20 mg to 200 mg; and more preferably from 50 mg to 150 mg.

11. The composition for use according to any one of claims 1 to 4 and 6 to 10, further comprising from 10 mg to 400 mg of Psidium guajava leaf and / or fruit extract per dosage unit; preferably from 20 mg to 200 mg; and more preferably from 50 mg to 150 mg.

12. Composition for use according to any one of claims 1 to 4 and 6 to 11, further comprising one or more ingredients selected from the group consisting of herbal derivatives in extract or powder form, ferments, probiotics, yeasts, bacterial lysates, postbiotics, prebiotics, oils, essential oils, polyphenols, bioflavonoids, berberine, phytosterols, phytostanols, fungi in extract or powder form, seaweed in extract or powder form, lipoic acid, glutathione, melatonin, resveratrol, GABA, coenzyme Q10, choline, amino acids, vitamins and minerals.

13. Composition for use according to one or more of claims 1 to 12, in the form of a swallowable tablet, a chewable tablet, a hard capsule, a soft capsule, granules or powder to be reconstituted with water in a bottle or sachet or taken directly orally, a solution, a suspension, a liquid, a bar or a sachet to be dissolved or dispersed in the mouth or a two-phase ampoule.

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