FIELD OF THE INVENTION
The invention provides novel yeast strains having accession numbers DRF-UDS-004/wf, DRF-UDS-016/wf and DRF-UDS-017/wf, a method for the propagation of the yeast strains and a method for manufacture of a whole range of oral herbal formulations, especially those mentioned in Ayurvedic literature and falling under the category 'Asava - Arishta' and any other proprietary formulations falling under the same category using the yeast strains. The novel method reduces the time consumed in conventional methods, increases productivity and minimises product rejections. Also, the novel method maintains the inherent natural quality and efficacy of these formulations/formulations.
BACKGROUND OF THE INVENTION
'Asavas' denote fermented infusions and 'Arishtas' denote fermented decoctions or herbal extracts. These are commonly used terms in 'Ayurveda', a traditional system of medicine using herbs or their extracts for treatment of diseases.
Asavas and Arishtas comprise an important group of liquid orals mentioned in ancient Ayurvedic literature. This group of products represents one of the highly evolved dosage forms, which offer the advantages of accessibility, palatability and product stability over the traditionally used decoctions, which are primitive in their nature. This particular group of liquid orals finds their use not only in Ayurveda, but also in other traditional systems of medicine practiced in neighboring oriental countries like Sri Lanka, Tibet, China and Bhutan. Few hundreds of Ayurvedic formulations described in ancient and mediaeval Ayurvedic literature fall under this group. Various Ayurvedic pharmaceutical companies in India do manufacture almost 50 different formulations falling under this range and some of these formulations are also being exported to other countries. To an estimate, every year Rs. 2000 millions worth of Asava & Arishta group of formulations are being consumed in India alone.
Present methods of manufacturing:
Presently, herbal formulations, classified as 'Arishtas' or 'Asavas' are manufactured in accordance with the procedures laid down in ancient texts. This procedure involves mainly
three phases: 1) Extraction 2) Preparation of fermentation medium 3) Inoculation and
i
fermentation. These three phases are summarized as below:
Extraction Phase: Water is used as solvent for extraction purposes. For Asavas, the extraction procedure involves a cold infusion technique, wherein the coarse powder of herbal material is soaked in water for a fixed duration at room temperature and a filtrate is obtained.
In case of Arsihtas, the extraction procedure involves an Open Pan Boiling Technique, wherein coarsely ground herbal material is charged to a boiling pan along with required quantity of water. This mixture is then heated using steam or firewood or some other fuel. The filtrate is then collected discarding the herbal marc.
Preparation of fermentation medium: To grow any microorganism nutrients are necessary. As the extract mainly contains active constituents coming from herbs, Jaggery was suggested as a source of complex nutrition in ancient literature. Accordingly, the Fermentation medium is to be prepared by dissolving Jaggery in the herbal extract obtained either by cold infusion for Asavas or by means of Open Pan Boiling Technique for Arishtas.
Inoculation & Fermentation: Ancient literature recommends two herbs-viz. Woodfordia fruticosa (Dhataki) and Madhuca latifolia (Madhuka) as the inoculum bearing herbs. One or both of these two inoculum-bearing herbs and other aromatic herbs in form of fine powders are topped to the fermentation medium. Fermentation is carried out in a closed fermentation

vessel, under controlled temperature conditions, at 30-35 C. Depending upon the type of herbal formulation, this phase of process may take anywhere between 30-45 days.
Thus, the prior art uses processes involving complex anaerobic fermentation of herbal extracts. The end product is expected to contain a self-generated alcohol in range of 7 to 14% v/v (volume/volume).
The herbal product range produced as per the above process are termed as Asava or Arishta as the case may be. The product line has certain specific advantages from clinical application and stability points of view and the same can be summarized as under:
♦ These preparations are generally palatable for the user with a sweet taste combined with a fine spicy aroma, which masks the unacceptable taste and odour of the active, herbal ingredients.
♦ Apart from the aqueous extract of the prime ingredients, the preparation brings the hydro-alcoholic extract of supportive ingredients used as powders added during fermentation.
♦ The process also brings, the extract of inoculum bearing herbs, which is considered to potentiate the activity of prime ingredients.
♦ The self-generated alcohol content in the product acts as a preservative and contributes for its prolonged shelf life.
♦ Self-generated alcohol is considered to enhance the bioavailability of all the herbal ingredients contained in the formulation.
