WO2010032269A2

WO2010032269A2 - Anti-inflammatory activity of the iridoid glycosides

Info

Publication number: WO2010032269A2
Application number: PCT/IN2009/000512
Authority: WO
Inventors: Maninder Karan; Inderpreet Kaur; Divya Chopra
Original assignee: Vasisht, Karan
Priority date: 2008-09-18
Filing date: 2009-09-18
Publication date: 2010-03-25
Also published as: WO2010032269A3

Abstract

The present invention provides method of treating mammals and humans having inflammation, pain and swelling with pharmaceutical compositions containing iridoid glycosides selected from barlerin. acetyl barlerin and shanzhisidc methyl ester or combination thereof isolated from the plant source or alcoholic extract of the plant source Barleria crislala. Further, the present invention also provides a process for the isolation of these iridoid glycosides.

Description

ANTIINFLAMMATORY ACTIVITY OF THE IRIDOID GLYCOSIDES FIELD OF THE INVENTION

The present invention provides a method of treating mammals and humans having inflammation, pain or swelling using the pure iridoid glycosides acetyl barlcrin, barlcrin and shan/hisidc methyl ester individually; or in combination thereof.

The present invention provides three iridoid glycosides namely, barlcrin, acetyl barlcrin and shanzhiside methyl ester, isolated from the plant source, preferably from Barlcria cristata and process of their isolation from the plant source.

The present invention further provides an anti-inflammatory activity of the pure iridoid glycosides isolated from plant source Barleria cristata namely: barlerin, acetyl barlcrin and shan/.hiside methyl ester, as well as bioactive fraction (alcoholic extract) of plant source containing these iridoid glycosides.

Particularly, the present invention provides anti-inflammatory activity of bioactive fraction (alcoholic extract) and the pure iridoid glycosides isolated from the plant source Barleria cristata var. dichotoma.

BACKGROUND OF THE INVENTION

Barleria cristata (and its var. dichotoma) belong to family Λcanthaccac and is well-known plant used in the indigenous system of medicine in India. Almost all parts of this plant are used as medicine. In Ayurveda, the plant is recommended for the treatment of fever, bronchitis, blood disease, pains and asthma. The roots and leaves of the plant arc used to reduce swelling and an infusion is given in cough. Various parts of the plant are found to contain chemical compounds, which arc studied and isolated.

Two iridoids, acetyl barlcrin and shanzhiside methyl ester have been isolated from the ethyl acetate extract of Barleria cristata. Apigenin, naringcnin, apigcnin glucuronidc and malvidin-3.5- diglucosidc have been identified in Barleria cristata having violet flowers while apigcnin-7- glucuronidc is isolated from Barleria cristata with white flowers (Barleria cristata var. dichotoma).

The first report on the chemical investigation of this plant appeared in 1972 when Subramanian and Nair reported the presence of apigcnin 7-glucuronidc from white (lowers. Bulletin of Pharmaceutical Sciences, Assiut University (1990), 13(1 ), 65-72 described the isolation of two iridoids namely acetyl barlcrin and shanzhiside methyl ester from the plant Barleria cnslata. However neither the activity of the plant extract nor of the isolated iridoid glycoside is disclosed in this journal. There are many varieties of the plant Barleria crislata known to exist in nature; the above journal does not disclose that which plant variety is taken for the isolation of the iridoids.

Inflammation is the complex biological response of vascular tissues to harmful stimuli including pathogens, irritants or damaged cells. It is an protective effort by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue. The most common therapy for the treatment of inflammatory disorders involves use of non-steroidal anti-inflammatory drugs (NSAIDs) e.g. naproxen, diclofenac, ibuprofen to alleviate symptoms such as pain. However, despite the widespread use of NSAIDs, many individuals cannot tolerate the doses necessary to treat the disorder over a prolonged period, as NSAIDs arc known to cause gastric erosions. Moreover. NSAIDs merely treat the symptoms of disorder and not the cause.

When patients fail to respond to NSAIDs, other drugs such as methotrexate, gold salts. D- pcnicillamine and corticosteroids are used. These drugs also have significant toxic eflects. Monoclonal antibody drugs such as infliximab, etanerccpt and adalimumab arc useful as antiinflammatory agents, but have drawbacks such as route of administration (only parenteral), high cost, allergy induction, activation of latent tuberculosis, increased risk of cancer and congestive heart disease. Hence, there is a need for the development of improved and alternative medicaments with reduced side effects for the prevention and treatment of inflammatory disorders. I hroughout history a wide variety of plants and herbs, and extracts of such plants and herbs arc used to alleviate aches and pains, improve immunity to infection, treat various illnesses, or even to induce relaxation or stress reduction.

