WO2011145105A2

WO2011145105A2 - Herbal composition for treatment of filariasis

Info

Publication number: WO2011145105A2
Application number: PCT/IN2011/000305
Authority: WO
Inventors: Premananda Das
Original assignee: Premananda Das
Priority date: 2010-05-18
Filing date: 2011-05-02
Publication date: 2011-11-24
Also published as: WO2011145105A3; AP2012006601A0

Abstract

Disclosed herein a standardized herbal extract composition derived from the tubers of Typhonium trilobatum along with one or more pharmaceutically acceptable excipients for treatment of filariasis.

Description

"HERBAL COMPOSITION FOR TREATMENT OF FILARIASIS" Field of Invention:

The present invention relates to a standardized herbal extract composition derived from Typhonium trilobatum for the treatment of parasitical diseases such as Filariasis, Elephantiasis and the like. More particularly, the present invention relates to the treatment of Lymphatic filariasis leading to elimination of the disability and morbidity and in patients suffering from such diseases.

Background and Prior Art:

Filariasis or Lymphatic Filariasis, popularly known as Elephantiasis is caused by threadlike, parasitic filarial worms of Wuchereria bancrofti, Brugia malayi and Brugia timori. Infection of filariasis causes permanent, long term and severe disability such as edema with thickening of the skin and underlying tissues (lymphoedema) or Elephantiasis. Filariasis affects different parts of the body such as legs, scrotum, breasts, arms and vulva etc.

Filariasis is the second largest disability factor, next only to mental illness. It had been estimated that about 1200 million people live in areas endemic to lymphatic filariasis. Around 450 million of people living in India are said to be exposed to the risk of contracting filarial infection. Such an infection greatly impacts productivity leading to loss in billion. Women affected with lymphoedema are often unable to carry out normal and day-to-day activities including participating in marketing and trading.

Diseases such as Filariasis, being common in developing and least developed countries, predominantly in tropics, affects large coastal population living below poverty line. Adequate medical facilities have not been provided for the treatment of people affected with Filariasis and cure with scientific evidence and logistics is lacking.

Conventional treatment of Filariasis uses diethyl carbamazine citrate and salts thereof which are primarily intended for the treatment of eosinophilia and other medical conditions. No other agent for the effective treatment of Filariasis is known. As such, Filariasis diseases are relatively ignored by researchers as well as pharma companies. No specific research or research findings are available which are directed towards treatment and cure of Filariasis.

Efforts have been made to treat Filariasis by use of synthetic compounds. However, most of the synthetic compounds have been found to have acute toxicity. Being fused rings, these organic compounds are also likely to be associated with carcinogenicity due to long term use.

KR 20040014813 discloses pharmaceutical composition of arsenic oxide for protection and treatment of filariasis.

WO/2005/113478 describes anthraquinones for the treatment of malaria, schistosomiasis and elephantiasis (filariasis) helminthic and other parasitic diseases.

Pharmaceutical composition of azarhodacyanine for the treatment of protozoa infection is described in US2009076067.

WO/2009/113569 discloses benzo[a]phenoxanthin compound for the treatment of parasitic protozoa.

WO/2004/019956 deals with organo phosphorous compounds for the treatment of helminthic infections.

In recent times, there have been a few reports of traditional Chinese medicines for the treatment of Filariasis. However, these reports have not been substantiated or supported with scientific data and clinical evidence.

CN101199575 discloses Chinese medicine preparation of calcite; lithospermum, radix isatidis, hairy holly root, purslane and arborvitae, indigo rhizoma ligustici wallichii and liquorice for treating filariasis. CN101152249 describes internal medicines of nuxvomicas, pangolin scales and stiff silkworms and external medicines of ephedras, lopseeds, light yellow sophora roots, sodium sulfates and salt for remedying the filariasis spargosis.

CN 1872036 discloses drop pills of agrimophol for treating filariasis and trichomoniasis.

An article titled "Antifilarial activity of Caesalpinia bonducella against experimental filarial infections" by R.L. Gaur, M.K. Sahoo et al, in Indian J. Med Res 128, July 2008, pp 65-70 discusses antifilarial activity of crude extract of seed kernel of C. bonducella and its various fractions against Litomosoides carinii (rodent filarial parasite) in cotton rats and to target human filarial parasite B. malayi in Mastomys coucha.

