WO2003080091A1

WO2003080091A1 - Herbal ophthalmic formulation for preventing cataract

Info

Publication number: WO2003080091A1
Application number: PCT/IN2002/000242
Authority: WO
Inventors: Suresh Kumar Gupta; Sujata Joshi; Sushma Srivastava; Deepa Trivedi; Nabanita Halder
Original assignee: All India Institute Of Medical Sciences; Department Of Science And Technology
Priority date: 2002-03-27
Filing date: 2002-12-24
Publication date: 2003-10-02
Also published as: AU2002353487A1

Abstract

The present invention relates to a herbal ophthalmic composition and a process for preparing the same. The ophthalmic composition is used for delaying the onset and progression of cataract and comprises of at least one aqueous extract of Ocimum sanctum and Curcuma longa and optionally adding a viscoelastic substance.

Description

Title of the Invention An herbal ophthalmic formulation for delaying the onset and progression of cataract and a process for preparing the same.

Field of the Invention

The present invention relates to an herbal ophthalmic formulation for delaying the onset and progression of cataract and a process for preparing the same.

Background of the Invention

Cataract is the leading cause of blindness in the world. In India alone the annual incidence of cataract blindness is about 3.8 million. The present level of surgical performance is in the order of about 1.6-1.9 million cataract operations as against 5-6 million required to clear the backlog. Although, cataract surgery is the most commonly performed procedure and is generally recognized as being one of the safest operations, there is a significant complication rate. For this reason, if an intervention is able to delay the onset of cataract by 10 years or so, the number of cataract operations can be reduced to nearly half.

Visual impairment in cataract results from opacity or light scattering produced most often by the formation of large protein aggregates in the lens. The process of aggregation involves variety of complex metabolic and physiological mechanisms, which act in combination to change the refractive index. Studies on lens proteins indicate that post translational modifications such as oxidation, glycation, Schiffs base formation, carbamylation, transamidation, phosphorylation, elevated calcium levels and proteolysis lead to lens opacification. These chemical changes can be regulated to maintain lens homeostasis and transparency. Thus, cataract is believed to be a disease that probably requires a chemical solution for its delay.

Although cataract is a multi-factorial disease, oxidative mechanisms through generation of reactive oxygen radicals are believed to play an important role in the progressive decline of vision and formation of cataract.

In vitro and in vivo studies with experimental animal models as well as the epidemiological studies conducted with selected human populations ' demonstrated a lower incidence of cataract in the groups consuming higher amount of ascorbate and other antioxidants. Reactive oxygen species (ROS) such as hydrogen peroxide, superoxide radical, singlet oxygen and hydroxyl radical are postulated to contribute to this process. ROS are generated in the eye both enzymaticaHy and photo-chemically. Endogenous defense mechanisms through scavenging of ROS by antioxidant enzymes like superoxide dismutase, glutafhione peroxidase, catalase and glutathione-S-transferase, simultaneously protect the lens from oxidative damage.

Besides, physiological antioxidants such as Vitamin C, Vitamin E and pyruvate also play an important role in protection of lens from oxidative damage.

Various substances with diverse chemical structures and properties have been reported to offer protection against cataract in different experimental models. However, the need of the long- term treatment with such agents pose a serious impairment for successful accomplishment of clinical trials. Medicinal plant based formulations are being used since long for a variety of diseases. Because of sufficient evidence that oxidative stress plays a role in mechanism of cataractogenesis, there has been an increasing interest in the development of suitable antioxidant products of plant origin, that could be effective in delaying or preventing the formation of cataract. Flavonoids and related poly-phenols are antioxidants and also potent inhibitors of aldose reductase. Effect of mefhanolic extract and the alkaloidal components from several plants have been studied on lens aldose reductase activitiy, the key enzyme in diabetic cataract. Further, a decreased risk of cataract has been associated with consumption of tea, a major source of flavonoid quercetin. Quercetin has been shown to inhibit hydrogen peroxide-induced oxidation of the lens proteins. Optimization of dietary intake of protective nutrients has been an effective approach towards reducing the incidence of cataract. A polyherbal preparation, Chyavanprash protects against steroid induced opacities in lens of chick embryo. Recently, possibilities of plant extracts iruhibiting xanthine oxidase, the enzyme responsible for super-oxide radical production, are under investigation for cataract. It is suggested that utilization of natural products may lead to better results with rrtinimum side effects.

