Description:TECHNICAL FIELD

[0001] The present invention relates to a statistically designed process for recovery of natural dye from the bark of Soymida febrifuga via Response Surface Methodology. The invention also relates to a method for sustainable harvesting of bark from Soymida febrifuga rather than irregular stripping of the bark which is detrimental to the health of the tree. Also, the present invention relates to

BACKGROUND ART

[0002] Forest is a great reservoir of phyto-diversity and a treasure-house of diverse natural products. One of such products is dye. Pigment from leaves, fruits, seed, wood and roots were used as dye stuff for textiles and as paint in art and craft. Natural dyes are environmentally friendly, hygienic, and user friendly than other colorants.

[0003] Though synthetic dyes are more affordable for the industry, the environment suffers a heavy price because they are dangerous and carcinogenic. An eco-friendly production model offered by natural dyes is a better alternative to synthetic dye. Natural dyes help in replacing the use of the hazardous and carcinogenic synthetic dyes.

[0004] Nevertheless, a fundamental challenge for the promotion of natural dyes is the sustainable availability of resources that include dye. It is challenging to extract dyes from tree bark, wood, and roots without harming the plant, so there is a need for a regulated and sustainable harvesting method that enables sustainable harvesting while maintaining tree health and healing. This will maintain tree health and lessen reckless cutting, both of which reduce deforestation.

[0005] Apart from the issue of sustainable supply of raw material as dye resources, the existing practices of dye extraction is a tedious process which involves large amount of natural raw materials, and solvent, more extraction time and low yield of dye which demotivates the industry/end users from using natural dyes.

[0006] Improvement in harvesting and dye extraction process will help in improving the livelihoods of many people involved in collection and extraction process. The environmentally friendly dye used in various finished products will be advantageous to both the industry and the consumer.

[0007] WO2011012471A2 discloses a method for producing a solution containing a dye and to a method for dyeing a substrate, characterized by the following steps: a. transferring the dye from biological material into an aqueous medium, b. precipitating the dye from the aqueous medium by the addition of a suitable precipitant, wherein at least a part of the precipitant forms a part of the dye precipitate, c. separating the dye precipitate from the aqueous medium and d. transferring the dye from the dye precipitate into an aqueous medium by adding a release agent and dyeing the substrate with an aqueous solution by releasing the dye from the dye precipitate by adding a release agent to the aqueous solution, characterised in that the release agent is a complexing agent, selected from the group of polycarboxylates, polycarboxylic acids, amino polycarboxylic acids, such as NTA or EDTA, oxalate, gluconate, citrate, phosphate, polyphosphate or combinations thereof.

[0008] EP0754734A1 discloses aprocess for producing dyestuffs from plant materials. The dried, cleaned and pulverized plant material is subjected to the step of extraction consisting of a first soaking in an aqueous solution of pH 4 to 10, followed by a second step of heating. The solution is filtered and concentrated.

[0009] Several natural dyes have been isolated from different parts of various plant species. However, there is an imperative need to assess the sustainable availability of the raw plant material from lesser known plant species to conserve and explore their bioprospecting potential. One such species is Soymida febrifuga (Roxb.).

[0010] Based on the traditional knowledge and published literature, bark of the species has application as dye. In nature, Soymida febrifuga oozes red color material from cut bark which could be a potential dye source. However, information available on the use of S. febrifuga bark as dye is lesser known, and method for recovery of dye from this bark is previously not known.

[0011] To address the aforementioned drawbacks of the sustainability of the raw material and the expensive as well as time-consuming dye extraction process, there is a need in the art to develop a method that uses a sustainable supply of the raw material, such as the bark of Soymida febrifuga, and enhances the recovery of dye in an economical way.

OBJECTS OF THE INVENTION

[0012] The principal object of the present invention is to overcome the disadvantages of the prior art.

[0013] An object of the present invention isto provides a method for sustainable harvesting of bark from Soymida febrifuga.

[0014] Another object of the present invention is to provide a statistically designed Box Behnken method that optimizes the dye extraction process from the bark of Soymida febrifuga.