Article titled "In vitro effect of four herbal plants on the motility of Brugia malayi microfilariae" in Indian Journal of Medical Research, May, 2008 by .N.Sahare, V. Anandharaman, et al discloses aqueous/methanol extracts of Vitex negundo L. (roots), Butea monosperma L. (roots and leaves), and Aegle marmelos Corr. (leaves) for treatment for human lymphatic filariasis.

However, currently available antifilarial drugs are not able to control the disease effectively. Filariasis, being one of the most neglected diseases, attention needs to be drawn to it to provide much needed medical facilities to the poor, to increase their quality of life and health and thus increase productivity.

Typhonium trilobatum which belongs to the family Araceae has now received much attention from the botanists for the treatment of parasitical disease such as filariasis. The plant occurs in Bangladesh, China, Thailand, Malaysia, Burma, Srilanka and also in India. Different species of the genus Trilobatum are used in traditional medicine. Tuber of T. flagelliforme is used in treatment of cough, reducing phlegm, and chronic bronchitis and externally used for traumatic injury and abscesses. T. blumei tuber is used for the treatment of traumatic injury, abscesses, snake and snakebites, lympho tuberculosis and vacuities. T. hunanense is used in swelling, abscesses and snake bites. T. trilobatum is known for its antibacterial activity. An article titled "Efficacy of Typhonium trilobatum (L.) Schott Tuber Extracts on Pathogenic Bacteria" by M. Kandhasamy et al in Electronic Journal of Natural Substances, 3, (2008), 1-7 discloses the use of ethanolic and methanolic, petroleum ether, hexane, benzene, and chloroform extracts of tuber of Typhonium trilobatum (L.) Schott, (family: Araceae) to inhibit the growth of multi drug resistant P. aeruginosa, and methicillin resistant S. aureus.

However, the use of Typhonium trilobatum in the treatment of filariasis is not yet known. In view of the above discussion, the present inventor felt a need to explore the effect of herbal extract of Typhonium trilobatum on parasitical diseases such as Filariasis. This remains the object of the invention.

Thus, in the present invention a standardized herbal extract composition which is known to be free from toxicity, being edible has been evaluated for its effect on parasitic diseases such as filariasis. The present invention shows great promise and expects to bring hope to millions who are otherwise disabled and unable to earn their livelihood being apostatized.

Summary of the Invention:

In one aspect of the invention there is provided a standardized herbal extract composition derived from tubers of Typhonium trilobatum along with one or more pharmaceutically acceptable excipients.

In another aspect of the invention, there is provided a process for preparation of a standardized herbal extract of Typhonium trilobatum.

In yet another aspect, the present invention provides characterization and standardization of the herbal extract derived from Typhonium trilobatum through HPTLC and TLC.

In yet another aspect, the present invention provides herbal extract composition delivered in pharmaceutically acceptable dosage forms such as granules, tablets, capsules, liquid and the like. In another aspect, the invention provides a method for treatment of parasitical diseases such as filariasis, Elephantiasis and the like.

Description of figures:

Fig.l. depicts decrease in swelling (in cm) of the Gastrocnemius of left and right leg respectively after treatment with Rid-Fil.

Fig. 2. depicts decrease in swelling (in cm) of the Soleus of left and right leg respectively after treatment with Rid-Fil.

Fig. 3. depicts decrease in swelling (in cm) of the Sural of left and right leg respectively after treatment with Rid-Fil.

Fig. 4. depicts decrease in swelling (in cm) of the Planar of left and right leg respectively after treatment with Rid-Fil.

Fig. 5. depicts rapid decline in Antigen Units(A.U.) due to treatment with herbal formulations as revealed by Serological tests

The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

Detailed Description:

The term 'biological composition' as used herein refers to a mixture of components obtained from the biological source i.e plant in the current context.

The term 'components' as used herein refers to chemical compounds, salts, complexes, ionic or molecular species found in nature.

The present invention relates to standardized herbal extract composition derived from tubers of Typhonium trilobatum along with one or more pharmaceutically acceptable excipients for treatment of lymphatic filariasis.

With the current trend of progression in practice of medical science and usage of herbal extract in pharmaceuticals, the present invention could find extended application in dosage form, delivery system and methods of treatment. The plant material used in the present invention, Typhonium trilobatum are collected from Bangladesh and cultivated in the inventor in his own medicinal garden and research center.

In an embodiment, the present invention provides a process for the preparation of standardized herbal extract from the tubers of Typhonium trilobatum for treatment of filariasis.