It is the object of the present invention to overcome the afore-mentioned disadvantages.

Further object of the present invention is to study and evaluate antioxidant and anti cataract potential of herbal extracts in experimental models of cataract.

Yet another object of the present invention is to formulate a herbal ophthalmic formulation with optimum anti. cataract activity. Summary of the invention

To achieve the aforesaid objectives the present invention provides a herbal ophthalmic composition for delaying the onset and progression of cataract comprising: - about 0.01- 10 % w/v of at least one aqueous extract selected from

Ocimum sanctum and Curcuma longa; about 0.1 - 5 % w/v of visco-elastic substance and q.s distilled water

The present invention further provides a process for preparing a herbal ophthalmic formulation for the delaying the onset and progression of cataract comprising the steps of: preparing aqueous extracts of Ocimum sanctum and Curcuma longa, - mixing at least one extract selected from Ocimum sanctum and

Curcuma longa in an amount between 0.01- 10 % w/v with 0.1 - 5 % w/v of visco-elastic substance; adding distilled water to obtain the herbal ophthalmic formulation and - sterilizing the composition

The extract of Ocimum sanctum is prepared from the fresh leaves and of Curcuma longa from the rhizomes.

The visco-elastic substance is selected from hydroxy propyl methyl cellulose (hpmc), sodium hyaluronate, Chondroitin Sulfate, polyacrylamide or a mixture thereof, preferably hydroxy propyl methyl cellulose (hpmc) The ingredients of the herbal ophthalmic formulation are in the following proportions: aqueous extract of Ocimum sanctum - 0.25 %w/v and visco elastic substance - 0.25 % w/v

The ingredients of the herbal ophthalmic formulation are in the following proportions: aqueous extract of Ocimum sanctum - 0.1 % w/v and visco elastic substance - 0.25 % w/v

The ingredients of the herbal ophthalmic formulation are in the following proportions: aqueous extract of Curcuma longa - 0.02 % w/v and visco elastic substance - 0.25 % w/v

The ingredients of the herbal ophthalmic formulation are in the following proportions: aqueous extract of Curcuma longa - 0.01 % w/v and visco elastic substance - 0.25 % w/v

The ingredients of the herbal ophthalmic formulation are in the following proportions: aqueous extract of Ocimum sanctum - 0.3 %w/v aqueous extract of Curcuma longa - 0.02 % w/v and - visco elastic substance - 0.25 % w/v

The ingredients of the herbal ophthalmic formulation are in the following proportions: aqueous extract of Ocimum sanctum - 0.15 %w/v aqueous extract of Curcuma longa - 0.01 % w/v and visco elastic substance - 0.25 % w/v

The ingredients of the herbal ophthalmic formulation are in the following proportions: aqueous extract of Ocimum sanctum - 0.03 w/v and aqueous extract of Curcuma longa - 0.02 % w/v

The ingredients of the herbal ophthalmic formulation are in the following proportions: aqueous extract of Ocimum sanctum - 0.01 w/v and aqueous extract of Curcuma longa - 0.02 % w/v

DETAILED DESCRIPTION OF THE INVENTION:

The following methodology was followed for the preparation of eye drops:

Step 1: Washing of the eye drop containers (glass vials) The glass vials were first washed with teepol solution and were rinsed with ordinary tap water, followed by distilled water. They were then dipped in distilled water containing anti-microbial agent for few hours. Vials were dried before use and kept in closed containers.

Step 2: Sterilization of glass-wares:

To avoid the contamination of the eye drops and thereby ocular infection, the glass-wares used in the preparation of the ophthalmic formulation were sterilized by autoclaving. The glass vials, filtering assembly, other glass wares etc., were sterilized by moist heat under pressure (15lbs ) for 15 minutes.

Step 3: Preparing extract of Ocimum sanctum and Curcuma longa Fresh leaves of Ocimum sanctum and rhizomes of Curcuma longa were procured, identified and processed for the preparation of their aqueous extracts. Ocimum leaves were weighed, washed, cut into small pieces and soaked in distilled water for three days. The contents were filtered and lyophilized and yield (w/w) was calculated. Extract once prepared in adequate quantity was stored desiccated in a refrigerator for carrying out further experiments. Curcuma longa rhizomes were powdered, weighed and dissolved in distilled water with constant stirring for 24 hrs. Solution was filtered. Undissloved part was dried and weighed to calculate the solubility.

