FORM 2
THE PATENT ACT 1970
(39 OF 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
TITLE OF THE INVENTION
BIOTRANSFORMATION OF BIOLOGICALLY ACTIVE COMPOUNDS FROM MEDICINAL PLANTS
APPLICANT(S)
(a) Name: Myko Tech Private Limited
(b) Nationality: Indian
(c) Address: #313 Vainguinnim valley,
Dona Paula,
Goa-403004,
India.

Priority Date
This complete specification claims priority from the provisional application number 3256/MUM/2010 filed at Indian Patent Office, Mumbai on 29-November-2010.
The following specification particularly describes the invention and the manner in which it is to be performed.
Field of the Invention
The present invention relates to a process for biotransformation of compounds, extracts or herbal extracts of medicinal plants using the enzyme laccase. It also relates to several products obtained through biotransformation of compounds, extracts or herbal powder of medicinal plants with improved biological activities against different disease conditions.
Background of the Invention
Since ages, plants have been the source of numerous compounds that are used in modern medicine, either in their native form or in the form of their synthetic analogues. There is further scope to develop novel drugs from traditionally used medicinal plants. In recent years a great interest has been revived in traditional medicinal plants and their active principles in order to treat various metabolic disorders such as diabetes, cancer, inflammation and others more effectively. For example, there are over 3,000 recognized medicinal plants in India, while actually twice the number of these plants in the country is actually believed to be used in various forms of traditional herbal medicine in the country (Dubey NK, R Kumar and P Tripathi. 2004. Global promotion of herbal medicine: India's opportunity. Current Science 86: 37-41). These plants are used in various forms of herbal preparations to treat diseases in traditional medicines, such as the Ayurveda, Unani and Siddha systems of India. In contrast to such traditional methods to treat diseases, modern medicine prefers to use single compounds for treatment of diseases because this approach has the benefit of repeatability. In addition, the precise mechanisms of action, as well as toxicology problems associated with it can be accurately determined. However, the use of active principles from traditional medicinal plants has also faced several problems. Thus, some of the compounds show low bioavailability when consumed. Many medicinal plants have yielded active principles, which have, however, shown low activity compared to those in use. It is also believed that some of the active principles may not produce the desired results when used alone, but that adjuvant substances in the plant enhance their activity through a synergistic action, either through protection of the active substance from degradation by enzymes, facilitation of transport across barriers such as cell and organelle walls, or by overcoming multidrug resistance (Gilbert B and L Alves. 2003. Synergy in plant medicines. Current Medicinal Chemistry 10: 13-20). One of the potential solutions to overcome these problems in using the active

principles is to increase the activity of the compound itself or further increase the efficacy of the herbal preparation to treat specific diseases. Such an improvement could be achieved by various means of chemical or biological transformation and catalysis, allowing the compound or the herbal preparation to be modified, or polymerized.
Biological transformation can be used by biocatalysis using enzymes. Biocatalysis using enzymes has the advantage over chemical synthesis because enzyme-based reactions are often highly enantitoselective and regioselective. These reactions can be carried out at ambient temperature and atmospheric pressure and are much more environmental friendly than harsh chemical methods (Kobayashi S, H Uyama and S Kimura, 2001, Enzymatic Polymerization. Chemical Reviews 101: 3793-3818). Patel R et alo., 2003. Enzymatic synthesis of chiral intermediates for pharmaceuticals. Journal of Industrial Microbiology and Biotechnology 30: 252-259).One of the tools available for biocatalysis is the use of the enzyme laccase.
Laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) belongs to the group of polyphenol oxidase enzyme or blue copper oxidases. It is widely distributed in higher plants and fungi. The enzyme oxidizes polyphenols, methoxy-substituted phenols, aromatic diamines and a range of other compounds, using oxygen, which is reduced to water (Baldrian P. 2005. Fungal laccases - occurrence and properties. FEMS Microbiological Reviews 30: 215-242). Because of its generally low substrate specificity, extracellular production and the fact that it does not need the addition of other cofactors for its activity, laccase is an extremely useful enzyme in biotechnology. Laccase has been widely used in a number of biotechnological applications, such as in bioremediation of effluents, textile industries, detoxification to pulp bleaching, removal of phenolics, food and beverage industries and others (Kunamneni A. et al. 2008. Laccases and their applications: A Patent Review. Recent Patent Biotehnology 2: 10-24).) Laccase activity can be further modified towards oxidation of non-phenolic substrates by including a mediator in the reactions. Such mediators include compounds such as 2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulfinic acid) or ABTS, violuric acid or VLA, 1-hydroxybenzotriazole or HOBt and N-hydroxyphthalimide or HPI (Baiocco P, AM Barreca, M Fabbrini, C Galli and P Gentili. 2003. Promoting laccase activity towards non-phenolic substrates: a mechanistic investigation with some laccase mediator systems. Organic Biomolecular Chemistry 1: 191-197).
Laccase has been used in a number of biocatalytic reactions to provide improved molecules for various applications. Laccase from the fungus Trametes versicolor has been used to generate the polymer polycatechol from catechol for various environmental applications (Aktas N and A Tanyolac. 2003. Reaction conditions for laccase catalyzed polymerization of catechol. Bioresource Technology 87: 209-214). A laccase-mediated biopolymerization method was used to generate polymers of 2,6-dimethylphenol in order to provide an alternative for production of conventional phenol resins (Ikeda R, J

