FIELD OF THE INVENTION
The present invention relates to a process for preparing herbal medicine. More particularly, the present invention relates to a novel process for preparing enzyme treated Asavarishta without fermentation.
BACKGROUND OF THE INVENTION
A shift towards a healthy lifestyle has increased the demand for natural products, such as extracts of herbs, vegetables, fruits and other plants. Herbal products are complex mixtures of organic chemicals that may come from any raw or processed part of a plant, including leaves, stems, flowers, roots, and seeds. As herbs are perceived as “natural” and therefore safe, there is a growing preference for herbal drugs and traditional medicine over synthetic drugs.
Asavarishtas are unique dosage forms used in the ayurvedic system of medicine in India. Both Asava as well as Arishta fall under the fermented products category. Generally, Asava is prepared by fermenting fresh juices (svarasa) while Arishta is prepared by fermenting decoctions (kvatha).

Sushruta Samhita, 21st Chapter of Sutra Sthana (2 AD), quotes the process of Sandhana i.e., fermentation with the combination of Kinva or Surabija (molasses / yeast), Udaka (water) and Pishta (rice flour). Under Sandhana Kalpa (fermented products), Sarngdhara Samhita Madhyama Khanda (13-14 AD), mentions naturally self-liberated alcoholic preparations, i.e., Asavas and Arishtas. Presence of water and alcohol aids in dissolving active ingredients from the herbs, thereby enhancing the efficacy of said herbal preparation. Asavarishtas, such as Abhayarishta, Amritarishta, Arjunarishta, Asavagandharishta, Ashokarishta, Babbulyadarishta, Dashmularishta and Balarishta are sold as compositions containing self-generated alcohol (6-12%).

Dashmularishta is a fermented formulation of various herbal ingredients, such as Shalparni (Desmodium gengeticum), Prishnparni (Uraria picta), Brihati (Solanum indicum), Kantakari (Solanum xanthocarpum), Goksura (Tribulus terrestris), Bilwa (Aegle marmelos), Shyonak (Oroxylum indicum), Gambhari (Gmelina arborea), Patala (Streptosperneum sauveolens), Agnimantha (Premna integrifolia), commonly referred to as Dashmool, in synergistic combination with other herbs (Formulation and Evaluation of tablet of a Classical Fermented Preparation of Dashmularishta, International Journal Of Pharma Professional’s Research, 6 (1), Jan, 2015). It is known to provide numerous health benefits, including promoting vitality and strength, restoring strength in pregnant women after child birth, improving milk production, removing toxins from body and nourishing body tissue. It contains alcohol in the range from 5-10% that is self generated over a period of time due to fermentation.
The conventional process for making Asavarishta comprises the following steps:
(a) preparing a decoction by boiling all the herbs in a specified volume of water;
(b) adding and dissolving cane sugar, jaggery and/or honey in the decoction;
(c) keeping the sweetened decoction for fermentation in an air tight vessel;
(d) separating the solvent containing the active ingredients from the raw material using various filtration methods; and
(e) evaporating the solvent to increase the concentration of the active ingredient in order to produce a concentrated liquid extract (Traditionally fermented biomedicines, arishtas and asavas from Ayurveda, Indian Journal of Traditional Knowledge, 7(4), October 2008, 548-556).
The above process suffers from drawbacks, such as uncontrolled fermentation (bacterial and/or fungal), prolonged manufacturing time, consumption of excessive raw material, degradation of the active herbal ingredients and reduced biological efficacy. Hence, there is a need for a novel process for preparing Asavarishta which not only saves time, overcomes the aforementioned drawbacks and delivers a consumer friendly product but also enhances its bio-efficacy.
SUMMARY OF THE INVENTION
The present invention relates to a novel process for preparing enzyme treated Asavarishta, without fermentation.
In accordance with an embodiment of the invention, there is provided a novel process for preparing enzyme treated Asavarishta, comprising the steps of;
a. extracting a plurality of herbs to obtain a herbal extract;
b. concentrating the herbal extract to obtain a concentrated herbal extract;
c. adding at least one enzyme to the concentrated herbal extract; and
d. incubating the composition of step c at a temperature not more than 50 °C for a pre-determined period of time to obtain enzyme treated Asavarishta.