[0015] Another object of the present invention is to provide a process which provides a high yield of dye via Response Surface Methodology.

[0016] Another object of the present invention is to provide a method that is efficient and cost-effective.

[0017] The foregoing and other objects of the present invention will become readily apparent upon further review of the following detailed description of the embodiments as illustrated in the accompanying drawings.

SUMMARY OF THE INVENTION

[0018] The present invention relates to an optimized process for extraction of dye in high yield from Soymida febrifuga bark. Box-Behnken design is applied for optimizing the conditions for extraction of dye in high yield.

[0019] The invention also relates to a harvesting method which ensures sustainable supply of dye yielding bark vis-a vis maintaining the health and growth of the tree.

[0020] According to an embodiment of the present invention, a sustainable method for harvesting of the bark comprises of following steps, i) selecting a tree i.e. Soymida febrifuga having girth at breast height (GBH) more than 50 cm, followed by collecting bark samples measuring 2x4 inches cut at 5 places in a tree, ii) Drying and chopping of the bark samples, followed by powdering using a pulverizer to obtain a powdered sample (material).

[0021] According to another embodiment of the present invention,iii) heating the powdered material in an aqueous solution at pH 11 on a boiling water bath, followed by filtering using a muslin cloth to obtain filtered solution; and iv) centrifuging the obtained solution at 2850-3150 rpm for 10-20 minutes at room temperature, followed by lyophilization using an Allied Frost Lyophilizer for 24-48 hrs. to obtain the natural red-brown dye.

[0022] While the invention has been described and shown with particular reference to the preferred embodiment, it will be apparent that variations might be possible that would fall within the scope of the present invention.

[0023] Figure 1 illustrates a pictorial representation of bark harvested from tree with 2 x 4 inches; and

[0024] Figure 2 illustrates a pictorial representation of drying of bark.

BRIEF DESCRIPTION OF DRAWINGS

[0025] So that the manner in which the above-recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may have been referred by embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

[0026] These and other features, benefits, and advantages of the present invention will become apparent by reference to the following text figure, with like reference numbers referring to like structures across the views, wherein:

DETAILED DESCRIPTION OF THE INVENTION

[0027] While the present invention is described herein by way of example using embodiments and illustrative drawings, those skilled in the art will recognize that the invention is not limited to the embodiments of drawing or drawings described and are not intended to represent the scale of the various components. Further, some components that may form a part of the invention may not be illustrated in certain figures, for ease of illustration, and such omissions do not limit the embodiments outlined in any way. It should be understood that the drawings and the detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the scope of the present invention as defined by the appended claim.

[0028] As used throughout this description, the word "may" be used in a permissive sense (i.e. meaning having the potential to), rather than the mandatory sense, (i.e. meaning must). Further, the words "a" or "an" mean "at least one” and the word “plurality” means “one or more” unless otherwise mentioned. Furthermore, the terminology and phraseology used herein are solely used for descriptive purposes and should not be construed as limiting in scope. Language such as "including," "comprising," "having," "containing," or "involving," and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents, and additional subject matter not recited, and is not intended to exclude other additives, components, integers, or steps. Likewise, the term "comprising" is considered synonymous with the terms "including" or "containing" for applicable legal purposes. Any discussion of documents, acts, materials, devices, articles, and the like are included in the specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention.

[0029] In this disclosure, whenever a composition or an element or a group of elements is preceded with the transitional phrase “comprising”, it is understood that we also contemplate the same composition, element, or group of elements with transitional phrases “consisting of”, “consisting”, “selected from the group of consisting of, “including”, or “is” preceding the recitation of the composition, element or group of elements and vice versa.

[0030] The present invention is described hereinafter by various embodiments with reference to the accompanying drawing, wherein reference numerals used in the accompanying drawing correspond to the like elements throughout the description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. In the following detailed description, numeric values and ranges are provided for various aspects of the implementations described. These values and ranges are to be treated as examples only and are not intended to limit the scope of the claims. In addition, several materials are identified as suitable for various facets of the implementations. These materials are to be treated as exemplary and are not intended to limit the scope of the invention.