Sugihara, H Uyama and S Kobayashi. 1996. Macromolecules 29:8702-8705). Laccase derived from the fungus Pycnoporus coccineus was used for the purpose. Laccase from the fungus Rhizoctonia praticola was used to cross-couple the environmental pollutant chloraniline in the presence of ferulic acid, in an investigation to incorporate chloranlinine with humic matter (Tatsumi K, A Freyer, RD Minard and J-M Bollag. 1994. Enzyme-mediated coupling of 3,4-dichloroaniline and ferulic acid: a model for pollutant binding to humic materials. Environmental Science Technology 28: 210-215). Laccase-mediated polymerization of new urushiol analogues for the preparation of artificial urushi, Japanese traditional coating known for its toughness and longevity has been demonstrated (Kobayashi et al., 2001). The flavonoid rutin, a compound with vasorelaxation, anti-inflammatory and hepatoprotective activities has been polymerized using laccase (Anthoni J et al., 2008. Investigations of enzymatic oligomerization of rutin. Rasayan Journal of Chemistry 1: 718-731). The antioxidant activity of the oligomers of rutin, however, was shown to decrease with molecular weight.
Laccase has been used to generate homo- or heteromolecular dimers through C-O, C-C and C-N bonds by means of phenolic oxidative coupling of various substrates (Mikolasch A and F Schauer. 2009. Fungal laccases as tools for the synthesis of new hybrid molecules and biomaterials. Applied Microbiology and Biotechnology 82: 605-624). The enzyme has been used to generate new antibiotics via phenolic oxidation, involving penicillin derivatives and phenolics. A variety of examples for derivatization of amino acids and production of polymers and biomaterials are also known.
WO0198518 (A2), WO0198518 (A3) and US2003180893 (Al) relate to a method to produce biologically active ingredients comprising additional functional groups, a modified spectrum of activity and modified application properties from medicaments or plant protection agents in the form of substrates which, as functional groups, carry at least one amino or hydroxyl group obtained by means of a one-electron reaction catalyzed using enzymes such as laccase.
US Patent 7538084 relates to the Synthesis of novel Cyclosporin A derivatives achieved biocatalytically by the use of enzymes from the laccase family.
Thus, there exists a great possibility to generate novel molecules with a variety of bioactive properties using laccase (Mikolasch and Schauer, 2009).
It would be useful if the biocatalytic properties of laccase could be used to generate novel and more useful compounds from active principles of traditional medicinal plants such that they could be practically used in modern medicines with greater efficacies than exist now.