Research on the biological activities of the plants during the past two centuries has yielded numerous compounds for the development of modern drugs. There is also several evidences that products derived from plants have potential pharmacological and therapeutic effects on mammals and tend to have less deleterious side effects than synthetic drugs.

To our knowledge, there is no report of any medicament containing extract of Barleria crislala for treatment of inflammatory disorders. To overcome the problems of side effects of present line of treatment, the present invention provides pure iridoid glycosides isolated from a plant source Barleria cristala var. dichotoma which arc effective in treating inflammation, pain and swelling. OBJECT OF THE INVENTION

The principal object of the invention is to provide method of treating mammals and humans having inflammation, pain and swelling with pharmaceutical composition containing pure iridoid glycosides barlerin, acetyl barlcrin and shanzhiside methyl ester individually or in combination thereof.

Another object of the invention is the isolation of pure iridoid glycosides from a plant source Barleria crislata. Particularly the main object of the invention is the isolation of pure barlerin from a plant source Barleria crislala along with acetyl barlerin and shanzhiside methyl ester.

Another object of the invention is to study the anti-inflammatory activity of pure iridoid glycosides: acetyl barlerin, barlerin and shanzhiside methyl ester isolated from a plant source of Barleria cristala.

Another object of the invention is to study the anti-inflammatory activity of bioactivc fraction i.c alcoholic extarct of plant source Barleria cristala.

Still another object of the invention is to provide the comparable data, which demonstrate that antiinflammatory activity of the isolated glycosides is better than the standard drug (ibuprofcn).

Yet another object of the invention is to provide use of the iridoid glycoside to prepare a pharmaceutical composition useful for treating inflammation, pain and swelling in mammals and animals.

SUMMARY OF THE INVENTION

According to one embodiment, the present invention provides a method of treating mammals and humans having inflammation, pain and swelling with pharmaceutical composition containing pure iridoid glycosides barlcrin, acetyl barlerin and shanzhiside methyl ester individually or in combination thereof.

According to one embodiment, the present invention provides the isolation of an iridoid glycoside barlcrin from the plant source Barleria cristala for the first time along with other two iridoid glycosides namely, acetyl barlerin and shanzhiside methyl ester.

According to another embodiment, the present invention provides a method for the isolation of the pure iridoid glycosides barlcrin, acetyl barlerin and shanzhiside methyl ester from a plant source Barleria crislata The process comprises the step of: a) mechanically disrupting the whole plant; b) extracting with alcohol or aqueous alcohol; c) concentrating the alcoholic extract; d) partitioning the concentrate between water and an organic solvent; e) optionally, repeating the step d); f) optionally, extracting the aqueous extract with butanol; g) optionally, concentrating the butanol extract, h) isolating pure iridoid glycosides using chromatographic techniques; and i) optionally, crystallizing the isolated iridoid glycosides with suitable solvent.

According to another embodiment, the present invention provides anti-inflammatory activity of pure iridoid glycosides: barlerin; acetyl barlerin and shanzhiside methyl ester isolated from plant source Barleria cristata.

According to another embodiment, the present invention provides a method of treating mammals and humans having inflammation, pain and swelling with pharmaceutical composition comprising alcoholic extract prepared from the plant source Barleria cristata which contains iridoid glycosides such as barlerin, acetyl barlerin and shanzhiside methyl ester along with other components.

According yet another embodiment, present invention provides a process for the preparation of alcoholic extract from the plant source Barleria cristata.

The process comprises the step of: a) mechanically disrupting the whole plant; b) extracting with alcohol or aqueous alcohol; and c) concentrating the alcoholic extract.

According to another embodiment, the present invention provides anti-inflammatory activity of the bioactive fraction (alcoholic extract) of the plant Barleria cristata containing three iridoid glycosides: acetyl barlerin, barlerin and shanzhiside methyl ester along with other components. According to one more embodiment, the present invention further provides a method oϊ treating mammals and humans having inflammation and pain with the isolated glycosides.

According to another embodiment, present invention provides the use of the iridoid glycosides. barlerin, acetyl barlerin, and shanzhiside methyl ester individually or in combination thereof to prepare a pharmaceutical composition useful for treating inflammation, pain and swelling in mammals and animals.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the term "Barleria crislata" refers to plant Barleria cristata and its various varieties. Preferably, Barleria cristata var. dichotoma is used.

According to one embodiment, the present invention provides first time the isolation of an iridoid glycoside barlerin along with two known iridoid glycosides, namely acetyl barlerin and shan/hiside methyl ester from the plant Barleria cristata.