[0031] The present invention relates to a method that optimizes the dye extraction process to get high dye yield from bark of Soymida febrifuga for further bioprospecting and/or value addition.

[0032] The present invention relates to a method for extraction of dye from bark of Soymida febrifuga by ensuringits sustainable harvesting without harming tree trunk. The tree having GBH more than 50 cm was chosen andcuts (width x length) measuring 2x4inches were made for proper isolation of bark.

[0033] In another embodiment the method for extraction of dye, comprises of following steps, i) selecting a tree having GBH more than 50 cm, followed by collecting bark piecesmeasuring 2x4 inches cut at 5 places in a tree; ii) drying and chopping of the bark pieces, followed by powdering using a pulverizer to obtain a powdered material; iii) heating the powdered material in an aqueous solution at pH 11 on a boiling water bath, followed by filtering using a muslin cloth to obtain filtered solution; and iv) centrifuging the obtained solution at 2850-3150 rpm for 10-20 minutes at room temperature, followed by lyophilization for 24-48 hrs. to obtain the natural dye.

[0034] Referring to figure 1, the data of fresh weight, dry weight and healing time were recorded and analyzed statistically. Among the three cuts tried, 2x4 inches cut was found better for proper harvesting and healing of tree within 6-9 months. Thus, a 2x4 inches cut at 5 places in a tree could be appropriate approach for the sustainable harvesting of bark as a natural dye source.

[0035] The obtained bark samples from the above harvesting method were sun dried, chopped and ground to fine powder. To optimized the dye extraction process, a Box-Behnken design has been used by considering three levels of factor viz. bark quantity as material, pH and extraction time. All the extractions were done in 100 ml distilled water on boiling water bath.

[0036] Further, the absorption maxima of the extracted dye were screened (200-750nm) in spectrophotometer and maximum absorption was found at 472 nm. All the experiments were done in triplicate and data were analyzed statistically based on yield of dye. Among the three factors, alkaline medium gave better red-brown dye yield from the bark. This may be attributed to effect of media which enhances phenolic extraction from bark.

[0037] The result revealed that the optimized conditions for extraction using material, extraction time, and pH for the better dye yield were found to be material (6 g), extraction time (45 minutes) and pH (11.0) because of the significant linear effect of the factors. At these optimized parameters for extraction, maximum yield achieved for dye was found to be 18.77% with maximum absorbance at 472nm.

[0038] Therefore, the present inventions provide an optimized extraction process to get natural dye in high yield for industries, and a sustainable technique for harvesting of bark from Soymida febrifuga for supply of raw material with proper healing of tree.

[0039] Overall, the 2x4 inches cut is suitable for sustainable harvesting of Soymida febrifuga bark while optimized dye extraction parameters are material (6 g), extraction time (45 minutes) and pH (11.0) with maximum yield 18.77%. This approach could also provide a future path for bioprospecting or value addition of the species.

[0040] Extensive field surveys were carried out to locate the Soymida febrifuga populations in Telangana. The harvesting method of bark from Soymida febrifuga was standardized in order to provide sustainable supply of raw material. Trees having GBH more than 50 cm were chosen from Narsapur and Amangal forest areas.

[0041] All necessary permissions were taken from Telangana State Forest Department, Telangana and concerned DFO/ROs of the Division/ranges. The plant material was authenticated by Botanical Survey of India, Hyderabad and specimen of the plant was deposited in the herbarium maintained at IFB, Hyderabad, India.

[0042] To achieve sustainable harvesting and maintaining tree health and growth, an experiment with three cut sizes (width x length) i.e., 2x4, 3x4 and 4x4 inches cuts and 1-2 cm deep were made in tree trunk (care was taken to extract only bark portion) to get bark samples for natural dye extraction.