Summary of the Invention
It is an object of the present invention to improve the bioactive properties of compounds or herbal powders derived from medicinal plants.
A further object of the present invention is to improve the bioactive properties of the compounds or herbal powders derived from medicinal plants used in the Ayurveda system of medicine.
A further object of this invention is to improve the bioactive nature of compounds or herbal powders derived from medicinal plants, particularly those used in the Ayurveda system of medicine through biocatalysis.
Yet another object of this invention is to disclose a process to improve the bioactive nature of compounds or herbal powders derived from medicinal plants, particularly those used in the Ayurveda system of medicine through biocatalysis, using the enzyme laccase.
A further object of this invention is to disclose a process to improve the bioactive nature of compounds or herbal powders derived from medicinal plants, particularly those used in the Ayurveda system of medicne through biocatalysis, using the enzyme laccase in a way that their various properties, such as anti-inflammatory, anticancer, antioxidant and others are enhanced. This is achieved by treating these with the enzyme laccase at a favourable pH and temperature, through which process the pure, biologically active compound of the medicinal plant or the herbal powder of the medicinal plant is chemically modified. The chemically modified compound or herbal powder has better biological activity, compared to the untransformed compound or herbal powder. Thus, such biotransformed compounds or plant products show enhanced activities against diseased conditions. . Some of the enhanced properties of these biotransformed products are anti-inflammatory activity, antioxidant activity, antibacterial activity and anti-diabetic activity.
Brief description of the drawings
Figure 1: illustrates the thin layer chromatography of standard catechol and biotransformed catechol.
Figure 2: illustrates the HPLC profile of standard cathechol and biotransformed catechol.
Figure 3: illustrates the anti-bacterial activity of control catechol and biotransformed catechol against different bacterial species.
Figure 4: illustrates the antioxidant activity of catechol, chemically derived bis-catechol and biotransforemed bis catechol

Figure 5: illustrates the thin layer chromatography of standard aloin and biotransformed aloin.
Figure 6: illustrates the Anti-inflammatory activity of standard and biotransformed aloin
Figure 7: illustrates the thin layer chromatography of standard and biotransformed curcumin.
Figure 8: illustrates the HPLC profile of standard and biotransformed curcumin.
Figure 9: illustrates the thin layer chromatography of standard vanillin and biotransformed vanillin.
Figure 10: illustrates the HPLC profile of standard and biotransformed vanillin.
Figure 11: illustrates the thin layer chromatorgraphy of control podophyllotoxin (left lane) and biotransformed podophyllotoxin (right lane lower r.f)
Figure 12: illustrates the HPLC profile of control podophyllotoxin (top), biotransformed podophyllotoxin using mediator HOBt (middle) and biotransformed podophyllotoxin without using mediator (below).
Figure 13: illustrates the anti-diabetic activity of standard and biotransformed herbal powder of Momordica charantia based on inhibition of the activity of the alpha-glucosidase enzyme.
Figure 14: illustrates the HPLC profiles of standard (above) and biotransformed extracts of the herbal powder of Boswellia serrata (below).
Figure 15: illustrates the anti-inflammatory activities of biotransformed and standard extracts of the herbal powder of Boswellia serrata based on inhibition of lipoxygenase activity.
Figure 16: illustrates the thin layer chromatorgraphy of biotransformed (left lane) and standard (right lane) of extracts of herbal powder of Boerhavia diffusa.
Figure 17: illustrates the HPLC profiles of standard (above) and biotransformed (below) extracts of herbal powder of Boerhavia diffusa.