It is preferred to isolate the above iridoid from the plant source Barleria cristata var. dichotoma

According to another embodiment, the present invention provides a process for the isolation of the iridoid glycosides: barlerin, acetyl barlerin and shanzhiside methyl ester from a plant source with genus Barleria, preferably from Barleria cristata.

The process of the isolation comprises, mechanically disrupting the whole plant followed by extraction with alcohol which is then concentrated. Thereafter the concentrate is partitioned between water and a organic solvent. Preferably the organic solvent employed is moderately polar organic solvent. Thereafter partitioning can be repeated with same or different solvent, it is advantageous to extract the obtained aqueous portion with butanol followed by concentration. I he resulting residue is then chromatographed to isolate pure iridoid glycosides, which can be rccrystallized with suitable solvent, wherever required.

The mechanically disrupting the plant comprises drying, grinding, crushing, and macerating the plant or a combination thereof. After disrupting the plant, it is extracted with an alcoholic solvent, preferably methanol, cthanol and the like. Marc of the plant is separated from the alcoholic solvent by suitable techniques such as filtration and the like. Marc can be again extracted with alcohol and this process can be repeated to completely extract the bioactivc fraction from the plant. All the alcoholic extracts arc combined and then concentrated by suitable techniques such as evaporation. thin film evaporator, lyophilization, spray drying and the like. Thin layer chromatography analysis of alcoholic extract shows the presence of iridoid glycosides.

Thus obtained alcoholic extract is also found to be active against inflammation, pain and swelling. The alcoholic extract can be used to prepare pharmaceutical composition for treating humans and mammals having inflammation, pain and swelling. This extract contains the iridoid glycosides barlcrin, acetyl barlerin and shanzhisidc methyl ester along with other components.

The concentrated alcoholic extract can be then partitioned between water and a organic solvent Organic solvent used for partitioning can be selected from the group consisting of petroleum ether, hcxane, dichloromethane, chloroform, diethyl ether, ethyl acetate, and the like. I he partitioning can be done with a series of solvents with increasing polarity. The preferred scries is hcxane. chloroform that can be followed by ethyl acetate. The alcoholic extract is first partitioned between water and hexane. Thereafter the resulting aqueous portion is successively washed with chloroform and ethyl acetate and is then extracted with n-butanol. The purpose of the partitioning is to be enrich the content of glycosides in the extract. The final butanol extract or extract of any stage (hcxane extract, chloroform extract or ethyl extract) can be subjected to suitable chromatographic methods I o isolate iridoid glycoside thereof. The suitable chromatographic methods include high-pressure liquid chromatography, medium pressure liquid chromatography or column chromatography, preparative thin layer and the like. Preferably the separation of the iridoid glycosides is performed by column chromatography. Column chromatography can be carried out using silica gel as stationary phase and an cluting solvent as mobile phase. Silica gel used can be of si/c ranging from 60-400 mesh and preferably, 60-120 mesh and 100-200 mesh arc used. Elution of the compound from the column depends upon both the polarity of the eluting solvent as well as polarity of the compound to be elutcd. Preferred cluting solvent is a mixture of chloroform and methanol. I he polarity of the cluting solvent can be increased by increasing the amount ol methanol in the mixture. The different fractions containing iridoid glycosides are collected and then concentrated.

The fractions elutcd in 10 -14 % methanol in chloroform from the column, contains acetyl barlerin having formula I, FORMULA I

and barlcrin having formula II.

FORMULA II

Next fractions clutcd with 15- 20 % methanol in chloroform contain shan/hisidc methyl ester having formula III.

FORMULA III

All the three active iridoid glycosides are cluted from the column with up to 20% of methanol in chloroform. The isolated iridoid glycosides can further be purified by repeating column chromatography or re-crystallization with a suitable solvent, wherever required. I he preferred solvent for rc-crystallization is a mixture of chloroform and methanol in any suitable proportion, preferred ratio is 85-95: 15-5, most preferred ratio is 90: 10 i.e. 10 % methanol in chloroform.