Operative Constraints:
While Asava - Arishta range of herbal formulations has certain distinct advantages; the method involves serious operative constraints. It must be underlined that, the conventional methods of producing Asavas and Arishtas were originally laid down in ancient textbooks and as such, the technique is meant for small batches. It is also noteworthy that, the present manufacturing methods continue to be more or less, the same. Perhaps, there are only, few changes that took place in their manufacturing methodology. For example, instead of firewood heating, today steam is used for extraction. Instead of earthen pots mentioned in the texts for fermentation, many manufacturers use stainless steel vessels. Ancient texts recommend the placement of fermenting vessel in a heap of paddy husk to maintain temperature during fermentation, while industries use temperature conditioning systems. Except for such mechanical variations, nothing has changed in terms of the process methodology. Thus, the process or the technique may prove to be stifling, in terms of process duration and other complexities.
♦ For example, it takes almost 30 - 45 days to complete the fermentation process in spite of any mechanization.
♦ As mentioned above, two kinds of dried flower buds are topped on the culture medium to
facilitate fermentation. Both these herbs are very bulky and occupy a large volume of the
j
fermenting vessels (as much as 25% of gross capacity), restricting the output volume of finished product.
♦ Over and above these two major issues, is the problem of fungal contamination of the
product during middle of fermentation. Most of the times, the aforesaid flower buds work
as carriers of fungal spores and if, such fungi predominate the culture - it will spoil the product. Such spoilage generally becomes evident only, after about 10 days of fermentation. Once this happens, nothing can be done further as the product becomes toxic for the users. The product and the process must be abandoned midway. On an average, 25% of batches face the risk of fungal contamination.
The above major constraints in the traditional process of Asava-Arishta group of Ayurvedic formulations set the background for the invention.
PRIOR-ART
In general, the range of Asava-Arishta formulations are produced as per the process laid down in the ancient and medieval texts. Over a period of time, stainless steel containers replaced the earthen pots and modern methods of are adopted to maintain process temperatures required for fermentation. Except for these minor modifications in the systems, nothing further was altered. Academic research institutes in India conducted few studies on the ancient process methodology for Asava-Arishta manufacturing. However, these studies did not pay attention to the above problems faced by industry in undertaking large-scale production. Published reports of these studies can be summarized as under:
One of the earliest studies, (C. Seshadri and P.N.Krishna Nambisan. 1977, Jour.Res. Ind.Med.Yoga & Homoeo. 2(1), pp: 29-33.) worked to understand the specificity of Jaggery in the culture medium. The investigators opined that, White Crystalline Sugar might not be good for this purpose as jaggery has certain nutritive minerals. The study also reported that, big ball shaped slightly blackish Jaggery is the most suitable nutrient for fermentation. Subsequently, Muzafer Alam et.al (Muzaffer Alam, P. Brindha, P.S.Nataraja Sarma and K.K.Purushothaman. 1975, Jour. Res. Ind. Med. 10(4), pp: 49-53.) worked to throw some light on the importance of flowers facilitating the fermentation. Their group isolated two fungi from one of these floral buds, Woodfordia fruticosa. The fungi were identified to be Rhizopus nigricans and Aspergillus niger. It was observed that neither of the organisms capable of fermenting sugars to generate alcohol in the formulations. Their group further worked to identify microflora of Woodfordia fniticosa. These investigations observed that Bacillus polymixa and Bacillus acetoethylicus isolated from the flower buds were responsible for fermentation. However, Muzafer Alam's group did not appreciate the utility of these organisms in fermentation of Asava - Arishta group of formulations.
Studies on Draksharishtam and Lohasavam (two herbal formulations) revealed that Bacillus sp brought about the fermentation (Muzaffer Alam, K.Sathira Vasan, B.Rakamni, Varadarajan, R.Bhima Rao and K.K.Purushothaman. 1979, Jour. Res. Ind. Med. Yoga & Homoeo. 14(3), pp: 89-91.), but the rate of fermentation was faster in Lohasavam than in Draksharishtam. It was also reported that the pH, solid content and specific gravity decreased during fermentation.
CK Atal et.al. (C.K.Atal, A.K.Bhatia & R.P.Singh, 1982, Jour. Res. Ay. Sid., Vol. HI, No. 3&4, pp: 193-199.) studied the role of Woodfordia fruticosa as a source of inoculum in Asavas and Arishtas. It was observed that the flowers were capable of carrying out alcoholic fermentation as normally achieved by the use of pure yeast culture. A yeast strain of Saccharomyces cereviceae was isolated from the flowers.