Detailed description of the Invention
The foregoing object of the present invention is achieved by the below described method. Pure compounds corresponding to the biologically active principles of medicinal plants or the herbal powder of the medicinal plant containing a mixture of compounds including the active principle are used as the starting material. Such compounds may show any of various activities, such as anti-inflammatory, anticancer, antioxidant, antidiabetes, etc. The following are some of the examples, as given in Example 1. However, the compounds are not restricted to these.
Catechol, pyrocatechol or 1,2-dihydroxybenzene, which is found in a variety of plants and vegetables and has several medicinal applications, such as against rheumatoid arthritis, periodontitis, arteriosclerotic plaques, osteoporosis, arthrosis, metastasis and neoangiogenesis of tumors, ulceration of the cornea.
3-methyl catecho!,which is a derivative of catechol, which is being used as a precursor to fine chemicals such as perfumes and pharmaceuticals. It is a common building block in organic synthesis. Several industrially significant flavors and fragrances are prepared starting from catechol.
Vanillin is found in the vanilla bean, Vanilla planifola and is used as a natural flavouring agent.
5, 7-dihydroxy 4 -methyl coumarin, a derivative of coumarin, which is found in the cinnamon plant, Cinnamomum aromaticum and many others and is a blood thinner used to keep blood flowing smoothly and prevent the formation of blood clots. It has clinical medical value as the precursor for several anticoagulants, notably warfarin. Coumarin has been used in the treatment of lymphedema and it has appetite-suppressing properties.
P-Coumaric acid is found in many plants.
Menthol is obtained from the peppermint, Mentha piperita and other plants and is a flavouring compound.
Hydroquinone, which is present in very trace quantities in plants,is a depigmenting agent or skin lightening agent.
Phyllanthin is obtained from the plant Phyllanthus niruri and has antiviral properties.
Resveratrol, is found in the skin of red grapes and has anti-oxidant properties.
Curcumin, is found in the turmeric plant, Curcuma longa and which has several medicinal properties, including antitumor, antioxidant, antiarthritic, antiamyloid, anti-ischemic, and anti-inflammatory properties.

Aioin, is found in several species of the aloe plant and has properties that prevent colon cancer.
Podophyllotoxin is found in the roots and rhizomes of Poddphyllum species, and which is used on the skin to treat external genital warts, caused Py some types of the human papillomavirus and whose derivatives such as etoposide are anticancer drugs.
Herbal preparations of Momordica charantia, or the bitter gourd, which has antihelminthic, antiviral, antimalarial, cardioprotective, anti-diabetic and anti-cancer properties.
Herbal preparations of Boswellia serrata, or 'Shallaki' in Sanskrit, a well known Ayurvedic plant and which has potent analgesic and anti-Jnflammatory effects that can reduce the pain and inflammation of joints.
Herbal preparations of Gymnema sylvestre, has anti-diabetic properties.
Herbal preparations of Boerhavia diffusa, known as Punamava Sanskrit and widely used ia Ayurveda medicuve and which has antibacterial antioxidant,, hepatoprotective.. anticancer, antiestrogenic, antiamoebic and immunomodulatory activities.
The compounds or the herbal powder are dissolved in a suitable solvent, such as water, ethanol, ethyl acetate, acetone, chloroform, DMSO, etc. Herbal powders may also be simply suspended in an appropriate buffer or water. An appropriate quantity of the laccase enzyme is added to the dissolved compound or Plant material. The laccase enzyme may be commercially obtained or obtained by growing a suitable microorganism, such as a wood-decomposing fungus in a suitable culture rnedium using well known standard methods. The enzyme is secreted into the culture medium and may be used directly or after concentration. Concentration of the enzyme may be achieved by filtering the culture filtrate of the fungus through molecular sieves that will help in concentrating the enzyme. The enzyme may be used fresh, or may be freeze-dried and used whenever needed. The enzyme may be further purified using size exclusion or ion exchange chromatography and others known in the prior art before being used. The laccase enzyme is diluted to a suitable concentration in an appropriate buffer for the biocatalysis reaction. For each reaction, various pH and temperature ranges may be tried. For example, the pH may vary between 3 and 9. Temperatures for reaction may vary from a room temperature of approximately 25°C to 65°C. The appropriate temperature is generally decided by the optimal temperature for enzyme activity. Likewise, different concentrations of the laccase enzyme, from 2 units to 50 units per ml of reaction mixture may be tried. A typical example is the use of approximately 2 Units of the laccase enzyme per ml of reaction mixture. The enzyme dilutions are prepared using a suitable buffer, corresponding to the pH at which the enzyme may act optimally. Generally, an acetate buffer of pH 5.0 is suitable. Biocatalysis of the active compound is carried out by