[0043] To isolate 2x4, 3x4 and 4x4 inches bark pieces, 2 places were selected in a tree. The trees with cuts were visited in interval of six month to record the wound healing status. The fresh and dry weight of each bark samples were recorded and analyzed statistically. The result of bark yield from different cut sizes is presented in table 1.
Table 1: Characteristics of different cut size bark
Cut size (in inches) 2 x 4 3 x 4 4 x 4
Average sample fresh weight/tree (two samples/tree) 88.3 176.1 226.9
Std.
Deviation 14.7 17.2 37.4
Std. Error 6.5 7.7 16.7
F 38.4
Average sample dry weight/tree (Two samples/
tree) 50.8 94.3 127.3
Std. Deviation 9 10.5 33.2
Std. Error 4 4.6 14.8
F 17.0
Recorded complete Healing Time Within 6-9 months Within 18-24 months Within 24-36 months

[0044] The difference in the yield from the three cut sizes is statistically significant. However, the wound healing time is fast and comparatively less in 2x4 inch cut (Fig2). In subsequent experiment the numbers of cuts of 2x4 inch size were increased to 5.

[0045] The yield could be increased to 220.75 g (fw) and 127.0g (dw) with no change in wound healing time which is also very close to 4x4 inch cut. Though the harvested bark yield for 3x4 and 4x4 inches cut were higher than the 2x4 inch cut but healing time is a faster in 2x4 cut.

[0046] Overall, among the three cuts trees 2x4 inches cut was found better for proper harvesting and healing of tree within 6-9 months. Thus, a 2x4 inches cut at 5 places in a tree could be an appropriate approach for the sustainable harvesting of bark as a natural dye source.

[0047] Response Surface Methodology (RSM) is a systematic and contemporary statistical device employed in bioprocess optimization and modeling strategies where diverse design sets are used for problem analysis. RSM is composed of different mathematical designs of which Box-Behnken design (BBD) has been applied successfully to attain the enhanced values for extraction in diverse botanicals.

[0048] Response variables possess linear, quadratic and interaction relations to elicit better yield of the targeted substances during the extraction process. Hence, to confirm the influenced extraction variables as well as to calculate the response’s internal variability, BBD is employed for computational analysis of the relations that exist among diverse response variables consisting of linear, quadratic as well as 2-way interaction model. Analyses for multiple regressions were carried out to produce the predicted response values (%) for dye yield (Y).Three levels of each factor were taken follows
1. Material (A: 2, 4 and 6g),
2. Extraction time (B: 15, 30 and 45 minutes) and
3. pH (3, 7 and 11)

[0049] The three factors were taken as above in order to achieve enhanced dye yield from Soymida febrifuga. Distilled water 100 ml as liquid ratio was kept constant for all the experimental. Boiling water bath was used for extraction. The extracted dye was filtered through muslin cloth and then obtained liquid was centrifuged at 3000 rpm for 15 minutes at room temperature. The obtained clear supernatant was lyophilized in Allied Frost Lyophilizer for 24-48 hrs to avoid any chemical changes in dye.