The plurality of herbs is extracted with a mixture of water and alcohol. The mixture contains water and alcohol preferably in a ratio in the range from 50:50 to 80:20 and more preferably in a ratio of 7:3. Preferably the alcohol used is ethyl alcohol.
Further, the herbal extract is concentrated at a temperature not more than 80 °C to obtain a concentrated herbal extract having moisture content not less than 25%.
The enzyme used in the present invention is selected from the group consisting of a-amylase, pectinase, cellulase, xylanase and combinations thereof. Preferably, 1500 units of the enzyme are added per gram of the concentrated herbal extract.
The mixture comprising the enzyme and the concentrated herbal extract is incubated in an incubator at a temperature ranging from 33 to 37 °C and preferably at a temperature of 35 °C.
In accordance with another embodiment of the invention, the enzyme treated Asavarishta is enzyme treated Dashmularishta, and wherein the enzyme treated Dashmularishta has better anti-oxidant potential, anti-osteoporotic potential and immune-modulatory or immune-stimulatory activity compared to Dashmularishta.
In accordance with yet another embodiment of the invention there is provided a herbal composition, prepared by the above process.

DETAILED DESCRIPTION OF THE INVENTION
Discussed below are some representative embodiments of the present invention. The invention in its broader aspects is not limited to the specific details and representative methods. The illustrative examples are described in this section in connection with the embodiments and methods provided. The invention according to its various aspects is particularly pointed out and distinctly claimed in the attached claims read in view of this specification.
It is to be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
The expression of various quantities in terms of “%” or “% w/w” means the percentage by weight of the total solution or composition unless otherwise specified.
All cited references are incorporated herein by reference in their entireties. Citation of any reference is not an admission regarding any determination as to its availability as prior art to the claimed invention.
The present invention, in its product and process aspects, is described in detail as follows:
Through scientific studies it has been shown that certain chemical moieties in Asavarishta undergo changes during conversion of decoction (Kvatha/Kashaya) to fermented product (Asava/Arishta), resulting in an increase in its bio-efficacy. Due to the drawbacks of the fermentation process involving micro-organisms, the inventors of the present invention have made use of enzymes.
Enzymes occur in all living organisms and catalyze biochemical reactions necessary to support life. Enzymes are ubiquitous in fresh and processed food and are consumed every day. The use of enzymes in processing herbal decoctions may result in many benefits that cannot be achieved with traditional processes. These often include higher product quality, lower manufacturing cost, less waste and reduced energy consumption. Because enzymes are catalysts, the amount added to accomplish a reaction is relatively small. For example, an enzyme in most food uses is equal to 0.1% (or less) of the product (The enzyme advantage, Enzyme technical association, June 2001). The degree to which a desired technical effect is achieved by an enzyme can be controlled through various means, such as dose, temperature, and time. Enzymes used in food processing are generally destroyed during subsequent processing steps and not present in the final food product. Accordingly, the inventors have developed a novel process for preparing Asavarishta by treating the herbal extract with enzymes.
In accordance with an embodiment of the invention, there is provided a novel process for preparing Asavarishta, comprising the steps of:
(a) extracting a plurality of herbs to obtain a herbal extract;
(b) concentrating the herbal extract to obtain a concentrated herbal extract;
(c) adding at least one enzyme to the concentrated herbal extract; and
(d) incubating the composition of step c at a temperature not more than 50 °C for a predetermined period (which is less than conventional period) to obtain enzyme treated Asavarishta.
Asavarishtas that may be prepared by the process disclosed in the invention include, but not limited to, Abhayarishta, Amritarishta, Arjunarishta, Asavagandharishta, Ashokarishta, Babbulyadarishta, Dashmularishta, Balarishta, Kutajaarishta, Draakshaarishta, Devadaarvaarishta, Jeerakaarishta, Khadirashta, Karpoorasavam, Pippalyaadi asavam, Punarnavaasavam, Usiraashtam, Aravindaarishta, and Patrangaasavam.