adding an appropriate amount of the enzyme to the solution containing the compound, following standardization at different levels. The mixture containing the solution of the compound or the plant material and the enzyme in buffer is incubated at an appropriate temperature for the required length of time. This may vary from ambient room temperature at about 25 degrees Celsius to 60 degrees Celsius or more. The incubation time may vary from 1 hour to 24 hours. The incubation time may be decided by following the reaction and examining any changes in the compound or the plant extract or herbal powder by various chromatographic methods, such as thin layer chromatography, high performance liquid chromatography, or other suitable methods. For complex herbal powders, the bioactivity of the enzyme treated substrate may be checked after regular intervals. Often, the biocatalysis is stimulated by adding a mediator that helps to catalyze reactions of the laccase with the compound. Such a mediator may include among others 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfinic acid) or ABTS, violuric acid or VLA, 1-hydroxybenzotriazole or HOBt and N-hydroxyphthalimide or HPI. ABTS is often a suitable mediator. The mediator is used at levels described in earlier prior art, generally ranging from 1 to 2 mg per ml of reaction mixture. The reaction conditions may be further promoted by providing oxygen to the reactants. This may be done by continuous shaking of the reaction mixture, such as by placing the reaction flasks on an orbital shaker. Alternately, the glass vessel containing the reactants may be flushed with oxygen and stoppered tightly, followed by reaction for the required time at a required temperature. The bio-catalyzed reactants may be assayed for various disease states, using state-of-the-art protocols. Following changes in the compound or plant material, the bio-catalyzed compound may be separated and purified from the rest using various methods known in prior art, such as column chromatography and high performance liquid chromatography. The compound may then be used in various pharmaceutical preparations for treatment of disease states. Alternately, the transformed herbal powder may be used in various pharmaceutical preparations for treatment of disease states,
In order that this invention be more fully understood, the following preparative and testing examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.
EXAMPLES Example 1
Laccase was successfully used to transform compounds given in Table 1.
Table 1 provides the list of compounds biotransformed and their respective reaction conditions. The reaction mixture consisted of about 20 mg of substrate in 0.2 ml of solvent with the substrate, 1.8 ml of a buffer with appropriate pH, 0.5 ml of buffer

containing laccase from an appropriate fungal source from the culture collection of the patentee organization and the presence or absence of 2 mg of ABTS as the mediator.

No. Compound Solvent pH Source of
enzyme and
Units used Mediator Other conditions
1 Hydro-quinone Ethyl acetate 5.0 MT-1669; 8 o 17 Units ABTS 25°C
2 4-methyl catechol Ethyl acetate 5.0 MT-903; 21 Units ABTS 25°C
3 Vanillin Ethyl acetate 5.5 MT-896; 4 to 8 Units ABTS 25°C
4 Phyllanthin Acetone 7.0 MT-1669; 17 units ABTS 25°C
5 Pyrogallol Water 7.5 MT-1324; 7.0 Units ABTS 37°C
6 5, 7-dihydroxy 4
-methyl
coumarin Methanol 5.0 MT-1669; 8 units None 25°C
7 Plumbagin Ethyl acetate 9.0 MT-1669; 8.4 units ABTS 29-38°C
8 Menthol Ethanol 6.5 MT-1669; 17 units HOBt 28°C
The various fungi producing the laccase enzyme were accessed from the culture collection of Myko Tech Private Limited, Goa, India. The basic protocol used in transforming these compounds was as follows. About 20 mg of the individual compounds were dissolved in 0.2 ml of their appropriate solvents, such as methanol, ethanol, ethyl acetate, acetone, DMSO and others. An appropriate buffer was then chosen, based on the optimal activity of the laccase enzyme that was used for the reaction. The pH of the buffer was 4.0, 5.5 or 7.0. A total of about 0.5 to 1.0 ml of buffer, containing 2 to 20 Units of the laccase enzyme, derived from a variety of fungi was added to this. Buffers of different pH, namely 3.0, 4.5 and 6.0 were tried for every substrate. The compound ABTS, which acts as the mediator for laccase enzyme was added to one set of the above samples, in amounts of 3.0 mg per sample as above. The mixture was incubated overnight on a rotary shaker. Presence or absence of transformation was verified by TLC, with solvents appropriate for the particular substrates. Substrates that indicated positive transformation in TLC were further tested by HPLC to compare the profiles of control and transformed products.
Example 2
Catechol was biotransformed using the following reaction conditions. Five to 20 mg of commercially available catechol was dissolved in 0.2 ml of methanol or ethyl acetate. A