Accordingly, the process for the preparation of standardized herbal extract from the tubers of the plant Typhonium trilobatum comprises the steps of; a) preparing tubers of Typhonium trilobatum for extraction,

b) extracting the said raw material from aq. organic solvent followed by heating in a water bath maintained at the temperature range of 45-55°C under vacuum for 48hrs,

c) concentrating the extract by distilling the solvent extract over temperature regulated bath at 70°C under vacuum at 600 psi followed by evaporation and drying,

d) applying sample of extract dissolved in lower alcohol to chromatography column,

e) eluting said column with halogenated hydrocarbon to obtain the elute, f) distilling on water bath for recovery of halogenated hydrocarbon and further concentrating the distillate, cooling, adding methanol to cooled concentrate and repeating the process till complete isolation of compounds.

The solvents used for the extraction process are selected from water and lower C1-C6 alcohols, lower aliphatic hydrocarbons, in the ratio of 40:60

According to the process of the current invention, freshly harvested tubers of Typhonium trilobatum are cleaned; the roots and the dead tissues are washed with water for removal of soil and foreign materials and thereafter cut into small pieces for extraction. The fresh tubers are then taken in a extraction flask in 40:60 water: methanol mixture placed over a temperature regulated water bath at 40-100°C followed by addition of lower aliphatic hydrocarbons such as n-butane, n-pentane, n-hexane etc, heated , distilled to obtain fresh tubers free from the solvent and extra cellular or foreign material.

The fresh tubers, obtained from above, is extracted with aq. organic solvent selected from C1-C6 alcohol and heated in a water bath maintained at the temperature range of 45-55°C under vacuum for 48hrs till complete extraction of the active ingredients in the solvent. The solvent containing the active ingredients is then concentrated by distilling the solvent extract over temperature regulated water bath and further evaporated, dried, powdered and stored in an air tight container.

Further, the sample of the extract dissolved in a solvent selected from lower alcohols is used for isolation of compounds using column chromatography. Accordingly, the extracted active ingredients are isolated through column chromatography by injecting the test solution in a chromatographic column and running the test chromatogram by using a mobile phase selected from halogenated hydrocarbon, preferably chloroform.

The elutes are collected, distilled on a water bath to remove the eluent, concentrated and collected and compared through TLC method. The procedure is repeated several times till the complete isolation of the active ingredients. For isolation of other compounds, gradient elution is used.

Characterization and standardization of the herbal extract is carried out through HPTLC and TLC. In the process, the dried powdered extract is dissolved in lower alcohol, heated in the water bath for better dissolution, filtered, evaporated over a water bath to retain 3ml of solution for TLC studies. On analysis using TLC a total of 7 spots are observed with 5% methanolic sulfuric acid. Further, HPTLC analysis of the extract from Typhonium trilobatum showed 7 distinct active ingredients, as given in Table 1. The HPTLC scanning is done at wavelengths 200 nm and 366nm. In another embodiment, the present invention relates to a therapeutic composition comprising standardized herbal extract from the tubers of typhonium trilobatum along with pharmaceutically acceptable excipients.

The composition of the present invention contains the standardized herbal extract in the range of 3-5% weight of the total raw material.

The standardized herbal extract composition of the present invention is used for the treatment of filariasis.

In another embodiment, the present invention provides a pharmaceutical composition comprising of a therapeutically effective amount of standardized herbal extract of Typhonium trilobatum along with one or more suitable pharmaceutical carriers / exicipients. Further, the pharmaceutical composition of the invention may be any pharmaceutical form which contains the standardized herbal extract of Typhonium trilobatum of the invention. The pharmaceutical composition may be a solid form such as tablets, powders, granules, capsules, along with any suitable carrier well known in the prior art.

Suitable excipients and the amounts to use may be readily determined by the formulation scientist based upon experience and consideration of standard procedures and reference works in the field, e.g., the buffering agents, sweetening agents, binders, diluents, fillers, lubricants, wetting agents, disintegrants, etc.

The invention further discloses pharmaceutical dosage consisting of standardized herbal extract that can be administered to a subject in single dose or multiple dose, preferably single dose in the amount of 100-200mg a day preferably post dinner. Such dose is incorporated into pharmaceutically acceptable dosage forms such as granules, tablets, capsules etc.

The present invention provides a method of treatment of filariasis in a human comprising administering the therapeutically effective amount of the standardized herbal extract. The administration dose may vary depending on the age, body weight of the patient, severity of the disease etc.