The isolated iridoid glycosides, acetyl barlcrin (Found: C, 51.6; H, 5.9; calc. for C₂₁ H₃₀O_1.3: C, 51 .4: H, 6.1 %), barlcrin (Found: C, 50.1 ; H, 6.2; calc. for Ci₉M_2SOi₂: C, 50.9; H. 6.3%) and shan/.hisiclc methyl ester (Found: C, 50.2; H, 6.2; cal. for Ci₇H₂₆On : C, 50.3; H, 6.4%) arc characterized by proton nuclear magnetic resonance (¹M NMR) and carbon nuclear magnetic resonance (' 'C NM R). NMR spcctras arc recorded in CD₃OD using a Spectroscopin & Brukcr spectrometer (400 M l I/ for ¹H-NMR and 400 MHz for ¹³ C-NMR)

Acetyl barlerin is characterized by following data: ¹H NMR (400 MHz, CD₃OD) (δ): 1.45 (H-IO) [s], 1.92 (OCOOCH₃) |.v|

1.95 (OCOOCH₃) [s], 1.99 (H-7) [d J= 5.52 Hz], 2.03 (H-7) [d, J= 5.56 Hz], 2.84 (H-9) [M, ./ -- 8.56 and 3.44 Hz], 3.09 (H-5) [dd, J = 7.98 and 1.12 Hz], 3.30 - 3.60 (H-2' - H-6') [m\, 3.58 (COOCH₃) |>], 4.77 (H-I ') |>], 5.16 (H-6) [d, J= 5.56 Hz], 5.74 (H-I ) [d, J = 3.48 Hz], 7.41 (1 1-3) |4 J= 1.48 Hz]

¹³C NMR (75 MHz, CD₃OD) (δ) :93.96 (C₁), 153.1 1 (C₂), 107.10 (C₄), 38.52 (C₅), 77.55 (C₆), 43.5.9 (C₇), 88.27 (C₈), 48.87 (C₉), 29.39 (C₁₀), 20.88 (CH₃), 20.43 (CH₃), 50.58 (CH₃O), 167.1 7(CO), 171.62 (CO), 171.00 (CO), 98.84 (C₁ '), 73.27 (C₂'), 76.52 (C₃'), 76.96 (C₅'), 70.23 (C₁') and 61 .55 (C₆')

Barlerin is characterized by following data:

¹H NMR (400 MHz, CD₃OD) (δ) : 1 .41 (H-10) |>], 1.90 (H-7) [d, J= 5.36 Hz], 1 .93 (H-7) \d, J = 5.4 Hz], 2.08 (OCOOCH₃) |>], 2.09 (H-9) [dd, J= 15.92 and 2.9 Hz], 3.08 (H-5) [dd, J= 8.5 and 0.92 Hz], 3.20 - 4.42 (H-2' - H-6') [m\, 3.62 (COOCH₃) |.v], 3.80 (H-6) [dd J = 12.08 and 6.04 Hz], 4.8 (H-F) [s], 5.82 (H-I ) [d, J= 2.32 Hz], 7.35 (H-3) [d, J= 1.36 Hz]

¹³C - NMR (75 MHz, CD₃OD) (δ) :94.29 (Ci), 152.40 (C₂), 108.37 (C₄), 40.85 (C₅), 74.53 (C₆). 46.29 (C₇), 88.42 (C₈), 48.49 (C₉), 29.41 (Ci₀), 20.8 (CH₃), 50.57 (CH₃O), 167.75 (CO), 1 72.02 (CO), 98.88 (Ci'), 73.24 (C₂'), 76.54 (C₃'), 76.88 (C₅'), 70.18 (C₄') and 61 .47 (C₆').

Shanzhiside methyl ester is characterized by following data:

¹H NMR (400 MHz, CD₃OD) (δ) : 1 .27 (H-10) [s], 1.84 (H-7) [dd, J= 7.24 and 6 Hz], 2.12 (1 1-7) [dd, J= 6.84 and 6.4 Hz], 2.63 (H-9) [dd, J= 7.64 and 2.54 Hz]], 3.01 (H-5) [dd, J = 7.04 and 3.06 Hz], 3.20 - 3.66 (H-2' - H-6') [m\, 3.75 (COOCH₃) |>], 4.05 (H-6) [m\, 4.97 (H-I ') |.v|, 5.59 (H-I) [d, J= 2.68 Hz], 7.42 (H-3) [Λ]

¹³C - NMR (75 MHz, CD₃OD) (δ) : 93.44 (C,), 151 .44 (C₂), 1 10.04 (C₄), 39.97 (C₅), 76.06 (C_ft)_; 47.83 (C₇), δ 76.95 (C₈), 50.34 (C₉), 23.31 (C,₀),50.55 (CH₃O), 168.42 (CO), 98.41 ((Y), δ 73.23 (C₂'), 76.56 (C₃'), 76.95 (C₅'), 70.22 (C₄') and 61.43 (C₆').

