WO2011112067A1

WO2011112067A1 - A herbal composition having anti-obesity property

Info

Publication number: WO2011112067A1
Application number: PCT/MY2010/000196
Authority: WO
Inventors: Ismail Zhari Bin; Hussain Khalid
Original assignee: Universiti Sains Malaysia
Priority date: 2010-03-11
Filing date: 2010-09-29
Publication date: 2011-09-15

Abstract

A herbal composition for inhibiting neovascularisation of adepocytes comprising extract or fraction derived from Piper sarmentosum.

Description

A HERBAL COMPOSITION HAVING ANTI-OBESITY PROPERTY

FIELD OF INVENTION

The present invention relates to a herbal composition having anti-obesity property. In more particular, the present invention relates to an anti-obesity product made of natural herbs, which inhibits neovascularisation to the fast growing adipocytes in adipose tissue, thus reducing the deposition of fat.

BACKGROUND OF THE INVENTION

Obesity is not merely a cosmetic problem but associated with co-morbidities such as risk of type 2 diabetes, cardiovascular diseases and certain cancers. In the Organization of Economic Cooperation and Development (OECD), the prevalence of overweight and obesity was found to be 33.7% and 13.5%, respectively, in year 2007. Obesity is found to be increasing worldwide at an alarming rate and estimated to be 1.5 billion by 2015.

Obesity is a complex disease involving various poorly understood pathways. As a result, the development of anti-obesity drugs is slower compared to other drugs. There are only a few drugs available in the market which can reduce weight by 5% to 10% when taken with restricted diet and exercise. Hence, there is a dire need to explore new sources for substances to be used to control obesity. In drug discovery, plants have the special place due to their biodiversity, but the main complication associated with natural products is difficulty in maintaining quality and efficacy.

Standardization is a process to maintain consistency in claimed efficacy of a formulation and its batch-batch reproducibility. Despite many proven benefits, natural products could not get wider acceptance in the main stream of pharmaceuticals due to lack of standardization. It is due to unavailability or inadequacy of standards and methods for standardization. The other factors making standardization of herbal medicines further tedious are complexity of constituents, unknown chemicals and variation in chemical composition due to heterogeneity of plant species, plant part, growth conditions, seasonal cycle, climate and age of the plant. The way of collection, processing, storage and interaction with other constituents are the additional contributing factors. Therefore, a natural product must be standardized to ensure the consistency in quality and efficacy resulting evidence-based medicines from folklore remedies.

Keeping in view the importance of plants in drug discovery, various local medicinal plants have been investigated and researched. Piperaceae (pepper) family is one of the herbaceous shrubs which has been aggressively investigated into as it is well known in South East Asian countries due to its medicinal properties.

There are a few patented technologies disclosed in the prior arts relating to anti- obesity products containing plant extracts from the Piperaceae family. A Japanese Patent No. JP8245410 discloses the use of Piper nigrum-L as an active ingredient in a lipolysis promoter. However, this application is complicated as the promoter is blended with one or two more kinds of substances selected from xanthine derivative, a beta-adrenergic stimulant, an adrenergic suppressor and a bipyridine derivative, to obtain the anti-obesity effect.

Besides, there is also an anti-adipogenic composition disclosed in PCT publication No. WO2009040824 for inhibition, amelioration or prevention of adipogenesis mediated diseases such as obesity, lipid storage disease and hyperlipemia. However, this herbal composition must have a biologically effective amount of extract or fractions from Piper betel in combination with one or more of the extracts or fractions derived from D. biflora, Commiohora mukul, Boerhaavia diffusa, tribulus terrestris and Zingiber officinale. This composition requires a bio-enhancing agent or bio- protecting agent as well.

U.S. Patent no. US5698199 relates to a lipolysis acceleration method which comprises orally administering a thistle series or a pepper-family plant or an extract thereof by local administration or as a bath medicine composition. This invention exhibits excellent effects for the control, prevention and improvement of obesity. However, there is no formulation suggested for a herbal composition made of the plant from the pepper family which has anti-obesity property.