To confirm the efficacy of the treatment of filariasis using the standardized herbal extract composition of the present invention, the present inventor performed various in vivo experiments on animals including human and verified remarkable effects which are given in Table 2 and Table 3 below.

It is observed that the pharmaceutical composition consisting of standardized herbal extract of the present invention can be used safely for a long time without any toxic effects or any other adverse side effects for the treatment of filariasis.

The present invention is explained by the following examples and experimental examples in more detail. The examples are intended to further illustrate the present invention and the scope of the invention cannot be limited thereby in any way.

Experimental:

Example: Preparation of standardized herbal extract of Typhonium trilobatum

Material and Methods:

Preparation of materials for extraction of active compounds:

Freshly harvested tubers of Typhonium trilobatum were cleaned off, the root and the dead tissues washed for removal of soil and other foreign materials, and thereafter cut into small pieces (1 cm³ approximately) for extraction of active compounds.

The fresh tubers (300g) were then taken in a soxhlet apparatus (Borosil, 2500 ml capacity) with 300 ml condenser and 300 ml extraction flask and placed over a temperature regulated water bath (40-100°C), 1500ml of n-hexane was taken in a soxhlet apparatus to remove any left over extra cellular and other foreign materials. Temperature of the water bath was regulated at 50°C. n-hexane was then removed after lh and distilled for recovery. The tubers were removed and dried to get rid of the left over hexane fraction. In the RB flask left over extract was collected for examination of any compound released in this process. Extraction of active principles:

The fresh tubers of Typhonium trilobatum were cut into small pieces and taken in a soxhlet apparatus to which 1.5 of 70% methanol (1050 ml methanol and 450 ml distilled water) was added and kept in the water bath at 50° C temperatures under vacuum. The extraction was carried for 48hrs till the total compounds were removed along with the methanol; the methanolic extract was distilled over temperature regulated water bath at 70°C under vacuum (promivac) at 600 psi to recover the methanol, which took approximately 1.5h. The concentrate of the whole extract was taken in a porcelain disc and kept on a water bath for evaporation of methanol and water, and then kept in a vacuum oven at 50°C for 24h. The dried product was powered using mortar and pestle and stored in a sterile dehumidified chamber in an air tight container to prevent microbial contamination.

Isolation of compounds:

The compounds were subsequently isolated individually through column chromatography. A column (30mm x 750mm) was washed thoroughly, cleaned dried and rinsed with methanol to remove impurity and again dried in the oven and was ready for column chromatography. A sample (12g) of the extract was taken in a beaker, dissolved in 15 ml methanol and then lOg silica gel (60/120 mesh, heated at 120°C) was added and kept on water bath for lh. For column packing 250g of silica gel (60/120 mesh) pre heated at 120°Cwas filled slowly. Then the prepared sample was filled in the column. Then glass wool was put to avoid disturbance to the column bed, and the column was ready for separation of different compounds in the sample.

Packed column was washed with pure chloroform. The elution rate was set 150 ml/h, chloroform level was maintained at 10 cm above the bed line. The elute was collected and distilled on water bath at 60°C to recover the chloroform; the concentrate in the distillation apparatus was taken in a test tube and evaporated on water bath. After cooling of the concentrate few ml of methanol was added. While adding methanol, crystallization of the compound was observed. Further, elutes were collected and compared through the TLC method. This procedure was repeated several times to fully isolate the compounds; for isolation of other compounds, gradient elution was used. Methodology of separation of active principles:

About O.lg of the extract was dissolved in 5ml methanol and slightly heated in the water bath for better dissolution and filtered; the filtrate was then evaporated over a water bath to retain 3ml of solution for TLC studies.

For thin layer chromatography (TLC) silica gel G (40g) was added to 80ml of water and thoroughly mixed for 2 min. the TLC plate (25 X 5 cm) were arranged in line on the Teflon sheet and the silica gels slurry was spread by the Stalhs mechanical spreader. Then the plates were air dried under room temperature for 30 min, arranged in a stand and kept in an oven at 110°C for lh, the plates were ready for TLC application. 25ug of the above sample was spotted on the TLC plate and the plate was kept in TLC chamber until the mobile phase (chloroform: methanol, 9:1) moved upto 3/4* (18 X 5 cm) of the plate. Plate was then taken out of the chamber, air dried and sprayed with 5% methanolic sulfuric acid. Again the plate was air dried and kept in the oven at 110°C for 15 minutes. The plate was taken out of the oven, photographed and the Rf values were measured. A total of 7 spots were observed with 5% methanloic sulfuric acid. Rf values were 0.160, 0.316, 0.416, 0.550, 0.725, 0.790 and 0.803.