According to another embodiment, the present invention provides anti-inflammatory activity of the alcoholic extract of the plant Barleria cristata containing three iridoid glycosides: acetyl barlerin, barlerin and shanzhiside methyl ester.

Anti-inflammatory activity of the isolated iridoid glycosides is tested on rats using ibuprofcn as a standard drug for the inflammation. Inflammation is induced in the rats by injecting the carragcenan in the sub-plantar region of left hind paw of rats. Carrageenan or carragccnins arc a family of linear sulphatcd polysaccharides extracted from red seaweeds. The name is derived from a type of seaweed that is abundant along the irish coastline. Gelatinous extracts of the Chrondus crispus seaweed have been used as food additives for hundreds of years, though analysis of carrageenan safety as an additive continues.

Carragcenans are large, highly flexible molecules, which curl forming helical structures. This gives them the ability to form a variety of different gels at room temperature. Carrageenan induces inflammation in human intestinal epithelial cells in tissue culture through a BCL 10 mediated pathway that leads to activation of NFkappaB and IL-8. The exposure of the human intestinal epithelial cells to Carrageenan triggers a distinct inflammatory pathway via activation of BCI , 1 0 with NFkappaB activation and up regulation of IL-8 secretion. Carrageenan induced edema of rat hind paw has been an extensively used model of inflammation.

The anti-inflammatory activity of the isolated iridoid glycosides and the alcoholic plant extract is described with the help of examples.

According to another embodiment, the present invention provides method of treating mammals and humans having inflammation, pain and swelling with pharmaceutical composition containing the pure iridoid glycoside barlcrin, acetyl barlerin or shanzhisidc methyl ester individually or in combination thereof. The pharmaceutical composition may contain any one of the three iridoids or two in combination or all of them.

The method of treating subjects having inflammation comprising administering a pharmaceutically effective dosage of iridoid glycoside isolated from a plant Barleria crislala, to mammals and humans. Preferably the said glycosides are isolated from Barleria crislala var. dichotoma

In one preferred embodiment, the present invention provides method of treating mammals and humans having inflammation, pain and swelling with pharmaceutical composition containing barlerin.

In another preferred embodiment, the present invention provides method of treating mammals and humans having inflammation, pain and swelling with pharmaceutical composition containing acetyl barlcrin.

In a preferred embodiment, the present invention provides method of treating mammals and humans having inflammation, pain and swelling with pharmaceutical composition containing shan/hiside methyl ester. In a preferred embodiment, the present invention provides method of treating mammals and humans having inflammation, pain and swelling with pharmaceutical composition containing barlcrin and acetyl barlerin.

In a preferred embodiment, the present invention provides method of treating mammals and humans having inflammation, pain and swelling with pharmaceutical composition containing acetyl barlcrin and shanzhisidc methyl ester.

In a preferred embodiment, the present invention provides method of treating mammals and humans having inflammation, pain and swelling with pharmaceutical composition containing barlcrin and shanzhiside methyl ester.

In another preferred embodiment, the present invention provides method of treating mammals and humans having inflammation, pain and swelling with pharmaceutical composition containing barlcrin, acetyl barlcrin and shanzhiside methyl ester.

According to another embodiment, present invention provides an anti-inflammatory activity of isolated iridoid glycosides and use of these iridoids for the preparation of medicament useful for treating inflammation, pain and swelling in mammals and animals.

In a preferred embodiment, the present invention provides anti-inflammatory activity of acetyl barlcrin and use of acetyl barlcrin for the preparation of medicament useful for treating inflammation, pain and swelling in mammals and animals.

In another preferred embodiment, the present invention provides anti-inflammatory activity ol barlcrin and use of barlerin for the preparation of medicament useful for treating inflammation, pain and swelling in mammals and animals.

In yet another preferred embodiment, the present invention provides anti-inflammatory activity of shanzhiside methyl ester and use of shanzhiside methyl ester for the preparation of medicament useful for treating inflammation, pain and swelling in mammals and animals.

According to one another embodiment, the present invention provides a method of treating subjects (wherein said subject is selected from mammals and humans) having inflammation, pain and swelling with pharmaceutical composition comprising alcoholic extract of the plant liarlcria crista! a.

The method of treating subjects having inflammation, pain and swelling comprising administering a pharmaceutically effective dosage of bioactive fraction containing iridoid glycosides acetyl barlcrin. barlerin and shanzhiside methyl ester along with other components prepared from a plant Barleria cristata, to the selected subject. Preferably, the said alcoholic extract is prepared from Barleria cristata var. dichotoma.

According to one embodiment of the present invention, the present invention provides the use of bioactive fraction to treat inflammation caused by carragccnan.