total of 1.8 ml of Acetate buffer of pH 5.5, containing 0.5 to 1.0 Unit laccase obtained from the fungi with the Accession Nos. MT1233 or MT1324 from the patentee's organization was added. The reaction was carried out at 28°C in an orbital shaker at 200rpm for 24 hrs. Enzyme reaction was monitored by analyzing samples collected every half an hour from the reaction medium, by TLC. Transformation was confirmed by thin layer (Fig. 1) and high performance liquid chromatography (Fig. 2). The conversion was observed within 2 hours. The product was dark brown in colour and was soluble in ethyl acetate. GC-MS analysis of the product determined the molecular weight of reaction product as 218 which was identified as bis-catechol. The HPLC (Rt-7.2) Spectrum of the product Bis-catechol was also identical with that of standard.
Example 3
Antibacterial activity of transformed catechol was studied as follows. The antibacterial screening was carried out using the agar diffusion method. The test bacteria were first inoculated into tubes of nutrient broth separately and incubated at 37°C for 24 hrs. A sterile well cutter was then used to make two wells of 8mm diameter on each of the Muller Hinton Agar plates containing cultures of the different test organisms. The substrate, catechol and the transformed product were dissolved in Dimethyl sulphoxide at a concentration of 10 mg/ml and 100µl of the extract was then introduced into the wells using micropipettes. The culture plates were allowed to stand on the working bench for 30 minutes for pre-diffusion and were then incubated at 37°C for 24 hrs. After 24 hrs antimicrobial activity was determined by measuring the diameter of zone of inhibition (mm) against the test organisms around each of the extract. Transformed catechol showed enhanced antibacterial activities compared to the standard (Fig. 3).
Example 4
The antioxidant activity of catechol was estimated as follows. The initial OD value of 0.1ml of DPPH solution (0.1 mM DPPH in methanol) and 0.8ml of methanol was taken at 517 nm in UV Spectrometer. 10 mg of the test sample was weighed and dissolved in 1 ml of 99% methanol with a final concentration of 10 mg/ml. In this assay, 100µl of test sample was mixed with DPPH solution and incubated for half an hour. The Optical Density (OD) of the solution was then measured at 517 nm in UV Spectrometer. The radical scavenging activity was represented as percentage inhibition of DPPH radicals.
The radical scavenging activity was determined using the formula,
(OD control - OD sample) x 100
(OD Control)
Transformed catechol showed up to 85 % antioxiant activity, similar to the 87 % activity

obtained for commercially obtained, chemically formed bis-catechol. Both showed higher antioxidan activities than catechol (Fig. 4).
Example 5
The compound aloin, obtained from Aloe vera was transformed using the following reaction conditions. A total of 10 mg of aloin was dissolved in 0.2 ml of methanol. A total of 2.3 ml of phosphate buffer of pH 7.0, containing 8.5 Units of laccase obtained from the fungus with the Accession Nos. MT1669 from the patentee's organization was added. A total of 2 mg of ABTS was added to this reaction mixture, which was then incubated at 28°C in an orbital shaker at l00rpm for 24 hrs. Enzyme reaction was monitored by analyzing samples collected every half an hour from the reaction medium, by TLC. Transformation was confirmed by thin layer (Fig. 5) and high performance liquid chromatography.
Example 6
The anti-inflammatory activity of transformed aloin was tested using the standard lipoxygenase inhibition assay, wherein the rate of oxidation of linoleic acid in the presence of lipoxidase enzyme alone in buffer, was compared with the rate of oxidation of linoleic acid in the presence of lipoxidase enzyme, as well as the test material in buffer. The rate of oxidation was estimated as the change in absorbance over a defined amount of time, for example 3 or 4 minutes. The anti-inflammatory activity, as percent lipoxygenase inhibition activity, was calculated as
((A Absorbance of control - A Absorbance of Test sample) / A Absorbance of control) / 100). It was observed that biotransformed aloin increased decreased the oxidation rate of linoleic acid by lipoxygenase by 10 % and thus had an increased anti-inflammatory activity, compared to controls (Fig. 6).
Example 6
The compound curcumin, obtained from turmeric powder from the dried rhizomes of the plant Curcuma longa was transformed as follows. The turmeric powder was made into a fine powder. A total of 100 grams of fine powder was soaked in 500ml of dichloromethane for 48 hours in one-liter conical flask. The extract was filtered using Whatman filter paper and the solvent was removed under reduced vacuum. The extract contains both the Curcumin and the Turmeron isomers. The Turmeron isomers were separated by repeated wash with hexane and re-crystallized curcumin with ether.
The curcumin was biotransformed using the following reaction conditions. Five to 10 mg of curcumin was dissolved in 0.2 ml of DMSO. A total of 2.3 ml of phosphate buffer at pH 7.0, containing 35 to 70 Units of laccase obtained from the fungus with the Accession Nos. MT903from the patentee's organization was added. The reaction was carried out at