Even though some patented technologies disclose the use of plants from the Piperaceae family in anti-obesity products, they are usually applied in combination with other types of herbs or in trace amount (as a minor additive). As the plants from the Piperaceae family are potential in expressing anti-obesity effect, it is desirable for the present invention to provide a herbal composition of a selected species from this family to overcome the drawbacks of the prior arts. Piper sarmentosum can be a good candidate for this herbal composition. It has been investigated for a number of pharmacological activities such as anti-amoebic, antibacterial, anti-neoplastic, neuromuscular blocking, hypoglycemic, anti-malarial, antioxidant, anti-tuberculosis, anti-angiogenic activity and anticancer properties, yet it is not suggested in any of the prior arts to use this species in the manufacture of an anti-obesity product with higher level of effectiveness as compared to the existing anti-obesity products.

SUMMARY OF INVENTION

The primary object of the present invention is to provide a herbal composition of a selected species from the Piperaceae family which is capable of exhibiting an anti- obesity property. Another object of the present invention is to provide a herbal composition of P. sarmentosum as an evidence-based product to combat obesity.

Still another object of the present invention is to diversify the medicinal uses of the P. sarmentosum.

Yet another object of the present invention is to develop a method for standardizing natural products in terms of their medicinal properties, safety, pharmacokinetics and stability.

At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiments of the present invention describes a herbal composition for inhibiting neovascularisation of adepocytes comprising extract or fraction derived from Piper sarmentosum.

In a preferred embodiment of the present invention, the extract or fraction is present in an amount of 25% to 50% by weight of the composition. The extract or fraction is preferably derived from leaves of P. sarmentosum.

Another preferred embodiment of the present invention discloses that the extract or fraction comprises rutin, flavonone, pellitorine, sarmentine, sarmentosine, polyphenols, amides and glycosaponins.

Still another embodiment of the present invention is an anti-obesity drug comprising a herbal composition as embodied herein.

Further embodiment of the the present invention is a method for inhibiting neovascularisation of adipocytes in a mammal comprising administering to the mammal a herbal composition having an extract or fraction derived from Piper sarmentosum. Preferably, the herbal composition administered into the mammal's body is in a range of lOOmg/kg to 2000mg/kg.

This anti-obesity drug is tested and compared with commercial anti-obesity drugs, and found to be 30% to 40% superior. Besides, the product is also tested for safety and stability.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiments described herein are not intended as limitations on the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of facilitating an understanding of the invention, there is illustrated in the accompanying drawing the preferred embodiments from an inspection of which when considered in connection with the following description, the invention, its construction and operation and many of its advantages would be readily understood and appreciated.

Figure 1 shows the ex vivo inhibition of neovascularisation of the anti-obesity drug as described in one of the preferred embodiments of the present invention, which is denoted as Nobesiti, in comparison to suramin (standard) and markers used for standardization of the product, Al (pellitorine), A2 (sarmentine), and A3 (sarmentosine).

Figure 2 shows the comparison of weight gain in 45 days in animals (Sprague

Dawley rats) of different groups such as control (normal), treated with the Nobesiti, treated with 100 mg/kg each Nobesiti and fat, treated with 100 mg/kg each of Xenical (a commercially obtained product) and fat, and treated with fat 100 mg/ kg. is the comparison of IC₅o of Nobesiti, required to inhibit neovascularisation and to inhibit the growth of human umbilical vascular endothelial cells (HUVAC). IC₅o for inhibition of neovascularisation is less than IC₅o for cytotoxicity. is the comparison of different markers in the control and the Nobesiti- treated rats for 45 days. shows the micrographs of hepatic tissue of Nobesiti-treated rat and control (untreated) rat, the hepatocytes were found to be normal in both the rats showing no signs of toxicity. shows the pharmacokinetic profile of Nobesiti after oral absorption with reference to marker compounds showing absorption and excretion profile.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the invention shall be described according to the preferred embodiments of the present invention and by referring to the accompanying description and drawings. However, it is to be understood that limiting the description to the preferred embodiments of the invention and to the drawings is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.

The present invention discloses a herbal composition for inhibiting neovascularisation of adepocytes comprising extract or fraction derived from Piper sarmentosum.