B.H. Kroes et al (B.RKroes, A.J.J.Vanden Berg, A.M.Abeysekera, K.T.D.Desilva, R.P.Labadi. 1993, Jour, of Ethnopharmocology. 40, pp: 117-125.); reported the impact of Woodfordia fruticosa on the immunomodulatory activity and alcohol & sugar content of the Nimba Arishta. The use of Woodfordia fruticosa resulted in a substantial increase of the inhibition of both human complement activity and chemiluminiscence generated by zymosan stimulated human polymorphonuclear leukocytes. The increased biological activity was attributed to the immunoactive constituents released from the Woodfordia fruticosa
While the above studies enabled researchers to look at few intricate details of an ancient process, the investigations remained inconclusive as regards to the operative constraints faced by the industry handling this line of Ayurvedic liquid oral formulations. The prior art publications related to the process, did not make any specified attempt to reduce process duration or to improve productivity or to reduce the product rejection rate.
OBJECTIVES OF THE INVENTION
The main objective of the invention is to develop a novel method for the manufacture of Asava -Arishta group of Ayurvedic formulations by which, the method duration is brought-down at least, by 50%.
Another objective of the invention is to develop a method to manufacture Asava-Arishta group of Ayurvedic formulations by which, the productivity can be enhanced.
Yet another object of the invention is to develop a method to manufacture Asava - Arishta group of Ayurvedic formulations by which, the risk of fungal contamination is controlled effectively.
Still another object of this invention is to maintain the biological efficacy of the herbal formulation at par with conventional process - while fulfilling the above objectives.
SUMMARY OF THE INVENTION
The invention provides novel yeast strains isolated from natural sources such as flowers of Woodfordia fruticosa and Madhuca latifolia. The yeast strains of the invention are sugar resistant and capable of producing alcohol to the extent of about 7 to 1 lv/v% continuously and consistently in a herbal extraction medium, overcoming any resistance conferred by the herbal and spicy ingredients. The overall biomass attained by the yeast strains may be about lto 5 g/1 . These strains have the ability to potentiate and enhance the therapeutic value of a given herbal formulation.
Further, the invention provides a novel method for the propagation of the yeast strains. The invention also provides a novel method whereby herbal formulations containing self-generated alcohol can be efficiently manufactured within a short span of time. The novel method uses the novel strains of the invention.
Thus, the invention provides a novel method for preparation of herbal formulations classified as 'Asavas & Arishtas'. This new method brings down the process time at least by at least 2/3 of the conventional time. Also, the method reduces the rejection rate almost to zero and at the same time enhance the productivity by 25% of the batch size. Also, the novel method maintains the efficacy of this product range at par with the conventional processes.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the applicants, during the course of investigation, have identified at least three strains of yeast cultures useful in producing Asavas/Arishtas range of formulations when used in an effective concentration. Also the invention provides novel method of using these two herbs- in any given formulation.
As such, the invention provides a novel yeast strain having accession No DRF-UDS 004/wf, deposited at the Repository maintained by the Department of Microbiology, University of Delhi, South Campus, said strain having the following characteristics:
a) being sugar resistant,
b) capable of producing alcohol to the extent of about 7 to 1 lv/v% continuously and consistently in a herbal extraction medium,
c) capable of overcoming resistance conferred by the herbal and spicy ingredients present in the medium,
d) capable of attaining biomass of about 1 to 4g/l and potentiating and enhancing the therapeutic value of the herbal formulation, and
e) exhibiting the following bio-chemical properties: i) urease test - positive,
ii) utilization of fructose test - negative, iii) starch hydrolysis test - negative, iv) xylose hydrolysis test - negative.
The biomass attainable by the above strain is about 1.7 g/1, preferably 1.73 g/1. The alcohol produced by the strain is about 9 v/v%, preferably 9.580 v/v%.
Similarly, the invention provides another novel yeast strain having accession No DRF-UDS 016/wf, deposited at the Repository maintained by the Department of Microbiology, University of Delhi, South Campus, said strain having the following characteristics:
a) being sugar resistant,
b) capable of producing alcohol to the extent of about 7 to 11.5v/v% continuously and consistently in a herbal extraction medium,
c) overcoming resistance conferred by the herbal and spicy ingredients in the medium,
d) capable of attaining biomass of about lto 4g/l and potentiating and enhancing the therapeutic value of the herbal formulation, and
e) exhibiting the following bio-chemical properties: i) urease test - negative,
ii) utilization of fructose test - positive, iii) starch hydrolysis test - positive, iv) xylose hydrolysis test - positive.