According to one embodiment of the present invention, the present invention provides the use of isolated glycosides to treat inflammation caused by carragccnan.

According to another embodiment, the present invention provides the percent protection exhibited by the dosage levels of the isolated glycoside in carragecnan induced hind paw edema in rats.

According to another embodiment, the present invention provides the percent protection in inflammation exhibited at the dosage levels of the isolated glycoside in carragccnan induced hind paw edema in rats.

According to one another embodiment of the present invention, dosage of isolated acetyl barlerin for the treatment of carrageenan induced hind paw edema in rats is 1 -10 mg/kg of the body weight.

According to one another embodiment of the present invention, the percent protection exhibited by the acetyl barlerin in carragccnan induced hind paw edema in rats is up to 64%.

According to yet another embodiment of the present invention, dosage of isolated barlerin for the treatment of carragecnan induced hind paw edema in rats is 1 -10 mg/kg of the body weight.

According to another embodiment of the present invention, the percent protection exhibited by the barlerin in carragecnan induced hind paw edema in rats is up to 84%.

According to yet another embodiment of the present invention, dosage of isolated shan/hisidc methyl ester for the treatment of carragccnan induced hind paw edema in rats is 1 -10 mg/kg of the body weight.

According to another embodiment of the present invention, the percent protection exhibited by the shanzhiside methyl ester in carrageenan induced hind paw edema in rats is up to 75%.

According to another embodiment of the present invention, the desired dosage is administered for the curative properties.

According to another embodiment of the present invention, isolated glycosides arc administered orally by clinically or medically accepted methods. According to another embodiment of the present invention, the dosage of potent anti-inflammatory activity of isolated iridoid glycoside is at a dose of 1 -10 mg/kg p.o.

According to one another embodiment the present invention provides medicaments like formulations, pharmaceutical preparations and dietary supplements which may be prepared with the isolated bioactivc iridoid glycosides and use of such pharmaceutical preparations and dietary supplements to treat inflammation, pain and swelling in mammals. These pharmaceutical compositions can be provided in the form of powder, granules, tablets, sugar coated tablets, capsules, liquid preparations or the like and can be administered pcrorally, parenteral!}', by inhalation, by pcrrcctum, locally or the like. The parenteral administration includes a subcutaneous injection, an intravenous injection, an intramuscular injection, an intranasal administration or injection. A dose is usually in a range of about 0.1 to about 50 mg/kg body weight, and an exact dose is determined depending on ages, body weights and symptoms of patients and also administration route, etc.

Major advantages realized in the present invention arc: Barlerin is isolated for the first time from the plant source Barleria crisiata. The biologically active iridoids: acetyl barlerin. barlerin and shanzhisidc methyl ester, were isolated for the first time from the plant source Barleria crislaia var. dichotoma. The biologically active iridoids: acetyl barlerin, barlerin and shan/hisidc methyl ester, isolated from Barleria cristata, var. dichotoma have significant anti-inflammatory activity which is better than the standard drug. Further marked anti-inflammatory activity has been observed at low dose levels. Yet another advantage lies in the present invention is the prolonged marked effect of pure isolated glycoside which shall reduce the multi dosing in the subject and increase the compliance.

Having thus described the invention with reference to particular preferred embodiments and illustrative examples, those in the art would appreciate modifications to the invention as described and illustrated that do not depart from the spirit and scope of the invention as disclosed in the specification. The examples arc set forth to aid in understanding the invention but arc not intended to, and should not be construed to limit its scope in any way. The examples do not include detailed descriptions of conventional methods. Such methods arc well known to those of ordinal skill in the art and arc described in numerous publications. All references mentioned herein are incorporated in their entirety. EXAMPLES Plant material

Barleria crisiata var. dichotoma was collected from the gardens of Panjab University, Chandigarh. The identity of the plant was confirmed by N1SCΛIR, New Delhi (R.cf. No. NISCAIR/RHMD/Consult/-2007-08/861/46). A sample of the plant is kept in the herbarium of the NlSCAIR and also deposited in Muscum-cum-Herbarium of University Institute of Pharmaceutical Sciences.

Extraction and Isolation

Example I: Isolation of the mcthanolic extract from the plant Barleria cristata var. dichotoma

The whole plant of Barleria cristata was coarsely grounded and dried. The dried plant material was macerated with methanol at the room temperature. The methanol was filtered out from the plant material and again plant material was again macerated with methanol. All the mcthanolic extracts were combined. A portion of it was tested for anti-inflammatory activity.

