WO2020261299A1

WO2020261299A1 - Method for preparing biodegradable staining composition

Info

Publication number: WO2020261299A1
Application number: PCT/IN2020/050557
Authority: WO
Inventors: Vasudha Kulkarni; DR. Ramesh B.R.
Original assignee: Rajiv Gandhi University Of Health Sciences; Dr. B. R. Ambedkar Medical College
Priority date: 2019-06-28
Filing date: 2020-06-27
Publication date: 2020-12-30

Abstract

The present invention relates to a cost-effective and eco-friendly technique of producing nuclear staining composition from Indian black berry fruit. The composition is as effective as its counterpart Hematoxylin in staining biological samples. The general composition includes black berry fruit extract, tea decoction and potash alum.

Description

METHOD FOR PREPARING BIODEGRADABLE STAINING COMPOSITION

The present invention is directed to a cost-effective and biodegradable method for preparing staining compositions, and more particularly to a method for preparing composition from black berry fruit for staining biological samples.

Hematoxylin is the most widely and routinely used nuclear stain for histo-chemical staining and histo-pathologic studies. Every day up to 2.5 to 3 million slides are stained with this technique. In particular, Hematoxylin stains the chromatin within nucleus of a cell to detect the presence of malignant and/or metastatic cells in a tumor biopsy sample.

Hematoxylin is a naturally-occurring compound and the stain is obtained from logwood of tree Hematoxylon campechianum. The shortage of dye has resulted in increase in its cost, costing up to Rs. 2500 for 5gms of Hematoxylin powder.

Various plant, insect, flower and fruit extracts such as saffron, carmine have been used in place of Hematoxylin as tissue dyes to stain cytological and tissue samples before the era of Hematoxylin. Significant results have been obtained in earlier studies such as use of aqueous crude extract from butterfly pea flowers (ClitoriaTernatea L) for staining animal blood smears, use of Roselle (Hibiscus Sabdariffa) on histologic section of testis, mulberry juice with mordant as an alternate to Hematoxylin, Methyl green counterstaining of tissue section as an alternate to Hematoxylin.

Therefore, the need of hour is to produce a cost-effective substitute for Hematoxylin to reduce brunt of histopathology cost that is borne by patients today.

Object Of The Invention

The primary objective of the present disclosure is to provide a cost-effective substitute for Hematoxylin by exploring Indian black berry juice.

In one other objective of the present disclosure, a cost-effective manner of producing histological slides is provided for various streams such as medical, dental, paramedical etc.

Another objective of the present disclosure is to standardize and quantify Indian black berry stain as an alternate stain for Hematoxylin.

In yet another objective of the present disclosure, efficacy of substitute stain with Hematoxylin is compared and tested.

In accordance with first aspect of disclosure, a method for preparing staining compositions is disclosed. Accordingly, the steps comprises of first boiling a fruit pulp obtained from a fruit to extract active ingredient thereof.
This is followed by blending the fruit pulp and thereafter filtering juice obtained therefrom. Next, the filtered juice is reconstituted with a combination of mordants, buffering agents, oxidising agents, alcohol or acid; and finally the solution is oxidized at a predetermined pH value for a predetermined time period.

In accordance with another aspect of present disclosure the fruit selected is Indian black berry and the active ingredient extracted therefrom is anthocyanin.

Before the present working principle of producing substitute of Hematoxylin is described, it is to be understood that this disclosure is not limited to the particular method for achieving so, as described, since it may vary within the specification indicated. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. The words "comprising," "having," "containing," and "including," and other forms thereof, are intended to be equivalent in meaning and be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. The disclosed embodiments are merely exemplary methods of the invention, which may be embodied in various forms.

The present disclosure envisages a detailed and descriptive study design for exploring Indian black berry juice as a cost-effective substitute of Hematoxylin for staining biological samples. At first, tissue sections are obtained from formalized cadaver specimens (lymph node, palatine tonsil, intestine, papillae of tongue etc.). Thereafter, according to one exemplary embodiment, twenty slides are prepared out of each tissue, are stained with standardized berry stain, and visualized under binocular compound microscope for obtainment of results which are then photographed and documented.

In order to obtain the histo-chemical stain, Indian black berry fruit is first procured, washed thoroughly, followed by extraction of its pulp for further processing. The fruit pulp so obtained is further processed by any of the chosen ways. In one exemplary embodiment of fruit pulp processing, the fruit pulp is first boiled in distilled water to extract its active ingredient anthocyanin. The pulp is then blended into fine paste and the filtered juice is boiled. The filtered pulp is, however, used without boiling.