The biomass attainable by this yeast strain is about 1.5 g/1, preferably 1.58 g/1. The alcohol produced by the strain is about 12 v/v%., preferably about 12.340 v/v%.
Further, the invention provides yet another novel yeast strain having accession No DRF-UDS 017/wf, deposited at the Repository maintained by the Department of Microbiology, University of Delhi, South Campus, said strain having the following characteristics:
a) being sugar resistant,
b) capable of producing alcohol to the extent of about 7 to 12v/v% continuously and consistently in a herbal extraction medium,
c) overcoming resistance conferred by the herbal and spicy ingredients in the medium,
d) capable of attaining biomass of about 1 to 3.5g/l and potentiating and enhancing the therapeutic value of the herbal formulation, and
e) exhibiting the following bio-chemical properties: i) urease test - negative,
ii) utilization of fructose test - positive, iii) starch hydrolysis test - positive, iv) xylose hydrolysis test - negative.
The biomass attainable by the strain is about 1.6 g/1, preferably 1.65 g/1. The alcohol produced by the strain is about 10.5 v/v%, preferably 10.520v/v%.
The invention also provides a method for the propagation of yeast cultures capable of being used for fermentation of plant extracts, said method comprising the steps of:
a) preparing an aqueous medium containing 10 to 40 % sucrose, derived from jaggery,
b) inoculating the medium with a yeast culture,
c) incubating the culture under aerobic conditions at a temperature in the range of 20-37°C for a period of 24 to 72 hours, and I
d) maintaining the culture to obtain a liquid containing 5 to 30 mg of yeast biomass per ml.
In an embodiment, the medium is inoculated with yeasts isolated from natural sources such as Woodfordia fruticosa and Mudhuca latifolia. The yeast culture is selected from
Saccharomyces strains such as DRF-UDS-004/wf, DRF-UDS-016/wf and DRF-UDS-017 or combinations thereof. The medium further contains minor ingredients like buffering agents, stabilizing and emulsifying agents etc. As such, the medium is self-sufficient in protein source, vitamins, minerals etc.
In another embodiment, the cultures are incubated at 25 to 32 °C.
In another embodiment, the medium of step (a) is sterilized by autoclaving at general pressure conditions before proceeding with other steps.
In still another embodiment, the temperature for sterlization is in the range of 100 to 130 °C and the pressure is maintained at 15pi.
In another embodiment, the process is carried out successively, to obtain larger volume of biomass or medium containing the yeast cultures.
In an embodiment, the aerobic conditions comprise continuous aeration or conventional method such as injecting prefiltered air.
In yet another embodiment, the medium can be used for production of proprietary as well as traditional herbal formulations. The medium of step (a) contains cell density of yeast cultures ranging from 1 x 106cfu per ml to 1 x 1010cfu per ml after 48 hours of incubation.
Further, the invention also provides a novel method for the manufacture of herbal formulations classified as 'asavas' and 'arishtas' containing self-generated alcohol, said method comprising the steps of:
(i) obtaining aqueous extract of plant(s),
(ii) adding an inoculum containing a yeast strain selected from DRF-UDS 004/wf, No DRF-UDS 016/wf and No DRF-UDS 017/wf or mixtures thereof which are deposited at the Repository maintained by the Department of Microbiology, University of Delhi, South Campus and (iii) allowing the culture to ferment under anaerobic conditions for 4 to 8 days to obtain the herbal formulation containing 7 to 14% self-generated alcohol.

In an embodiment, the inoculum added is a liquid or a solid culture.
In another embodiment, the yeast culture is propagated in an aqueous jaggery medium containing 10 to 40% sucrose.
In still another embodiment, the extract of the plant/s of the herbal formulation is obtained along with one or more flowers selected from Woodfordia fruticosa and Mudhuca latifolia.
In yet another embodiment, the above method is capable of being applied to any proprietary and traditional herbal formulation.
In another embodiment, the method is capable of being applied to herbal formulation cited in traditional Ayurvedic texts or customised herbal formulation having similar properties.