Step 4: Weighing the extracts accurately on a sensitive balance.

The herbal extracts were weighed accurately using an electronic Mettler balance specifically for lower concentration ranges.

Step 5: Use of double distilled water Distilled water used for the eye drops was double distilled with the help of a distillation apparatus.

Step 6: Preparation of solution in laminar hood

The herbal extracts were accurately weighed, poured in a beaker and dissolved in the required volume of autoclaved distilled water under sterile conditions in a larninar hood. A sterile (autoclaved) stirrer was used for mixing the drugs and the solution was stirred occasionally until properly mixed. The beaker was covered with an aluminum foil. I t. It. ML I

Step 7: Adding of visco elastic substance

Visco elastic substance in the range of 0.1 to 5 % was accurately weighed and dissolved in autoclaved distilled water using autoclaved glass stirrer. It was stored at 4 deg C for 24 hours. To a predetermined amount of herbal extracts was added and dissolved, stirring with autoclaved glass stirrer in a laminar hood. The solution was stirred occasionally until properly mixed. The beaker was covered with an aluminum foil.

_f Step 8: Sterilization of eye drops using micropore filtration

Micropore filters having diameter 7 mm and size 0.2 μm were used to filter the eye drop solution. Filtration was carried out using an autoclaved Millipore filtration unit in a laminar hood.

The present invention will now be illustrated with the following examples but without intending to imply any limitation thereon.

Example 1:

To 0.1 % of aqueous extract of Ocimum sanctum 0.25 % w/v of Hydroxy propyl methyl cellulose was added.

Onset and progression of galactose cataract was delayed significantly by instillation of one eye drop two times per day as compared to control. The O.I on 7^Λ day was 0.75 and on 30^th day it was 2.81 in comparison to 1 and 4 in control respectively (Table-3) Example 2:

To 0.3 % of aqueous extract of Ocimum sanctum 0.25 % w/v of Hydroxy propyl methyl cellulose was added.

Onset and progression of galactose cataract was delayed significantly by instillation of one eye drop two times per day as compared to control. The O.I on 7^th day was 0.5 and on 30^th day it was 2.42 in comparison to 1 and 4 in control respectively (Table-I)

Example 3:

To 0.15 % of aqueous extract of Ocimum sanctum 0.25 % w/v of Hydroxy propyl methyl cellulose was added.

Onset and progression of galactose cataract was delayed significantly by instillation of one eye drop two times per day as compared to control. The O.I on 7^lh day was 0.5 and on 30^th day it was 2.31 in comparison to 1 and 4 in control respectively (Table-2)

Example 4: To 0.25 % of aqueous extract of Ocimum sanctum 0.25 % w/v of Hydroxy propyl methyl cellulose was added.

Example 5:

Aqueous extract of Curcuma longa was prepared, and to 0.02 % w/v Curcuma longa 0.25 % w/v of Hydroxy propyl methyl cellulose was added. Distilled water was added and the composition was sterilized to obtain the herbal formulation. Onset and progression of galactose cataract was delayed significantly by instillation of one eye drop two times per day as compared to control. The O.I. on 7^th day was 0.86 and on 30^th day it was 2.66 in comparison to 1 and 4 in control respectively (Table-4)

Example 6:

Aqueous extract of Curcuma longa was prepared, and to 0.01 % w/v Curcuma longa 0.25 % w/v of Hydroxy propyl methyl cellulose was added. Distilled water was added and the composition was sterilized to obtain the herbal formulation.

Onset and progression of galactose cataract was delayed significantly by instillation of one eye drop two times per day as compared to control. The O.I on 7^th day was 0.9 and on 30^th day it was 2.9 in comparison to 1 and 4 in control respectively (Table-4)

Example 7:

Aqueous extract of Curcuma longa was prepared, and to 0.01 % w/v Curcuma longa 0.25 % w/v of sodium hyaluronate was added. Distilled water was added and the composition was sterilized to obtain the herbal formulation.

Example 8:

Aqueous extract of Curcuma longa was prepared, and to 0.02 % w/v Curcuma longa 0.25 % w/v of polyacrylamide was added. Distilled water was added and the composition was sterilized to obtain the herbal formulation.