Example 2: Isolation of pure iridoids: acetyl barlerin, barlerin and shan/.hisidc methyl ester from the methanolic extract of the plant Barleria cristata var. diehotoma

The methanolic extract as obtained in Example 2 was concentrated under vacuum to obtain a residue. The residue obtained, was suspended in water and was washed successively with hcxane. chloroform and ethyl acetate. Thereafter, the aqueous suspension was extracted with butanol. I he butanol extract was concentrated and residue so obtained was subjected to column chromatography with chloroform: methanol as cluting solvent.

The TLC studies of the fraction obtained from column chromatography indicated the presence of iridoids such as acetyl barlerin, barlerin and shanzhisidc methyl ester. Acetyl barlerin was eluted first, followed by barlerin and finally shanzhisidc methyl ester was clutcd by increasing the polarity of cluting solvent.

The fractions containing individual glycoside were collected and concentrated. The fractions containing mixture of two glycosides were further purified by repeated column chromatography/crystallization with a mixture of chloroform and methanol. The pure glycosides isolated above were then used for performing pharmacological studies. Pharmacological studies

I. Animals for pharmacological studies

The pharmacological studies are conducted on Wistar albino rats (150-200 g) of cither sex procured from Central Animal House of Panjab University. The protocol to conduct anti-inflammatory studies on animals was duly approved by the Institutionals Animal Ethics Committee of Panjab University before the start of the experiment.

After procurement, all the animals are divided into different groups and left for one week for acclimatization to experimentation room and maintained on standard conditions (23+ 2 ⁰C. 60-70% relative humidity and 12-hours photo period). The animals arc fed with standard rodents pellet diet and water ad libitum. There arc five animals in each group.

II. Carragcenan induced inflammation

Inflammation was induced in the rats with the help of carragcenan. Acute edema was induced by injecting freshly prepared solution of carragcenan (Type IV, 1 %, 0.1 ml), under plantar region of the left hind paw of rats. Carrageenan causes a reproducible inflammatory reaction and remains a standard chemical for examining acute inflammation and effects of anti-inflammatory drugs. Carragcenan induced edema of rat hind paw has been an extensively used model of inflammation

I 11. Preparation of the test drug and standard drug for the administration

AU test substances for the administration were suspended in the aqueous solution of 0.5% carboxymethylcellulosc (CMC). Ibuprofen at a dose of 50mg/kg was used as a standard drug.

IV. Administration of the test drug, standard drug and vehicle

Different doses of freshly prepared solution of the test drug, standard drug and the vehicle were administered orally to the animals using a tuberculin syringe fitted with oral canula. The test and standard substance for the administration were suspended in the aqueous solution of 0.5% carboxymethylcellulosc (CMC). Control animals were given the corresponding amount of vehicle (0.5% carboxymethylcellulosc (CMC))

Determination of anti-inflammatory activity

Anti-inflammatory activity was determined using carragcenan induced paw edema model in the rats The animals were divided in to different groups of control, standard (ibuprofen) and tcsl. All animals are starved overnight. Both, the right and left hind paw is marked at the level of the lateral malleolus and immersed in the solution up to this mark to note the paw volume. I^'hc control group received only the vehicle whereas standard and the test received ibuprofcn and test substance respectively.

I^'hc increase in the paw volume was measured using plethysmomctcr (water displacement. UGO BΛSILE, italy) at 0/1 , 3, 6 and 9 hours after carrageenan challenge. The percentage protection was calculated as % P.

Percent protection is calculated by the following equation:

%P = 100 (l -V,/V_t) where V_t is edema volume of the treated group

V_c is edema volume of the control group.

Results were expressed as mean ± SEM (standard error mean). The significance of the difference in the response of treated and control group was assessed by one way analysis of the variance (ΛNOVA) followed by Dunnctt's t-test. The statistical analysis was done using the Jandcl Sigma Stat statistical software version 2.0.

I. Anti-inflammatory activity of mcthanolic extract from Barleria cristata var. dichotoma

Mcthanolic extract of the whole plant of Barleria cristala var. dichotoma was administered at two dose levels of 100 mg/kg and 400 mg/kg to the test group animals 30 minutes before the injection of carrageenan. The paw volume was measured immediately and at prc determined different time intervals after the injection of carrageenan. The mean paw volumes of the test group (mcthanolic extract) were compared with the control group and percent protection was calculated. I he results of the control, standard drug and the mcthanolic extract arc tabulated in the Table 1 .

Table 1 : Λnti-inflammatory activity of mcthanolic extract of Barleria cristata var. dichotoma

Mean difference in paw volume ± S. E. M with n = 5 " Significant at p < 0.05 when compared to control.