In an embodiment, the invention pertains to a range of classical Ayurvedic dosage forms having a significant financial stake to an estimate of Rs. 2000 million of Asava/Arishta formulations are produced in India by various manufacturers.
In an embodiment, the novel method of the invention uses specific cultures of yeast strains isolated from the flowers of Woodfordia fruticosa and/or from the flowers of Madhuka latifolia
In yet another embodiment, the invention identified these specified cultures from a total of 72 cultures isolated from the samples of Woodfordia fruticosa and/or from the flowers of Madhuka latifolia collected from different parts of the country.
In yet another embodiment, the invention seeks to grow and propagate selected yeast strains useful for the productivity of Asavas/Arishtas in a simple microbiological medium prepared from Jaggery.
In an embodiment, the invention seeks to use the flowers of Woodfordia fruticosa and/or from the flowers of Madhuka latifolia during the phase of extraction so as to maintain the efficacy of the Ayurvedic formulations produced by this novel method.
The inventive steps were designed to address all the operative constraints involved in mass-scale production of Asava-Arishta group of formulations taking a cue from existing knowledge on two main plant species viz. Woodfordia fruticosa and Madhuca latifolia. In order to accomplish the specified objectives, an extensive screening of microflora isolated from these two flower species was carried out.
Principles of Invention:
♦ Many strains of Saccharomyces species can produce alcohol when they are allowed to grow in a medium containing sugar under anaerobic conditions. Saccharomyces cereviceae, Saccharomyces uvarum and Saccharomyces exiguus, Saccharomyces bayanus are three species widely used to produce industrial alcohol or to produce alcoholic beverages and wines. Of these three species S. cereviceae is more often employed in production of industrial alcohol using molasses as starting material. It is also, a noteworthy fact that, all strains of these specific yeast species might not produce alcohol under identical conditions. Each of these strains can produce alcohol from the medium, under very specified operative conditions. These operative conditions include the initial levels of sugar, temperature, pH, aeration conditions etc.
♦ For example, different strains or species will be needed to produce beer using Barley extract as medium, for industrial alcohol using molasses and producing wines from grape juice medium. Therefore, every manufacturer identifies most suitable species and their strains of Saccharomyces or other alcohol producing organisms to suit the specific applications.
♦ For preparation of Asava - Arishta formulations, the micro-organisms used must be tolerant to high level sugars (touching 40% w/v) and a highly acidic medium (with pH around 4.0). Over and above these factors, the organism must be able to grow under hostile conditions created by herbs and spices. The inventors established through a series of investigations that, some of the yeast strains used for production of beer or wines might not survive in any medium of Asava-Arishta group. Similarly, certain other strains of yeast were found to produce alcohol too rapidly, breaking the ancient principles of a low paced alcohol generation. Therefore, Asava - Arishta process requires a new set of culture strains, which can survive in high sugar, low pH and the hostile culture environment created within the medium, by herbs and spices. /
♦ The role of flower buds (viz. Woodfordia fruticosa or Madhuca latifolia) used for fermentation is an important factor in the production of Asava - Arishta group. Often, it must be going beyond the production of alcohol in the formulation. Such additional role might be traced to enhanced therapeutic effect as identified by Kroes group or to undefined enzymatic processes triggered during fermentation by specialized strains of yeast hosted by these flower buds.
♦ While the flower buds are essentially needed, they also constitute the source of one major operative problem. Both the flower buds viz. Woodfordia fruticosa and Madhuca latifolia are known to host yeast strains and a range of bacterial and fungal strains as well. If the fungal strains predominates the yeast during fermentation, the product is contaminated and such contamination may often, be hazardous.
The experiments were carried-out in the following phases:
> Collection of flower buds & Isolation of yeast strains using microbiology techniques. (Isolation Phase)
> Pre-screening for fermentation profile in sucrose medium & short-listing the strains (Prescreening Phase)
> Screening for fermentation profiles in Asava - Arishta media and identification of viable organisms (Screening Phase)
> Reproducibility & Feasibility studies (Feasibility Phase)
> Evaluation of Invented process in terms of biological activity
> Characterization & Identification of yeast strains (Characterization phase)
> Development of methods for culture propagation (Propagation phase)
The details of the above phases of experiments are summarized as under:
Isolation Phase:
The samples of both kinds of flower buds, viz. Woodfordia fruticosa and Madhuca latifolia
were collected from different parts of India spanning from Jammu & Kashmir in north to
Andhra Pradesh in south and from Madhya Pradesh in centre to Maharshtra in west during
the active flowering season of the herbs. From a total of 49 samples of Woodfordia fruticosa,
25 samples and one sample of Madhuca latifolia was taken-up for isolation of yeast strains.