Further, the herbs that may be used in the present invention include, but not limited to, Bilva (Aegle marmelos), Shayonaka (Oroxylum indicum), Gambhari (Gmelina arborea), Patala (Stereospermum suaveolans), Agnimantha (Premna integrifolia), Shalaprani (Desmodium gangeticum), Prishniparni (Uraria picta), Brihati (Solanum indicum), Kantakari (Solanum surattense), Gokshura (Tribulus terrestris), Yashti (Glycyrrhiza glabra), Bharangi (Clerodendrum serratum), Kapittha (Feronia limonia), Bibhitaka (Terminalia belerica), Rakta punarnava (Boerhavia diffusa), Chavya (Piper chaba), Priyangu (Callicarpa macrophylla), Shveta sariva (Hemidesmus indicus), Pippali (Piper longum), Puga (Areca catechu), Shati (Hedychium spicatum), Haridra (Curcuma longa), Shatava (Anethum sowa), Padmaka (Prunus cerasoides), Nagakesara (Mesua ferrea), Karkatashringi (Pistacia integrrima), Jivaka (official substitute-Pueraria tuberosa), Rishbhaka (official substitute-Pueraria tuberosa), Meda (official substitute-Asparagus racemosus), Mahameda (official substitute-Asparagus racemosus) Draksha (Vitis vinifera), Dhataki (Woodfordia fruticosa), Kankola (Piper cubeba), Hrivera (Coleus vettiveroides), Shveta chandana (Santalum album), Jatiphala (Myristica fragrans), Lavanga (Syzygium aromaticum), Tvak (Cinnamomum zeylanicum) and combinations thereof.
The herbs are washed with water, dried, mixed and passed through miracle mill/ hammer mill to obtain a coarse to moderately fine ground powder. Thereafter, the powdered herbs are extracted using a suitable solvent. The solvent is a mixture of water and alcohol preferably in a ratio in the range from 50:50 to 80:20, and more preferably in a ratio of 7:3. Preferably, the alcohol is ethyl alcohol. The herbal powder may be extracted by boiling the powdered herbs in the suitable solvent or using a soxhlet apparatus.
Further, the herbal extract is concentrated to obtain a concentrated herbal extract. Concentration may be carried out under reduced pressure or at atmospheric pressure. The moisture content of the concentrated herbal extract is not less than 25%.
Still further, the concentrated herbal extract is treated with an enzyme selected from the group consisting of a-Amylase, pectinase, cellulase, xylanase and combinations thereof. Preferably the enzyme used is a-Amylase enzyme. The amount of enzyme used in the present invention is preferably 1500 units per gram of the concentrated herbal extract.
Thereafter, the mixture comprising the concentrated herbal extract and the enzyme is incubated in an incubator at a temperature in the range from 33 to 37 °C, preferably at a temperature of 35 °C, for a period not exceeding 24 h.
Examples
The present invention is more particularly described in the following examples that are intended as illustrations only, since numerous modifications and variations within the scope of the present invention will be apparent to those of skill in the art. Unless otherwise noted, all parts, percentages and ratios reported in the following examples are on a weight basis and all reagents used in the examples were obtained or are available from the chemical suppliers.