Next, almost fifteen different solutions are made by reconstituting fruit juice extract with:

Mordants such as Aluminum potassium chloride, ferric chloride, lugol’s iodine, tea decoction, aluminum ammonium chloride, phenol(carbolic acid)
Buffering agents such as Sodium hydroxide flakes, sodium acetate flakes, Sodium chloride, Caustic soda
Oxidizing agents such as Potassium dichromate, potassium iodide
Absolute alcohol, glacial acetic acid

After the above preparation is completed, about 370 slides are stained with solution made from Indian black berry fruit extract. Some of these solutions exhibited positive results as they successfully stained nervous tissues such as spinal cord and cerebellum. In one specific embodiment, one of the solutions which is naturally oxidized for three months and contained fruit extract, tea decoction, potash alum and aluminium ammonium chloride stained the nervous tissue sections such as spinal cord and cerebellum. Generally speaking, the duration of staining has now been reduced from 31 days to 8-9 days. Eventually, the stains are compared with Kluverberrara stain of nervous tissue for efficacy, clarity and cost effectiveness.

It is to be noted that the flavonoid which is responsible for staining of tissues by Indian black berry is anthocyanin. In accordance with one exemplary embodiment of present disclosure, two techniques can be attempted to increase quality of active ingredient Anthocyanin, available for tissue staining. In one such instance, almost 100 gms of Indian black berry fruit powder is obtained from deseeded fruit pulp by hot air drying technique. Then, approximately 200 gms of fruit pulp powder is processed from freeze drier system. The latter technique gave effective results on staining the tissues.

For qualitative assessment of Anthocyanin available from fruit powder processed through both the techniques, high performance liquid chromatography can be performed. Yield of Anthocyanin pigment from high performance liquid chromatography was 4.074 %. The Anthocyanin were characterized by two absorption bands Band I475560 nm (Visible region) Band II275280 nm (UV region) The broad peak of absorbance was observed at wavelength 526 nm.

In one working embodiment of present disclosure, the general composition of Indian black berry fruit for histo-chemical staining of a biological sample includes fruit extract, tea decoction, and potash alum. In particular, 70ml of fruit extract, 10 ml of tea decoction and 50mgm of potash alum can be used for producing the aforementioned staining composition. The solution is then allowed to naturally oxidize for 40 days while the pH of solution is maintained at 7.2.

In a general procedural methodology, the Indian black berry fruit is first processed to obtain a powder thereof from spray drying technique. Thereafter, the Indian black berry stain is produced using the aforementioned technique. The tissues are then stained with Indian berry stain and counter stained with Eosin. The general advantages of preparing nuclear stains from Indian black berry fruit is highly cost-effective, biodegradable, eco-friendly, is easy to prepare and can be obtained from renewable sources.

The foregoing description is a specific embodiment of the present disclosure. It should be appreciated that this embodiment is described for purpose of illustration only, and that numerous alterations and modifications may be practiced by those skilled in the art without departing from the spirit and scope of the invention. It is intended that all such modifications and alterations be included insofar as they come within the scope of the invention as claimed or the equivalents thereof.

Claims

A method for preparing staining compositions, comprising:
boiling a fruit pulp obtained from a fruit to extract active ingredient thereof;
blending the fruit pulp and filtering juice obtained thereof;
reconstituting the filtered juice with a combination of mordants, buffering agents, oxidising agents, alcohol or acid; and
oxidising the reconstituted filtered juice at a predetermined pH value for a predetermined time period.
The method for preparing staining compositions in accordance with claim 1, wherein the fruit is Indian black berry.
The method for preparing staining compositions in accordance with claim 1, wherein the active ingredient extracted from the fruit pulp is anthocyanin.
The method for preparing staining compositions in accordance with claim 1, wherein the filtered juice is boiled prior to reconstitution.
The method for preparing staining compositions in accordance with claim 1, wherein the mordant used for reconstitution is selected from a group comprising of Aluminum potassium chloride, ferric chloride, lugol’s iodine, tea decoction, aluminum ammonium chloride, phenol (carbolic acid) or a combination thereof.
The method for preparing staining compositions in accordance with claim 1, wherein the buffering agent used for reconstitution is selected from a group comprising of sodium hydroxide flakes, sodium acetate flakes, sodium chloride, caustic soda or a combination thereof.
The method for preparing staining compositions in accordance with claim 1, wherein the oxidising agent used for reconstitution is selected from a group comprising of potassium dichromate, potassium iodide, lugol’s iodine or a combination thereof.
The method for preparing staining compositions in accordance with claim 1, wherein the alcohol is absolute alcohol and acid is glacial acetic acid.
The method for preparing staining compositions in accordance with claim 1, wherein the predetermined pH level is 7.2.
The method for preparing staining compositions in accordance with claim 1, wherein the predetermined time period for oxidation is 40 days.