28°C in an orbital shaker at 200rpm for 24 hrs. Enzyme reaction was monitored by analyzing samples collected every half an hour from the reaction medium, by TLC. Transformation was confirmed by thin layer (Fig. 7) and high performance liquid chromatography (Fig. 8). The conversion was observed by 23 hours.
Example 7
The compound vanillin was transformed using the following reaction conditions. Ten to 20 mg of vanillin was dissolved in 0.2 ml of methanol. A total of 1.8 ml of acetate buffer at pH 5.0 containing 17.6 to 35.4 Units of laccase obtained from the fungus with the Accession No. MT1261 from the patentee's organization was added. A total of 2 mg of ABTS was added to the reaction mixure. The reaction was carried out at 28°C in an orbital shaker at 200rpm for 24 hrs. Enzyme reaction was monitored by analyzing samples collected every half an hour from the reaction medium, by TLC. Transformation was confirmed by thin layer (Fig. 9) and high performance liquid chromatography (Fig. 10).
Example 8
The compound podophyilotoxin was transformed as follows. Podophyllotoxin was obtained from Sigma-Aldrich. A stock solution of the compound was prepared by dissolving 4.5 mg of the compound in 500 µl ethanol, which was then dissolved in 10 ml of 0.1 M acetate buffer at pH 4.5. The reaction mixture contained 3 ml of the podophyllotoxin solution as above, 11 ml of acetate buffer at pH 4.5, containing 16 Units of laccase from the fungus with an Accession No. MT1669 from the culture collection of the patentee organization containing 500 µM of hydroxy benzothiazole or HOBt. The reaction flasks were kept on a shaker. Samples were taken periodically at 15 min, 2 h, 5 h and 24 h. Transformation was confirmed by thin layer chromatography was performed, using the solvent system chloroform: methanol (9:1) (Fig. 11). HPLC profiles further confirmed the transformation (Fig. 12).
Example 9
The extract obtained from Momordica charantia was transformed as follows. Initially, 2 g of the herbal powder of Momordica charantia, obtained from commercially available capsules (Himalaya Drugs, India) were suspended in 25 ml of methanol overnight, followed by centrifugation to remove the insoluble material. A total of 2.5 ml of the methanolic extract and 6 ml of acetate buffer at pH 4.5 containing nearly 7.5 units of laccase obtained from the fungi with Accession No. MT-1259, MT-1321 and MT-1079 were added. The reaction flasks were kept on a shaker for 24 h. This treated material was used for further assays on anti-diabetic activity.