Significant at p < 0.001 within group.

Significant at p < 0.01 within group.

Note: Values in the parenthesis indicate the percent protection. II. Anti-inflammatory activity of pure barlcrin

The anti-inflammatory activity of the pure isolated compound barlcrin was evaluated at three different dose levels of 1 , 5 and 10 mg/kg using the carragcenan induced hind paw edema model. The mean paw volumes of each test group were compared with the control group and the percent protection was calculated. The results are tabulated in the Table 2.

Table 2: Anti-inflammatory activity of isolated pure barlcrin

Mean difference in paw volume ± S. E. M with n = 5 "Significant at p < 0.05 when compared to control. Significant at p < 0.001 within group. Significant at p < 0.01 within group. ® Significant at p < 0.05 within group.

Note: Values in the parenthesis indicate the percent protection. III. Anti-inflammatory activity of pure acetyl barlcrin

The anti-inflammatory activity of the pure isolated compound acetyl barlerin was evaluated at three different dose levels of 1 , 5 and 10 mg/kg using the carragecnan induced hind paw edema model. The mean paw volumes of each test group were compared with the control group and the percent protection was calculated. The results are tabulated in the Table 3.

Table 3: Anti-inflammatory activity of isolated pure acetyl barlcrin

Mean difference in paw volume ± S. E. M with n = 5 " Significant at p < 0.05 when compared to control. A Significant at p < 0.001 when compared to control. Significant at p < 0.001 within group. Significant at p < 0.01 within group. ® Significant at p < 0.05 within group.

Note: Values in the parenthesis indicate the percent protection IV. Anti-inflammatory activity of pure shanzhiside methyl ester

The anti-inflammatory activity of the pure isolated compound shanzhiside methyl ester was evaluated at three different dose levels of 1 , 5 and 10 mg/kg using the carrageenan induced hind paw edema model. The mean paw volumes of each test group were compared with the control group and the percent protection was calculated. The results are tabulated in the Table 4.

Table 4: Anti-inflammatory activity of isolated pure shan/.hisidc methyl ester

Mean difference in paw volume ± S. E. M with n = 5 " Significant at p < 0.05 when compared to control. Significant at p < 0.001 within group.

Significant at p < 0.01 within group. Note: Values in the parenthesis indicate the percent protection

The anti-inflammatory activity of butanol extract was not promising. The anti inflammatory studies on the methanolic extract and isolated glycosides reveals that these glycosides arc showing better efficacy against inflammation in the very dilute solution. This indicates that glycosides arc more effective at low concentration.

Claims

WE CLAIM:

1. A method of treating mammals and humans having inflammation, pain and swelling with pharmaceutical composition containing pure iridoid glycoside barlcrin; acetyl barlcrin and shanzhiside methyl ester individually or in combination thereof.

2. The method according to claim 1 , wherein iridoid glycosides arc isolated from plant source genus Barleria.

3. The method according to claim 2, wherein iridoid glycosides arc isolated from plant source Barleria cristata.

4. The method according to claim 3, wherein iridoid glycosides arc isolated from plant source Barleria crisiala var. dichoioma by the process comprising the steps of: a) mechanically disrupting the whole plant; b) extracting with alcohol or aqueous alcohol; c) concentrating the alcoholic extract; d) partitioning the concentrate between water and an organic solvent; c) optionally repeating the step d); f) optionally, extracting the aqueous extract with butanol; g) optionally, concentrating the butanol extract; h) isolating pure iridoid glycosides using chromatographic techniques; and i) - optionally, crystallizing the isolated iridoid glycosides with suitable solvent.

5. The process according to claim 4, wherein in step b) alcohol is selected from methanol, cthanol and the like.

6. The process according to claim 4, wherein in step d) organic solvent is selected from petroleum ether, hexanc, dichloromcthanc, chloroform, diethyl ether, ethyl acetate, and the like.

7. The process according to claim 4, wherein in step i) suitable solvent is selected from chloroform and methanol in any suitable proportion.

8. Λ method of treating mammals and humans having inflammation, pain and swelling with pharmaceutical compositions with alcoholic extract prepared from the plant source Barleria cristata var. dichotoma containing the iridoid glycoside barlerin; acetyl barlcrin and shan/.hisidc methyl ester along with other components.

9. A process for the preparation of alcoholic extract according to claim 8, comprising the step o\\ a) mechanically disrupting the whole plant; b) extracting with alcohol or aqueous alcohol; and c) concentrating the alcoholic extract.

10. The process according to claim 9, where in step b) alcohol is selected from methanol, cthanol and the like.