[0050] The yield of dye was calculated and presented in percentage content. Table 2and3 exhibits the BBD design and dye yield which ranged from 7.7 to17.50%.
Table 2 Box Behnken Design (BBD) with three response variables: Material, Time, and pH
Replication Response variable Dye absorption and yield
Material (g) Time (min) pH λ max (nm) Yield (%)
3 6.0 45.0 7.0 0.072 10.35
1 2.0 30.0 11.0 0.17 15.90
2 6.0 45.0 7.0 0.069 10.20
2 4.0 30.0 7.0 0.052 8.47
1 2.0 45.0 7.0 0.044 10
2 2.0 45.0 7.0 0.036 10.75
1 4.0 15.0 11.0 0.217 16
1 2.0 30.0 3.0 0.021 11.30
3 6.0 30.0 11.0 0.527 17.50
1 6.0 45.0 7.0 0.071 10.25
2 2.0 15.0 7.0 0.029 7.7
1 4.0 30.0 7.0 0.051 8.30
2 2.0 30.0 11.0 0.175 16.10
3 2.0 30.0 3.0 0.019 11.40
3 4.0 30.0 7.0 0.053 8.50
1 6.0 15.0 7.0 0.072 8.60
3 4.0 30.0 7.0 0.053 8.50
3 4.0 15.0 3.0 0.028 10.15
3 4.0 30.0 7.0 0.053 8.50
1 4.0 45.0 3.0 0.039 12.57
1 6.0 30.0 3.0 0.044 13.30
1 4.0 15.0 3.0 0.029 10
1 4.0 30.0 7.0 0.051 8.30
3 6.0 15.0 7.0 0.073 8.66
2 4.0 30.0 7.0 0.052 8.47
3 2.0 45.0 7.0 0.043 10.50
2 2.0 30.0 3.0 0.021 11.55
3 2.0 30.0 11.0 0.182 16
2 4.0 15.0 11.0 0.22 16.30
2 6.0 30.0 11.0 0.524 17.16
2 4.0 30.0 7.0 0.052 8.47
1 6.0 30.0 11.0 0.531 17.50
3 2.0 15.0 7.0 0.024 7.60
3 4.0 15.0 11.0 0.219 16.15
1 2.0 15.0 7.0 0.03 7.45
3 4.0 45.0 11.0 0.369 17.67
2 6.0 15.0 7.0 0.074 8.61
2 4.0 45.0 3.0 0.042 12.62
2 4.0 45.0 11.0 0.395 17.80
1 4.0 45.0 11.0 0.356 17.55
2 4.0 15.0 3.0 0.028 10.32
3 6.0 30.0 3.0 0.041 13.21
1 4.0 30.0 7.0 0.051 8.30
3 4.0 45.0 3.0 0.04 12.60
2 6.0 30.0 3.0 0.043 13.18

Table 3: ANOVA TABLES AND RELATED INFERENCES
Factor Mean/Interc. Material(L) Material
(Q) pH (L) pH (Q) time (L) time (Q)
Effect 12.62639 1.01833 -0.58375 4.9525 -5.50125 2.10583 -0.21958
Std.Err. 0.069686 0.170695 0.125628 0.170695 0.125628 0.170695 0.125628
t(38) 181.1902 5.9658 -4.6467 29.0138 -43.79 12.3368 -1.7479
P 0 0.000001 0.00004 0 0 0 0.088561
-95.00% Cnf.Limt 12.48532 0.67278 -0.83807 4.60695 -5.75557 1.76028 -0.4739
+95.00% Cnf.Limt 12.76746 1.36389 -0.32943 5.29805 -5.24693 2.45139 0.03474
Coeff. 12.62639 0.50917 -0.29188 2.47625 -2.75063 1.05292 -0.10979
Std.Err. Coeff. 0.069686 0.085347 0.062814 0.085347 0.062814 0.085347 0.062814
-95.00% Cnf.Limt 12.48532 0.33639 -0.41904 2.30347 -2.87779 0.88014 -0.23695
+95.00% Cnf.Limt 12.76746 0.68194 -0.16471 2.64903 -2.62346 1.22569 0.01737

Note: For Yield Percentage, except the quadratic effect of time, all other effects are significant.

Factor ANOVA; Var.:yield per; R-sqr=.98729; Adj:.98528 (Spreadsheet1)
3 3-level factors, 1 Blocks, 45 Runs; MS Residual=.1748202
DV: yield per
SS df MS F P
Material(L) 6.2220 1 6.2220 35.591 0.000001
Material(Q) 3.7746 1 3.7746 21.591 0.000040
pH (L) 147.1635 1 147.1635 841.800 0.000000
pH (Q) 335.2292 1 335.2292 1917.566 0.000000
Time (L) 26.6072 1 26.6072 152.198 0.000000
Time (Q) 0.5341 1 0.5341 3.055 0.088561
Error 6.6432 38 0.1748
Total SS 522.6705 44

Note: For yield percentage, significant differences in mean were noticed for all except the quadratic effect of time.

Factor ANOVA; Var:yield per; R-sqr.=.98729; Adj:.98528 (Spreadsheet1)
3 3-level factors, 1 Blocks, 45 Runs; MS Residual=.1748202
DV: yield per
SS Df MS F p
Material L+Q 9.9966 2 4.9983 28.591 0.000000
pH L+Q 482.3928 2 241.1964 1379.683 0.000000
Tme L+Q 27.1413 2 13.5706 77.626 0.000000
Error 6.6432 38 0.1748
Total SS 522.6705 44

On the whole, all the mean yield percentage was significantly different for material, pH and time.