Example 10
The anti-diabetic activity of transformed extracts of Momordica charantia was tested as follows. The anti-diabetic activity of treated extracts of Momordica charantia was tested using the standard alpha-glucosidase inhibition assay. In this assay, the unit activity of the alpha glucosidase enzyme is calculated in terms of the amount of para-nitrophenol released from para-nitrophenol alpha-glucoside by the enzyme alpha glucosidase. The percent reduction in the units of the alpha glucosidase enzyme activity in the presence of the test substrate gives an indication of its anti-diabetic activity. Momordica charantia extracts treated with laccase from the fungi MT-1079 and MT-1321 reduced the activity of the alpha glucosidase by nearly 90 %, thus revealing significant anti-diabetic activity, compared to the control, untreated extracts (Fig. 13).
Example 11
The extract obtained from Boswellia serrata was transformed as follows. A total of 2.0 g of crude powder of Boswellia serrata (Himalaya Drugs) was suspended in 20 ml of DMSO overnight. This was then centrifuged and the supernatant was collected. The reaction mixture contained 2.5 ml of the DMSO extract of Boswellia serrata as above, 2.5 ml of acetate buffer at pH 4.5 containing a total of 5 units of laccase from the fungus with the Accession No. MT1669, obtained from the patentee's organization, 5 ml of acetate buffer at pH 4.5 and 8 mg of ABTS as mediator. The reaction flasks were kept on a shaker for 24 h. Transformation was confirmed by HPLC (Fig. 14).
Example 12
The anti-inflammatory activity of transformed extracts from herbal powder of Boswellia serrata was tested as given in Example 6. Transformed extracts yielded increased antiinflammatory activities (Fig. 15).
Example 13
The extract obtained from Boerhavia diffusa was transformed as follows. One gram of the powder crude Boerhavia diffusa powder (Himalaya Drugs) was soaked overnight in 15 ml methanol. The reaction mixture contained 2.5 ml of the extract as above, 6 ml of 0.2 M Acetate Buffer at pH 4.5, containing 5 Units of laccase from the fungus with an Accession No. MT903 obtained from the culture collection of the patentee organization and 8 mg ABTS. The reaction flasks were kept on a shaker. Samples were taken periodically at 15 min, 2 h, 5 h and 24 h. Thin layer chromatography was performed, using the solvent system chloroform: methanol: formic acid (35: 6:1). Spots with lower r.f.s (arrow in the right panel) appeared after 24 h, suggesting polymerization (Fig. 16). Transformation was confirmed by thin layer and high performance liquid chromatography (Fig. 17).

Claims
What is claimed is:
1. A biotransformed product obtained by a process involving the conversion of the substances derived from medicinal plants mediated by an enzyme in order to enhance the biological activity of the said biotransformed product.
2. The biotransformed product according to claim 1, wherein the said product comprises of transformed biologically active compound or herbal powder.
3. The biotransformed product according to claim 2, wherein the biologically active compounds are cathecol and its derivative, coumarin and its derivative, menthol, vanillin, curcumin, phyllanthin, pyrogallol, plumbagin, aloin, and podophyllotoxin.
4. The biotransformed product according to claim 2, wherein the herbal powders are Boerhavia diffusa, Boswellia serrata, Gymnema sylvestre and Momordica chorontia.
5. The process to prepare biotransformed product according to claim 1, wherein the process comprises the steps of:
a. dissolving the biologically active compound or herbal powder in a suitable
solvent;
b. adding the isolated enzyme, wherein the said enzyme is diluted in
appropriate buffer on for biocatalysis reaction;
c. incubation of the obtained mixture from step a and b at a temperature in
the range of 25°C to 65°C and pH in the range of 3 to 9 for a period of 1
hour to 24 hours in order to obtain the desired biotransformed product.
d. Optionally, a mediator is added during the course of the reaction in order
to catalyze the reaction.
6. The biotransformation process according to claim 5(a) wherein the solvent is selected from the group consisting of ethanol, ethyl acetate, acetone, chloroform, water or dimethyl sulfoxide (DMSO).
7. The biotransformation process according to claim 1 and claim 5(b), wherein the enzyme is laccase.
8. The biotransformation process according to claim 5(b), wherein the buffer is acetate.

9. The process according to claim 5(d), wherein the mediator is selected from the group consisting of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfinic acid) or ABTS, violuric acid or VLA, 1-hydroxybenzotriazole or HBT and N-hydroxyphthalimide or HP1.