P. sarmentosum is a herbaceous shrub which may be creeping along the ground or erect and grows up to 50cm to 60cm. This plant is well known in the traditional therapeutic system, as its leaves can be used to treat fever and indigestion, sooth headaches or mitigate chest pain according to the different practices in different regions. Whilst, its roots can be used to relieve toothaches, treat dermatomycoses, pleurisy, cough, asthma and toothaches. Besides, its fruits can be used as an expectorant. In the present invention, the leaves of P. sarmentosum is employed for use in combating obesity.

The plant has been selected on the basis of the research on in vitro inhibition of vascularization on diverse types of extracts of different parts of the plant. Every plant of a genera has some chemical constituents different from other members. P. sarmentosum contains many compounds which are not present in other species of Piperaceae.

The main constituents of the P. sarmentosum includes amide alkaloids, phenylpropanoids, pyrones, flavonoids, sterols and neolignans. There are more than 67 compounds identified from the essential oil of the P. sarmentosum and the main compounds obtained are 2, 4, 5-trimethoxy-l-propenylbenzene (23.20%), cis- caryophyllene (13.33%), 1 , 2-dimethoxy-4-(l-propenyl)-benzene (12.63%), 1, 3- benzodioxole-4-methoxy-6-(2-propenyl) (5.71%) and delta-cadinene (3.03%). The main components of the essential oil obtained from leaves of this plant are 10-Epi-γ- eudesmol which made up of 21.0%, a-cadenine which made up of 18.8%, seychellene which made up of 12.6%, and (E, E)-farnesol which made up of 10.5% by weight of the essential oil.

According to the preferred embodiment of the present invention, the extract or fraction is for the herbal composition is prepared from leaves of P. sarmentosum. Preferably, petroleum ether and chloroform is used to obtain the extract. The present invention is preferably to contain both the extract and fraction of P. sarmentosum in 2 : 1 ratio, as the extract/fraction is found to be superior to that of other extracts of the same part.

The extract of P. sarmentosum contains various types of chemical components, which include β-sitosterole, dihydrocinnamic acid, l-(3,4-methylenedioxyphenyl)-lE- tertadecene, N-(3-phenylpropanoyl)-pyrrole, N-isobutyl-2E,4E-decadienamide or pellitorine, N-[7-(3,4-methylenedioxyphenyl)-2E,6E-heptadienoyl]-pyrrolidine or sarmentosine, sarmentine or N-(2E,4Edecadienoyl)-pyrrolidine, caryophyllene, dodecane, β-pinene, limonine, safole, lallyl-2,5-dimethoxy-4,5- methylenedioxybenzene, 1 -(3 ,4-methylenedioxyphenyl)- 1 E-tetradecene, acids (palmitic, stearic, oleic, linoleic and linoleic), l-allyl-2,6-dimethoxy-3,4- methylenedioxybenzene, l-allyl-2,4,5 trimethoxybenzene, l-(lE-propenyl)-2,4,5- trimethoxybenzene, lallyl-2-methoxy-4,5-methylenedioxybenzene, N-(2- methylbutyl)-2E,4E-decadienamide, sesamin, horsfieldin, guineensine, brachhystamide B, sarmentamide A, B and C. In these constituents, sarmentosine and sarmentine are the major constituents, the preparation is active as a whole.

In a preferred embodiment of the present invention, the extract or fraction is present in an amount of 25% to 50% by weight of the composition. Another preferred embodiment of the present invention discloses the extract or fraction comprises various active compounds in different percentages, for instance, rutin (0.20 mg/g), flavonone (0.32 mg/g), pellitorine (0.043 mg/g), sarmentine (0.006 mg/g) and sarmentosine (0.005 mg/g). It is also found to contain polyphenols (7%), amides (12%) and glycosaponins (3%). and total amides (28-30%). In addition, the herbal composition further comprises starch, carboxy methylcellulose, lactose and croscarmellose sodium.