For this purpose, 1 gm of buds were washed with sterile distilled water, suspended in saline (0.85%) and shaken in an incubator shaker at 30 °C, 200 rpm for 3hrs. The supernatant liquid was used to inoculate any suitable culture medium like, MPYG Broth and incubated at 30 °C for 40 to 48 hrs. Dilution (10"1 to 10'5) of the MYPG broth were spread on MYPG agar plates (Tilbury, R.H. 1980: in Biology & Activities of Yeasts, eds. Skinner, F.A., S.M. Davenport, R.R., Academic Press, London, p-153.), pH-4. In order to avoid fungal contamination the medium was supplemented with ampicillin (60|ig/ml) and sodium propionate (250|ig/ml).
For further isolation, colonies of the yeast Saccharomyces cereviceae appearing on the culture plates were subjected sub-culture techniques' (Tilbury, R.H. 1980: in Biology & Activities of Yeasts, eds. Skinner, F.A., S.M. Davenport, R.R., Academic Press, London, p-153). The distinct colony that appeared after incubation were picked up and streaked on suitable medium for example, MYPG agar plates and then on to slants. The colony morphology and microscopic observation were recorded for all the isolates. The pure cultures of these yeast isolates are maintained on MYPG slants by 4 °C by reviving periodically. Preservation of isolates is also being done in glycerol at -20 °C.
By the above method, a total number of 67 isolates of Saccharomyces cereviceae were obtained from 25 samples of Woodfordia fruticosa and 5 isolates were obtained from 2 samples of Madhuca latifolia. Each of these isolates is assigned a Strain Number.(72 strains in all)
Prescreening Phase
The second phase of experiments were designed to examine the ability of each of the culture to produce alcohol in a medium containing high concentration of Sugars.
A fermentation medium was prepared by dissolving Jaggery in water so as to provide 40%
(w/v) sucrose. 100 ml of medium was dispensed in 250 ml flasks and were sterilized at 10
psi, for 20 minutes. The flasks were agitated at for 6 hrs at 200 rpm and then incubated under
i static condition, for 7 days covering their tops with aluminum foil. At the end of incubation
period, the content of the flasks was centrifuged at 10,000 rpm for 20 minutes. The
supernatant portion was collected for estimation of alcohol and residual sugar contents. The
pellet was washed with distilled water, followed by centrifugation, and to constant weight
dried at 80°C for determination of biomass.
Alcohol content was determined by gas chromatography using 1 % Ethanol as the standard. Residual sugar was determined by using refractometer and also by using anthrone reagent.
The results of this phase of investigations are summarized in Table-1. This table provides the alcohol produced and biomass attained by the yeast isolates of Saccharomyces cereviceae.
(TABLE REMOVED)

As seen from Table - I, of the 72 strains of Yeast isolated in prescreening phase, only 10 isolates were found capable of producing alcohol effectively, in a medium containing higher concentrations of Sugar. These strains are shown in bold lettering in the table. These strains were recognized as potential candidates for further screening under phase- in of investigations.
Screening Phase:
A total of 10 isolates of yeast were taken-up for screening under phase - HI of experiments. The objective of these investigations was to ascertain whether these strains could play their role in Asava-Arishta medium or not. Since, Dashmularishta is one of the most of complex formulation in the category; initial experiments were carried-out in this direction, using Dashmularishta as a case study. The following technique was employed for this phase of investigations.
Preparation of Medium: The formulation of Dashmularishta was based on description given in Bhaishajya Ratnavali (Ambikadutta Shastri, Kaviraj (1987): in Hindi translation & commentary to Bhaishajya Ratnavali, ed. by Rajeshwaradutta Shastri, Chaukhambha Sanskrit Sansthan, Varanasi, 8th edition, p-796.) and the formulation contains 67 ingredients in all. An extract was prepared from the herb as laid down in the textbook. The flowers of Woodfordia fruticosa meant for use as inoculum bearer herb was also combined in this extraction procedure. Requisite quantity of Jaggery was dissolved in this extract. However, the spice powder portion was omitted in this set of experiments.