Process for preparing Dashmularishta
Wash, dry and powder the Kwatha (decoction) ingredients, i.e. Bilva (Aegle marmelos), Shayonaka (Oroxylum indicum), Gambhari (Gmelina arborea), Patala (Stereospermum suaveolans), Agnimantha (Premna integrifolia), Shalaprani (Desmodium gangeticum), Prishniparni (Uraria picta), Brihati (Solanum indicum), Kantakari (Solanum surattense), Gokshura (Tribulus terrestris), Yashti (Glycyrrhiza glabra), Bharangi (Clerodendrum serratum), Kapittha (Feronia limonia), Bibhitaka (Terminalia belerica), Rakta punarnava (Boerhavia diffusa), Chavya (Piper chaba), Priyangu (Callicarpa macrophylla), Shveta sariva (Hemidesmus indicus), Pippali (Piper longum), Puga (Areca catechu), Shati (Hedychium spicatum), Haridra (Curcuma longa), Shatava (Anethum sowa), Padmaka (Prunus cerasoides), Nagakesara (Mesua ferrea), Karkatashringi (Pistacia integrrima), Jivaka (official substitute-Pueraria tuberosa), Rishbhaka (official substitute-Pueraria tuberosa), Meda (official substitute-Asparagus racemosus), Mahameda (official substitute-Asparagus racemosus), etc individually and pass through the sieve number 10 to 12 to obtain coarse powder. Add water, soak overnight, heat, reduce volume to half and filter through muslin cloth to obtain Kwatha. Wash and crush the Draksha (Vitis vinifera) add water, soak overnight, heat, reduce to one fourth and filter through muslin cloth to obtain Kwatha. Collect the two Kwathas into one clean container and mix to form a homogenous liquid. Clean, dry and powder the Prakshep dravya ingredients, i.e., Kankola (Piper cubeba), Hrivera (Coleus vettiveroides), Shveta chandana (Santalum album), Jatiphala (Myristica fragrans), Lavanga (Syzygium aromaticum), Tvak (Cinnamomum zeylanicum), etc individually and pass through the sieve number 20 to obtain fine powder. Add Guda (Jaggery, 41.7% w/v) to the Kwatha, dissolve and filter through a muslin cloth. Transfer the filtrate to a clean container; add Madhu (honey), Dhataki (Woodfordia fruticosa, a known source of Saccharomyces species) and other finely powdered Praksepa Dravyas and close the mouth of the container. Shift the container to the fermentation room and constantly check for the signs of completion of fermentation process. Filter the fermented material through a clean muslin cloth. Pack in air tight containers and allow for maturation. It should be noted that herbs other than those mentioned in the current specification may be used in the above process.

Process for preparing enzyme treated Dashmularishta.
Wash, and dry Kwatha (decoction) ingredients i.e. Bilva (Aegle marmelos), Shayonaka (Oroxylum indicum), Gambhari (Gmelina arborea), Patala (Stereospermum suaveolans), Agnimantha (Premna integrifolia), Shalaprani (Desmodium gangeticum), Prishniparni (Uraria picta), Brihati (Solanum indicum), Kantakari (Solanum surattense), Gokshura (Tribulus terrestris), Yashti (Glycyrrhiza glabra), Bharangi (Clerodendrum serratum), Kapittha (Feronia limonia), Bibhitaka (Terminalia belerica), Rakta punarnava (Boerhavia diffusa), Chavya (Piper chaba), Priyangu (Callicarpa macrophylla), Shveta sariva (Hemidesmus indicus), Pippali (Piper longum), Puga (Areca catechu), Shati (Hedychium spicatum), Haridra (Curcuma longa), Shatava (Anethum sowa), Padmaka (Prunus cerasoides), Nagakesara (Mesua ferrea), Karkatashringi (Pistacia integrrima), Jivaka (official substitute-Pueraria tuberosa), Rishbhaka (official substitute-Pueraria tuberosa), Meda (official substitute-Asparagus racemosus), Mahameda (official substitute-Asparagus racemosus) Draksha (Vitis vinifera) and Dhataki (Woodfordia fruticosa) individually.
Clean and dry Prakshep dravya ingredients, i.e., Kankola (Piper cubeba), Hrivera (Coleus vettiveroides), Shveta chandana (Santalum album), Jatiphala (Myristica fragrans), Lavanga (Syzygium aromaticum), and Tvak (Cinnamomum zeylanicum).
Mix all the above ingredients and pass through miracle mill/ hammer mill to get coarse to moderately fine ground powder. Take 250 g of ground powder in a soxhlet apparatus and pour 300 mL of extraction solvent, i.e. water and ethyl alcohol in a ratio in the range from 50:50 to 80:20, preferably 7:3, on dry packed column. Maintain the temperature of solvent in the range from 70 - 80 °C to achieve boiling point and to avoid bumping of solvent. Continue extraction/ siphoning till the siphoning solvent is colourless. Filter the herbal extract to remove any raw particle and concentrate at a temperature not more than (NMT) 80 °C to form a concentrated herbal extract having moisture content not less than 25% w/w.