HPTLC analysis of active principles:

Thin layer chromatography (TLC) on silica gel coated glass (Altech 0.2 X 100 X 100 mm HPTLC silica gel 60 plates) using CAMAG TLC Scanner3 "Scanner3_0700408" S N 070408(1) separated compounds in the lipophilic fraction by using chloroform/methanol/water (17.5:12.5:1.5). TLC plates were viewed at wavelength 200nm and 36nm using CAMAG TLC Scanner3 with automatic detector (sensitive 52).

HPTLC analysis of the extract from Typhonium trlbobatum (200ug) showed 7 distinct major peaks at wavelengths 200nm and 366nm (Table 1) Table 1: The identity of the different compounds from Typhonium trilobatum extracts detected through 4HPTLC analysis.

Pharmacological investigation:

The study was carried out in adult albino wistar rats of 6-8 weeks old. The animals were procured from the standard breeder and they were acclimatized to the laboratory conditions for a week prior to the test. The animals were kept in a temperature (22 ± 3°C) and humidity (30 to 70%) controlled room with 12h light, 12h dark cycle through out the experimental period. The animals were fasted over night prior to the administration of drug. The test material (solid) was dissolved in water and administered in a single dose orally to the test animals in the required doses by gavages using a suitable incubation canola. After treatment, the food was withheld for 4 hours. Throughout the experimental period, standard laboratory diet and water were provided ad libitum. The test animals were kept under observation for the period of 14 days after dosing. The feed intake and body weight were monitored daily for the observation period, whereas, mortality, sign and symptoms if any were recorded twice on the day of dosing and once every day, thereafter. At the end of the test period, surviving animals were weighed and sacrificed. The results of the experiment are given in the Table 2.

Table 2: Acute toxicity study of Typhonium trilobatum extract in albino wistar rat by oral route:

Table 3: Acute oral toxicity study of Typhonium extract at 2000 mg/kg dose in both male and female o of

Male Female

days

Body Feed Intake Body Weight Feed intake Mortal it

Mortality

weight (g) (g) (g) (g) y

MeaniSD Mean ± SD Mean±

Mean ± SD Mean ± SD Mean ± SD

SD

1. 142.2 ± 4.2 14.2 ±0.8 0 140.4 ±7.6 13.2 ±2.3 0

2. 141.4 ±4.5 13.0 ±3.8 0 142.0 ±8.0 11.6± 1.8 0

3. 142.2 ± 7.32 13.0 ±8.8 0 148.2 ± 5.8 13.8 ±3.8 0

4. 150.2 ±8.49 160.0 ±2.0 0 154.0 ± 10.1 14.4 ±0.8 0

5. 157.0± 10.9 16.4 ±2.1 0 156.6 ±8.8 15.4 ± 1.9 0

6. 160.0 ±6.3 15.8 ±3.1 0 156.6 ± 13.7 13.4 ±2.3 0

7. 166.8 ±6.2 16.0 ±0.7 0 159.6 ± 11.4 13.8 ± 1.9 0

8. 166.6 ±8.2 14.2 ± 1.9 0 163.0 ± 10.7 13.4 ± 1.5 0

9. 167.0 ±8.8 14.4 ± 4.0 0 168.8 ± 12.1 13.8 ±2.4 0

10. 171.2 ±5.2 14.6 ±3.3 0 177.0 ± 11.8 15.4 ± 1.5 0

11. 175.4 ±7.0 15.6 ±2.0 0 181.6 ± 14.6 16.4 ±2.4 0 12. 178.8 ± 9.8 16.6 ± 2.3 0 185.4 ± 11.6 17.0 ± 1.0 0

13. 185.4 ± 7.7 18.0 ± 1.2 0 185.8 ± 12.6 15.0 ± 2.3 0

14. 186.0 ± 9.2 16.6 ± 0.8 0 186.0 ± 8.4 14.4 ± 1.9 0

Values are means of 5 animals for male and female.

The LD₅o value for Typhonium trilobatum extract in rat by oral route was >2000mg/kg body weight. No toxic signs, symptoms and mortality were noted in the treated animals.