Example 9:

A herbal ophthalmic formulation of the present invention comprises: Aqueous extract of Ocimum sanctum - 0.3 % w/v aqueous extract of Curcuma longa - 0.02 % w/v and sodium hyaluronate - 0.25 % w/v

Example 10:

A herbal ophthalmic formulation of the present invention comprises: aqueous extract of Ocimum sanctum - 0.15 w/v aqueous extract of Curcuma longa - 0.01 % w/v and

Polyacrylamide - 0.25 % w/v

Example 11:

A herbal ophthalmic formulation of the present invention comprises: aqueous extract of Ocimum sanctum - 0.03 w/v and aqueous extract of Curcuma longa - 0.02 % w/v

Example 12:

A herbal ophthalmic formulation of the present invention comprises: aqueous extract of Ocimum sanctum - 0.01 w/v and aqueous extract of Curcuma longa - 0.02 % w/v

Effect of herbal eye drops

Antioxidant nature of herbal extracts was established by monitoring lipid peroxidation by determination of thiobarbituric acid reacting substances (TBARS), glutathione (GSH) levels and antioxidant enzyme activities like superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione-S-transferase (GST) in isolated rat lenses exposed to osmotic/ oxidative stress. Incorporation of 30 mM galactose in culture medium resulted in significant hydration, osmotic swelling and accumulation of polyols in the lens. Oxidative insult to lens was induced by incubating the lenses in tissue culture medium with either riboflavin (lOμM), or hydrogen peroxide (0.2mM) or sodium selenite (lOOμM). Effect of these agents on lens clarity as well as on antioxidant biochemical parameters was observed. To study preventive role of the herbal formulations (test compound), the medium was also supplemented with different concentrations of these. Photodocumentation of the lens was achieved and then were analyzed for biochemical parameters.

The activity of herbal ophthalmic formulation was evaluated in vivo, in experimentally induced galactose cataract in rats. The rats were equally divided into control and test groups. Wistar rats (60-80g) of either sex were used for the study and fed with 30 % galactose in diet and water ad libitum to induce galactose cataract.

For the herbal formulation comprising Ocimum sanctum and the visco- elastic substance as well as herbal formulation comprising Curcuma longa and the visco-elastic substance, the rats in the test group were instilled with 1 drop of the herbal extract twice a day from the day of the cataractogenic insult and was continued till the end of the experiment. At regular intervals, cataract stages in both the groups were graded according to the classification of Sippel (1960) using a slit lamp bio-microscope after dilation of the pupil.

At stage 0 lens is similar to the normal lens; stage 1= faint peripheral opacity; stage D irregular peripheral opacity and slight involvement of the lens at the center; stage IH= irregular opacity involving entire lens, stage IV= pronounced opacity readily visible as white spot Onset and stage wise progression of cataract was recorded for groups. An overall grade point average was calculated to compare the rate of progression in test group with that of control.

For doing so normal eyes were given no points, stage I-one point; stage Il-two points; stage HI- three points and stage IV-four points. The sum of points in each group was divided by the number of eyes to get opacity index (OI) of that group. The statistical significance between the two groups was determined using two tailed, unpaired students 't' test and 'p' value less than 0.05 was considered to be significant.

The results of the test as shown in table 1, 2 &3 confirm that instillation of herbal ophthalmic formulation comprising Ocimum sanctum and the visco- elastic substance significantly delayed the onset and the progression of galactose induced cataract as compared to control.

The results of the test as shown in table 4 confirm that instillation of herbal ophthalmic formulation comprising Curcuma longa and the visco-elastic substance significantly delayed the onset and the progression of galactose induced cataract as compared to control.