The concentrated herbal extract was treated with a-Amylase enzyme (1500 units of enzyme per gram of concentrated herbal extract) with stirring for uniform mixing, and the bulk was kept for incubation at 35±2 °C for 24 h in an incubator to obtain enzyme treated Dashmularishta. Finally, the enzyme treated Dashmularishta was packed under aseptic conditions to avoid any microbial contamination. It should be noted that herbs other than those mentioned in the current specification may be used in the above process. Although the above process is for the preparation of enzyme treated Dashmularishta, it can be used for the preparation of any enzyme treated Asavarishta.
In-vitro estimation of free radical scavenging activity of Dashmularishta and enzyme treated Dashmularishta.
The Dashmularishta and enzyme treated Dashmularishta used in the present study were prepared by above process. The present study was conducted to compare the free radical scavenging activity of Dashmularishta and enzyme treated Dashmularishta using ABTS (2,2'- azinobis-3-ethylbenzothiozoline-6-sulphonic acid) assay in vitro. Trolox, a compound with well reported antioxidant properties, was used as a positive control.
ABTS assay is the most widely reported method for the estimation of antioxidant activity of test compounds/plant extracts. The radical scavenging activity of Dashmularishta and enzyme treated Dashmularishta was evaluated using PBS solution of the stable free radical, ABTS. A freshly prepared ABTS solution exhibits green colour with a maximum absorption at 734 nm, which decreases in presence of compounds with antioxidant potential. Antioxidant compounds can quench ABTS free radicals and convert them to colourless product, resulting in a decrease in absorbance at 734 nm. Change in absorbance values in presence of Dashmularishta and enzyme treated Dashmularishta was determined with reference to blank and reflected its anti-oxidant potential.
Dashmularishta, was tested for its anti-oxidant potential at various concentrations ranging from 0.01%-20% and enzyme treated Dashmularishta at concentrations ranging from 0.01% - 17.5%. It was observed that Dashmularishta and enzyme treated Dashmularishta exhibited strong antioxidant potential with Trolox equivalent antioxidant capacity (TEAC) values of 0.55 and 1.07 respectively. Therefore, it is suggested that Dashmularishta and enzyme treated Dashmularishta offer protection against various diseases mediated by oxidative stress. Hence, based on the data obtained in the current study, it can be suggested that the antioxidant potential of enzyme treated Dashmularishta is greater than that of Dashmularishta.

Evaluation of anti-osteoporotic potential of Dashmularishta and enzyme treated Dashmularishta using human osteoblast like cell line.
The present study compared the anti-osteoporotic potential of Dashmularishta and enzyme treated Dashmularishta by assessing its estrogenic potential using human osteoblast like cell line. An increase in cell proliferation and alkaline phosphatase (ALP) enzyme activity were considered as markers of bone health. MG-63 cell line represents human osteoblast phenotype with high levels of alkaline phosphatase enzyme activity. MG-63 cell line is widely used for studying the potential of compounds to improve bone health.
The present study assessed the potential of Dashmularishta, and enzyme treated Dashmularishta to improve bone health using human osteoblast cell line, MG-63. MG-63 cells were treated with Dashmularishta and enzyme treated Dashmularishta at concentrations ranging from 0.005% - 2% and 0.1 µg/mL to 1000 µg/mL respectively. Thereafter, cell proliferation was measured using MTT assay and alkaline phosphatase enzyme activity was measured by monitoring the hydrolysis of p-nitrophenyl phosphate to p-nitrophenol using colorimetry based method.
Dashmularishta led to an increase in cell proliferation at all the concentrations tested. An up to 18.4% increase in cell proliferation with respect to untreated cells was observed at the concentration corresponding to 0.5 % (volume/volume concentration in cell growth medium). Also, an increase in ALP enzyme activity (up to 15.8 %) was observed in Dashmularishta at tested concentrations.