Clinical trials:

Clinical trials were conducted in two coastal districts of Orissa with 209 persons suffering from filariasis: the patients were diagnosed to have the disease, past history was also noted and medicine used. There were 102 male and 107 female patients. Amongst 209 persons, 92 had swelling on their left leg, 65 on their right leg, 44 on both legs, while 6 had hydrocel, 2 had swelling in their left hand. Detailed clinical studies in 30 cases (Table 4), which fell mostly in the age group of 20-50 years, revealed spectacular effects in alleviating the sufferings of the patients. In all the 30 cases, the patients had swelling of different dimensions (Grade 1 Oedema 1; Grade 2 Oedema 3 Grade 3 Oedema 25 and Grade 4 Oedema 1). After 15 days of the use of the herbal extract as prescribed by the doctor, 21 (70%) patients showed 10-25% reduction in the swelling and after 30days of treatment, 25 (83%) cases had reduced swelling. All the patients felt light and comfortable. In 23 out of 30 cases who reported to have been suffering from fever before the use of the herbal formulation. 20 (86%) were relieved after 15 days and all the patients (23 out of 23 i.e. 100%) did not have fever after 30 days of use of the said herbal formulation. Out of 30, 21 patients reported to have pain in their limbs and the body. After 15 days use 18 (86%) and after 30 days of use, all the 21 (100%) patients got relieved from the pain by using the herbal formulation. Only 8 out of 30 patients reported having lymphadenitis and all of them got completely cured 15 days after the use of herbal formulation. Table 4: Effect of Typhonium trilobatum extract on the clinical symptoms of filariasis

Case Study: Spectacular reduction in swelling of different parts of both left and right leg (Gastrocnemius, Soleus, Sural, Plantar) was observed in 90 days of treatment. This is given below in Table 5:

Table 5:

Serological investigations also showed gradual decline in antigen units in 90 days indicating reduction in microfilariae population; fever and pain were eliminated in 30 days of treatment. The Decrease in Antigen Units (A.U.) due to the treatment with Rid Fil as revealed by Serological Tests (Og4C3, Filaria) is given below in Table 6 below: Table 6:

Advantage:

The results of clinical evaluation of composition of standardized Typhonium trilobatum extract for the treatment of filariasis as seen above, has been extremely encouraging and rewarding. The elimination of disability in acute filariasis patients has been completed within 30days of the treatment with the herbal extract composition. The in-house availability of Typhonium trilobatum by sustainable development through large scale cultivation by the inventor and the successfiil use and method of the treatment of filariasis, has opened up a new ray of hope for millions of patients suffering from filariasis, lymphatic filariasis and the like, which causes serious disabilities and substantially restricts mobility and the ability to pursue income earning and fruitful employment opportunities, for the patients.

Claims

I claim,

1. A standardized herbal extract composition derived from the tubers of Typhonium trilobatum along with one or more pharmaceutically acceptable excipients for treatment of filariasis.

2. The standardized herbal extract composition according to claim 1, wherein, the standardized herbal extract is in the range of 3-5% weight of the raw material.

3. The standardized herbal extract composition according to claim 1, wherein the pharmaceutically acceptable excipients are selected from buffering agents, sweetening agents, binders, diluents, fillers, lubricants, wetting agents, disintegrants, etc.

4. The standardized herbal extract composition according to claim 1, wherein the composition is formulated into acceptable dosage forms such as granules, tablets, capsules, liquid and the like.

5. A process for the preparation of standardized herbal extract from the tubers of

Typhonium trilobatum comprising the steps of;

a) preparing tubers of Typhonium trilobatum for extraction,

d) applying sample of extract dissolved in methanol to chromatography column,

e) eluting said column with chloroform to obtain chloroform elute, f distilling on water bath for recovery of chloroform and further concentrating the distillate, cooling, adding methanol to cooled concentrate and repeating the process till complete isolation of compounds.

6. The process according to claim 5, wherein the solvents used for the extraction process are selected from water and lower C1-C6 alcohols, lower aliphatic hydrocarbons, in the ratio of 40:60.

7. A method of treating filarial infection comprising administering to the subject a therapeutically effective amount of the standardized herbal extract composition along with pharmaceutically acceptable excipients.

8. The method of treating filarial infection according to claim 7, wherein, the subject is administered a single dose in the amount ofl00-200mg a day.

9. Use of standardized herbal composition according to claims lto 8, for the treatment of filariasis.