The treatment with herbal formulation comprising Ocimum sanctum, Curcuma longa and visco-elastic substance was started simultaneous to galactose challenge and was continued till the end of the experiment. To evaluate the anti-cataract potential of herbal extracts against galactose cataract, the formulation was adniinistered in the form of an eye drop (1 drop two times a day or once a day) Table 5 shows the anti-cataract activity effect of the herbal ophthalmic formulation comprising Ocimum sanctum, Curcuma longa and visco-elastic substance

While the present invention has been particularly shown and described with reference to the preferred embodiment thereof, it will be understood by those skilled in the art that various changes in form and details may be effected therein without departing from the spirit and scope of the invention as defined by the appended claims

Table - 1

Topical effect of 0.3% Ocimum sanctum in galactose cataract

OI = Opacity Index n = number of eyes

**p <0.01 as compared to control

***p <0.001 as compared to control Table - 2

Anticataract effect of Ocimum sanctum 0.15% eyedrop with hpmc (0.25%)

OI = Opacity Index n = number of eyes

**p <0.01 as compared to control

***p <0.001 as compared to control Table - 3

Topical effect of 0.1% Ocimum sanctum in galactose cataract

OI = Opacity Index n = number of eyes *p <0.05 as compared to control

**p <0.01 as compared to control ***p <0.001 as compared to control Table- 4

Anticataract effect of C.longa eye drops with hpmc (0.25%)

n - number of eyes OI = Opacity Index *p<0.05, **p<0.01 and ***p<0.001 as compared to control Table - 5

Anticataract effect of OS + CL eye drops with hpmc (0.25%)

n = number of eyes

***p <0.001 as compared to control

OI = Opacity Index

Claims

We claim:

1. A herbal ophthalmic composition for delaying the onset and progression of cataract comprising: - about 0.01- 10 % w/v of at least one aqueous extract selected from

Ocimum sanctum and Curcuma longa and q.s distilled water and optionaUly about 0.1 - 5 % w/v of visco-elastic substance

2. A herbal ophthalmic composition for delaying the onset and progression of cataract as claimed in claim 1 comprising: about 0.01- 10 % w/v of at least one aqueous extract selected from Ocimum sanctum and Curcuma longa about 0.1 - 5 % w/v of visco-elastic substance and - q.s distilled water

3. A herbal ophthalmic composition of claim 1 wherein the extract of Ocimum sanctum is prepared from the fresh leaves and of Curcuma longa from the rhizomes.

4. A herbal ophthalmic formulation of claim 1 wherein the visco-elastic substance is selected from hydroxy propyl methyl cellulose (hpmc), sodium hyaluronate, Chondroitin Sulf ate or polyacrylamide or a mixture thereof.

5. A herbal ophthalmic formulation of claim 4 wherein the visco elastic substance is hydroxy propyl methyl cellulose (hpmc)

6. A herbal ophthalmic formulation of claim 1 wherein the ingredients are in the following proportions: aqueous extract of Ocimum sanctum - 0.25 %w/v and visco elastic substance - 0.25 % w/v

7. A herbal ophthalmic formulation of claim 1 wherein the ingredients are in the following proportions:

- aqueous extract of Ocimum sanctum - 0.1 % w/v and

- visco elastic substance - 0.25 % w/v

8. A herbal ophthalmic formulation of claim 1 wherein the ingredients are in the following proportions: aqueous extract of Curcuma longa - 0.02 % w/v and visco elastic substance - 0.25 % w/v

9. A herbal ophthalmic formulation of claim 1 wherein the ingredients are in the following proportions: aqueous extract of Curcuma longa - 0.01 % w/v and visco elastic substance - 0.25 % w/v

10. A herbal ophthalmic formulation of claim 1 wherein the ingredients are in the following proportions: aqueous extract of Ocimum sanctum - 0.01 % w/v and aqueous extract of Curcuma longa - 0.02 % w/v

11. A herbal ophthalmic formulation of claim 1 wherein the ingredients are in the following proportions: aqueous extract of Ocimum sanctum - 0.03 % w/v and aqueous extract of Curcuma longa - 0.02 % w/v

12. A herbal ophthalmic formulation of claim 1 wherein the ingredients are in the following proportions: - aqueous extract of Ocimum sanctum - 0.3 % w/v aqueous extract of Curcuma longa - 0.02 % w/v and visco elastic substance - 0.25 % w/v

13. A herbal ophthalmic formulation of claim 1 wherein the ingredients are in the following proportions: aqueous extract of Ocimum sanctum - 0.15 %w/v aqueous extract of Curcuma longa extract- 0.01 % w/v and visco elastic substance - 0.25 % w/v

14. A herbal ophthalmic formulation of claim 12 wherein the ingredients are in the following proportions: aqueous extract of Ocimum sanctum - 0.3 % w/v aqueous extract of Curcuma longa - 0.02 % w/v and sodium hyaluronate - 0.25 % w/v