Desirability function parameters (Spreadsheet1) Desirability function settings for each dependent variable
Variable Low value Desirability value Medium value Desirability value High value Desirability value s Param. t Param.
yield_per 7.45 0 12.625 0.5 17.8 1 1 1

Current factor settings and predicted responses (Spreadsheet1) Predicted responses at the current level of each factor in the model
Level of material Level of time Level of pH Predictedyield per Desirability Value (-)95%CI yield per (+)95%CI yield per
6 45 11 18.76625 0.865602 18.37352 19.15898

[0051] The optimize values of the levels of the factors are given above. The overall desirability value is 0.87 which is close to 1, which is essential for arriving at values close to true values. The predicted response is also given in the table 3. Overall, the optimized conditions for extraction using material, extraction time, and pH for the better dye yield were found to be material (6 g), extraction time (45 minutes) and pH (11.0) because of the significant linear effect of the factors. At these optimized parameters for extraction, maximum yield achieved for dye was found to be 18.77% with maximum absorbance at 472nm.

[0052] Overall, present invention provides a process for sustainable harvesting of bark from Soymida febrifuga for regular supply of raw material (bark) with proper healing of tree. The presented optimized dye extraction process could also be a leading economical approach to get natural dye for industries. This approach could also provide a future path for bioprospecting or value addition in the species.

[0053] Further, while one or more operations have been described as being performed by or otherwise related to certain modules, devices or entities, the operations may be performed by or otherwise related to any module, device or entity. As such, any function or operation that has been described as being performed by a module could alternatively be performed by a different server, by the cloud computing platform, or a combination thereof.

[0054] Further, the operations need not be performed in the disclosed order, although in some examples, an order may be preferred. Also, not all functions need to be performed to achieve the desired advantages of the disclosed system and method, and therefore not all functions are required.

[0055] Various modifications to these embodiments are apparent to those skilled in the art from the description and the accompanying drawings. The principles associated with the various embodiments described herein may be applied to other embodiments. Therefore, the description is not intended to be limited to the 5 embodiments shown along with the accompanying drawings but is to be providing the broadest scope consistent with the principles and the novel and inventive features disclosed or suggested herein. Accordingly, the invention is anticipated to hold on to all other such alternatives, modifications, and variations that fall within the scope of the present invention and appended claims.
, Claims:I/We Claim:
1) A process for recovery of natural dye from sustainably harvested bark of Soymida febrifuga,comprises the following steps:

i) Selecting a tree having GBH more than 50 cm, followed by collecting bark pieces measuring 2x4 inches cut at 5 places in the tree;
ii) Drying and chopping of said pieces, followed by powdering to obtain a powdered material;
iii) Heating said powdered material in an aqueous solution at pH 11 on a boiling water bath, followed by filtering using a muslin cloth to obtain filtered solution; and
iv) Centrifuging said solution at 2850-3150 rpm for 10-20 minutes at room temperature, followed by lyophilization for 24-48 hrs to obtain said dye.

2) The method as claimed in claim 1, whereinsaid tree is more preferably a Soymida febrifuga.

3) The method as claimed in claim 1, wherein said bark of tree is selected for extraction of dye.

4) The method as claimed in claim 1, wherein said powdering of the dried and chopped bark is performed by a pulverizer.

5) The method as claimed in claim 1, wherein said powdered bark is heated in an aqueous solution at pH 10-12 on a boiling water bath for 40-50 minutes followed by filtering using a muslin cloth to obtain filtered solution

6) The method as claimed in claim 1, wherein said filtered solution is centrifuged at 2850-3150 rpm for 10-20 minutes at room temperature followed by lyophilization for 24-48 hrs to obtain the said dye

7) The method as claimed in claim 1, wherein said lyophilization is performed using anAllied Frost Lyophilizer.