Still another embodiment of the present invention is an anti-obesity drug comprising a herbal composition as embodied herein. The method of preparation includes collection of plant material, drying and extraction. Chloroform extract fractionated with hexane and chloroform sequentially, then the extract and fraction in 2 : 1 ratio were combined with excipients as set forth in the preceding description. Preferably, the anti-obesity drug is orally administered into a mammal's body in an mount of 1 OOmg/kg to 2000mg/kg by weight of the mammal's body.

This anti-obesity drug is subjected to a series of evaluation tests and studies. Analytical studies includes physicochemical analysis, isolation of markers which include sarmentine, pellitorine and sarmentosine; and qualitative and quantitative analysis. Pharmacodynamics of the drugs are also investigated whereby the standardized extracts can be evaluated using in vitro and in vivo models. This can be followed by a safety profiling process, in which animals are used to examine for acute and chronic toxicity. Pharmacokinetic studies including adsorption, distribution, metabolism and excretion are also conducted. Further studies involved can be accelerated stability studies such as shelf life and chemical kinetics. These evaluation studies are further demonstrated in the following examples.

The efficacy studies of the anti-obesity drug can also be performed. This anti-obesity drug is tested and compared with commercially obtained anti-obesity drugs, and found to be 30% to 40% superior because of the potent activity of the preparation. The test methodologies and results are further described in the examples.

Further embodiment of the the present invention is a method for inhibiting neovascularisation of adepocytes in a mammal comprising administering to the mammal a herbal composition having an extract or fraction derived from Pipe sarmentosum. Preferably, the effective amount of the composition is lOOmg/kg to 2000mg/kg by weight of the mammal's body. The product can be suspended in water and administered to animals orally using feeding needle.

The present disclosure includes as contained in the appended claims, as well as that of the foregoing description. Although this invention has been described in its preferred form with a degree of particularity, it is understood that the present disclosure of the preferred form has been made only by way of example and that numerous changes in the details of construction and the combination and arrangements of parts may be resorted to without departing from the scope of the invention.

EXAMPLE

Examples are provided below to illustrate different aspects and embodiments of the present invention. These examples are not intended in any way to limit the disclosed invention, which is limited only by the claims.

Example 1

Efficacy test was carried out using an ex vivo model. The anti-obesity drug as described in the preceding preferred embodiments were denoted as Nobesiti. Suramin was used as the standard, and markers used for standardization of the product includes Al (pellitorine), A2 (sarmentine), A3 (sarmentosine). The ex vivo inhibition of neovascularisation of Nobesiti is shown in Figure 1.

Example 2

Efficacy test was carried out using an in vivo model. Figure 2 shows the comparison of weight gain in 45 days in Sprague Dawley rats of different groups such as control (normal), treated with Nobesiti, treated with 100 mg/kg each Nobesiti and fat, treated with 100 mg/kg each of commercially obtained Xenical and fat, and treated with fat 100 mg/ kg. The group of animals treated with Nobesiti recorded the lowest percentage in weight gain. In 45· days, weight gain with Nobesiti along with fat 100 mg/kg/day was lesser as compared to combination of Xenical and fat 100 mg fat.

Example 3

Cytotoxicity test was conducted using human umbilical vascular endothelial cells. Figure 3 shows the comparison of IC₅o of Nobesiti which is required to inhibit neovascularisation and to inhibit the growth of human umbilical vascular endothelial cells. As shown in Figure 3, IC₅o for inhibition of neovascularisation is less than IC₅o for cytotoxicity.

Example 4

The safety test was also conducted to examine the acute oral toxicity of the drug. The extract of the P. sarmentosum was evaluated for acute oral toxicity using "Up and down procedure" described in OECD guidelines. Female Sprague Dawley rats weighing 250 g ± 20 and age 8-12 weeks were used. The LD₅o of the Nobesiti was found to be more than 2000 mg/kg body weight because all the animals survived at limit dose (2000 mg/kg) without any signs and symptoms of toxicity. Besides, subacute toxicity test was also carried out by administering the rats with Nobesiti at an oral dose of 100 mg/kg per day for 45 days. Then, the animals were sacrificed to take plasma and liver. Plasma was analyzed for certain markers while liver was used for histology and to prepare liver homogenate, which was analyzed for certain markers to assess any toxicity. The results were compared with that of normal rats. Micrographs of hepatic tissue of Nobesiti-treated rat and control (untreated) rat are illustrated in Figure 5. The hepatocytes were found to be normal in both the rats showing no signs of toxicity.