15. A herbal ophthalmic formulation of claim 13 wherein the ingredients are in the following proportions: aqueous extract of Ocimum sanctum - 0.15 w/v aqueous extract Curcuma longa - 0.01 % w/v and - Polyacrylamide - 0.25 % w/v

16. A herbal ophthalmic formulation of claim 13 wherein the ingredients are in the following proportions: aqueous extract of Ocimum sanctum - 0.15 w/v aqueous extract of Curcuma longa - 0.01 % w/v and

Hydroxy propyl methyl cellulose - 0.25 % w/v

17. A process for preparing a herbal ophthalmic formulation for the delaying the onset and progression of cataract comprising the steps of: preparing aqueous extracts of Ocimum sanctum and Curcuma longa, mixing at least one extract selected from Ocimum sanctum and Curcuma longa in an amount between about 0.01- 10 % w/v adding distilled water to obtain the herbal ophthalmic formulation Optionally adding about 0.1 - 5 % w/v of visco-elastic substance, and sterilizing the composition

18. A process for preparing a herbal ophthalmic formulation for the delaying the onset and progression of cataract of 17 comprising the steps of: preparing aqueous extracts of Ocimum sanctum and Curcuma longa, - mixing at least one extract selected fro Ocimum sanctum and

Curcuma longa in an amount between about 0.01- 10 % w/v with about 0.1 - 5

% w/v of visco-elastic substance adding distilled water to obtain the herbal ophthalmic formulation and - sterilizing the composition

19. A process of claim 17 wherein the extract of Ocimum sanctum is prepared from the fresh leaves and the extract of Curcuma longa is prepared from the rhizomes.

20. A process of claim 17 wherein the visco-elastic substance is selected from, hydroxy propyl methyl cellulose, sodium hyaluronate, Chondroitin Sulf te, polyacrylamide or a mixture thereof.

21. A process of claim 17 wherein the visco elastic substance is hydroxy propyl methyl cellulose (hpmc)

22. A process of claim 17 wherein the ingredients are mixed in the following proportions:

Ocimum sanctum extract - 0.25 %w/v and - visco elastic substance - 0.25 % w/v

23. A process of claim 17 wherein the ingredients are mixed in the following proportions:

Ocimum sanctum Extract - 0.15 % w/v and - visco elastic substance - 0.25 % w/v

24. A process of claim 17 wherein the ingredients axe mixed in the following proportions:

Curcuma longa extract - 0.02 % w/v and - visco elastic substance - 0.25 % w/v

25. A process of claim 17 wherein the ingredients are mixed in the following proportions: Curcuma longa extract - 0.01 % w/v and visco elastic substance - 0.25 % w/v

26. A process of claim 17 wherein the ingredients are mixed in the following proportions:

Ocimum sanctum extract - 0.3 % w/v

Curcuma longa extract - 0.02 % w/v and visco elastic substance - 0.25 % w/v

27. A process of claim 17 wherein the ingredients are mixed in the following proportions: aqueous extract of Ocimum sanctum - 0.01 % w/v and aqueous extract of Curcuma longa - 0.02 % w/v

28. A process of claim 17 wherein the ingredients are mixed in the following proportions: aqueous extract of Ocimum sanctum - 0.03 % w/v and aqueous extract of Curcuma longa - 0.02 % w/v

29. A process of claim 17 wherein the ingredients are mixed in the following proportions:

Ocimum sanctum extract - 0.15 % w/v

Curcuma longa extract - 0.01 % w/v and visco elastic substance - 0.25 % w/v

30. A process of claim 26 wherein the ingredients are mixed in the following proportions:

Ocimum sanctum extract - 0.3 % w/v Curcuma longa extract - 0.02 % w/v and sodium hyaluronate - 0.25 % w/v

31. A process of claim 29 wherein said ingredients are mixed in the following proportions:

Ocimum sanctum extract - 0.15 w/v

Curcuma longa extract - 0.01 % w/v and

Polyacrylamide - 0.25 % w/v

32. A process of claim 29 wherein the ingredients are mixed in the following proportions:

Ocimum sanctum - 0.15 w/v

Curcuma longa - 0.01 % w/v and

Hydroxy propyl methyl cellulose - 0.25 % w/v