Enzyme treated Dashmularishta led to an increase in cell proliferation at all the concentrations tested. An up to 36.9% increase in cell proliferation with respect to untreated cells was observed at concentration corresponding to 500 µg/mL of enzyme treated Dashmularishta. Also, an increase in ALP enzyme activity up to 17.3% was observed in cells treated with enzyme treated Dashmularishta at tested concentartions.
From the above data it is evident that compared to Dashmularishta, enzyme treated Dashmularishta possesses greater potential to improve bone health by increasing the number of osteoblast cells and the concentration of alkaline phosphatase enzyme.
Evaluation of immune-modulatory activity of Dashmularishta and enzyme treated Dashmularishta using natural killer cells assay
The present study was conducted to compare the immune-modulatory or immune-stimulatory activity of Dashmularishta, and enzyme treated Dashmularishta on natural killer (NK) cells.
Immune-stimulatory activity of an agent involves an increased proliferation of splenic lymphocytes. Splenocytes are rich source of NK cells, which function as effector cells in NK cells activity. YAC-1 cell line has been widely used as target cells for NK cells. NK cells mediated killing and lysis of YAC-1 cells is well established end point for estimation of NK cells activity for determining immune-stimulatory activity.
NK cells were treated with Dashmularishta and enzyme treated Dashmularishta at non-cytotoxic concentrations ranging from 0.01%-1% v/v and 0.01 µg/mL to 300 µg/mL, respectively for 24 h.
The results have shown that Dashmularishta and enzyme treated Dashmularishta stimulated basal level of NK cells up to 27.8% and 36.3% respectively. However, the percentage increases in comparison to control were 18% and 26.5% respectively. Increased activity of NK cells suggests potential immune-stimulatory activity of Dashmularishta and enzyme treated Dashmularishta.
Hence, based on the data obtained for NK cells activity in the current study, it can be suggested that the immune-stimulatory potential of enzyme treated Dashmularishta is greater than that of Dashmularishta.
While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is thereof intended to cover in the appended claims such changes and modifications that are within the scope of the invention.

We claim:
1. A process for preparing enzyme treated Asavarishta, comprising the steps of;
a. extracting a plurality of herbs to obtain a herbal extract;
b. concentrating the herbal extract to obtain a concentrated herbal extract;
c. adding at least one enzyme to the concentrated herbal extract; and
d. incubating the composition of step c at a temperature not more than 50 °C for a pre-determined period of time to obtain enzyme treated Asavarishta.
2. The process for preparing enzyme treated Asavarishta, as claimed in claim 1, wherein the plurality of herbs are extracted with a mixture of water and alcohol.
3. The process for preparing enzyme treated Asavarishta, as claimed in claim 2, wherein water and alcohol are preferably in a ratio in the range from 50:50 to 80:20 and more preferably in a ratio of 7:3.
4. The process for preparing enzyme treated Asavarishta, as claimed in claim 1, wherein the concentration is carried out at a temperature not more than 80 °C and wherein moisture content of the concentrated herbal extract is not less than 25%.
5. The process for preparing enzyme treated Asavarishta, as claimed in claim 1, wherein the enzyme is selected from the group consisting of a-amylase, pectinase, cellulase, xylanase, and combinations thereof.
6. The process for preparing enzyme treated Asavarishta, as claimed in any of the claims 1 to 5, wherein the enzyme is used in an amount of 1500 units per gram of the concentrated herbal extract.
7. The process for preparing enzyme treated Asavarishta, as claimed in claim 1, wherein the pre-determined period of time ranges upto 24 h.
8. The process for preparing enzyme treated Asavarishta, as claimed in claim 1, wherein the step of incubation is carried out at a temperature in the range from 33 to 37 °C and preferably at 35 °C.
9. The process for preparing enzyme treated Asavarishta, as claimed in any of the claims 1 to 8, wherein the enzyme treated Asavarishta is enzyme treated Dashmularishta, and wherein the enzyme treated Dashmularishta has better anti-oxidant potential, anti-osteoporotic potential and immune-modulatory activity compared to Dashmularishta.
10. A herbal composition, prepared by the process as claimed in any of the claims 1 to 8.