Example 5

Pharmacokinetic profile of Nobesiti was studied after oral absorption with reference to marker compounds showing absorption and excretion profile. Cumulative excretion of pellitorine, sarmentine and sarmentosine in fecal matter and urine volume in experimental and control groups (Con) after oral administration of Nobesiti, each value represents the mean ± S.D of three rats is given in Table 1.

Table 1

Excretion parameters Urinary excretion

Pellitorine

Rat-1 Rat-2 Rat-3 Mean SD

Cumulative amount (0 - 72 h. ug)

0.0910 0.0564 0.0845 0.0773 0.0183

Percent of dose (%)

0.0008 0.0006 0.0009 0.0007 0.0001

Peak time (h) 48 48 48 48 0.00

Maximum excretion rate (ug/h)

0.00189 0.00117 0.00176 0.00161 0.00038

Sarmentine

Cumulative amount (0 - 72 h. ug)

1.2176 0.866 0.844 0.976 0.2093

Percent of dose 0.0041 0.0036 0.0035 0.0037 0.0003

Peak time (h) 24 24 24 24 0.00

Maximum excretion rate (ug/h) 0.02584 0.02814 0.02498 0.02631 0.00164

Sarmentosine

Cumulative amount (0 - 72 h. ug) 5.20551 3.04516 4.88136 4.37734 1.16503

Percent of dose 1.11658 0.65318 1.04705 0.93894 0.2499

Peak time (h) 24 24 24 24 0.00

Maximum excretion rate (ug/h) 0.1549 0.0782 0.1476 0.1269 0.0423

Renal excretion

Rat-1 Rat-2 Rat-3 Mean SD

Cumulative urinary volume

14.52 18.35 16.75 16.54 1.92361 (0-24 h, ml)

Maximum urine flow rate (ml/h) 0.605 0.765 0.697 0.689 0.08029

Control Control Control Mean SD

1 2 3

Cumulative urinary volume 15.67 17.34 14.43 15.8133 1.46028 (0-24 h, ml)

Maximum urine flow rate (ml/h) 0.653 0.723 0.602 0.65933 0.06074

Example 6

The accelerated stability of the Nobesiti was conducted by shelf life test. Shelf life of Nobesiti with reference to markers at different storage conditions is tabulated in Table 2.

Table 2

Marker

25 °C 30 °C/60%RH 40 °C/75%RH 60 °C/85%RH

Pellitorine 16.94 11.29 5 1.24

Sarmentine 16.67 10.94 4.77 1.07

Sarmentosine 16.15 11.80 6.44 2.17

Example 7

In an example of the formulation of Nobesiti, the product was found to contain active ingredients of rutin (0.20 mg/g), flavonone (0.32 mg/g), pellitorine (0.043 mg/g), sarmentine (0.006 mg/g), sarmentosine (0.005 mg/g), total polyphenols (7%), total amides (12%) and total glycosaponins (3%).

Claims

1. A herbal composition for inhibiting neovascularisation of adepocytes comprising extract or fraction derived from Piper sarmentosum.

2. A composition according to claim 1, wherein the extract or fraction is present in an amount of 25% and 50% by weight of the composition.

3. A composition according to claim 1, wherein the extract or fraction is derived from leaves of Piper sarmentosum.

4. A composition according to claim 1, wherein the extract or fraction comprises rutin, flavonone, pellitorine, sarmentine, sarmentosine, polyphenols, amides and glycosaponins.

5. An anti-obesity drug comprising a herbal composition according to claim 1.

6. A method for inhibiting neovascularisation of adepocytes in a mammal comprising administering to the mammal a herbal composition having an extract or fraction derived from Piper sarmentosum.

7. A method according to claim 6, wherein the herbal composition administered into the mammal's body is in a range of lOOmg/kg to 2000mg/kg.