100 ml of extract each was dispensed into Erlenmayor flasks for 9 different cultures in triplicate. The yeast isolates selected for screening were inoculated into these flasks and they were agitated at 30 °C for 6 hrs and then shifted to static conditions for seven days. Samples were drawn from each flask from day-2 onwards and were examined for alcohol content and
TSS levels. The results of these two tests at different time intervals are summarized in the following tables.
(TABLE REMOVED)

i As seen from the above tables, only four strains (shown in bold lettering in both the above
tables) were found to be able to produce alcohol in Dashmularishta medium. Rest of the
cultures was discarded for further experiments.
In the subsequent experiments, three cultures (isolated from Woodfordia fruticosa) and their combinations and also the culture isolated from the Madhuka latifolia were re-examined for their capability to survive and produce alcohol in Dashmularishta medium using a similar protocol. This time, the spicy powder mix (called as "Prakshep Powder") as given in the textbook was prepared and was used for topping the culture medium. These results are summarized in Table- III.
(TABLE REMOVED)

The above three yeast strains having accession Nos DRF-UDS 004/wf, DRF-UDS 016/wf and DRF-UDS 017/wf are deposited at the Repository maintained by the Department of Microbiology, University of Delhi, South Campus. These cultures are available to the public for all purposes. These cultures are sugar resistant and are capable of producing alcohol to the extent of about 7 to llv/v% continuously and consistently in a herbal extraction medium. They are capable of overcoming resistance conferred by the herbal and spicy ingredients in the medium containing herbal extracts. The biomass attained by them is about 1 to 4g/l. The cultures can potentiate and enhance the therapeutic value of herbal formulations.
As said earlier, the above three strains are specific isolates of Saccharomyces cereviceae.
i Some of their morphological characteristics are as under:
1. These strains are microscopic oval cells, exhibiting budding in Malt-yeast-protein-glucose
medium.
2. These strains produce ascospores in malt-extract-agar medium. The ascospores are seen
as tetrads in the ascads.
3. As a colony, they appear as white spherical, pin-head mucoid, convex, opaque colonies.
Feasibility Phase:
The experiments detailed above clearly indicate that, the processing methodology of Asava-Arishta range can be modified using yeast strains isolated from flower-buds of Woodfordia fruticosa. Such process was demonstrated in small volumes of Dashmularishta medium. To establish the feasibility of the new process further experiments were carried out in increased volumes of 1- 4 It. batch. Further, the experiments also examined the utility of new process in other formulations of Asava-Arishta group. Feasibility studies were also conducted using variable volume of culture inoculum. All these experiments invariably proven that, three strains of yeast isolated from the flower-buds of Woodfordia fruticosa are capable to work as inoculum for carrying-out, fermentation in any of the Asava-Arishta formulations.
As a part of these feasibility investigations, the competency of organisms was also compared to well-known yeast strains generally used for alcoholic products. These investigations distinctly indicated that, the new cultures of yeast identified by the above inventive steps; are much more superior to these strains with reference to their utility for the intended purpose i.e. in the manufacturing Asava-Arishta range of Ayurvedic formulations. These specific advantages are summarized as below:
The yeast strains used normally, for alcohol production might not survive in any medium containing sucrose content exceeding 15 - 20%. However, a typical medium of Asava-Arishta range contains over 35% w/v sugar content. Contrary to this phenomenon, the yeast strains identified by this invention are novel and can thrive well upon, higher concentration of sugars.
Some other strains like, Baker's yeast was observed to produce alcohol in Asava-Arishta medium comfortably. However, the speed of alcohol generation is too rapid. Ayurvedic medicines falling under Asava-Arishta category should ideally processed in a slow pace enabling the organisms to carryout underlying enzymatic processes. On the other hand, the new cultures identified by the invention carryout the fermentation at a medium pace, which, is not very slow as taken by the conventional process or too rapid as taken by certain other strains.
Characterization Phase:
Once the utility of organisms is established by various experiments, needful investigations were carried-out to establish the identity of yeast strains isolated by the invention. These methods included but not limiting to Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and biochemical characterization.
Propagation Phase:
The investigations in this phase were directed to develop suitable methods to propagate the yeast strains isolated during the inventive steps. By these procedures, it was possible to obtain a good volume of biomass using a Complex Jaggery Medium when grown under aerobic culture conditions. It was also observed that, three different isolates can be grown in the same medium comfortably and the strains do not compete with each other, under the specified culture environment.
The invention is further described and illustrated by the following examples which should not be construed as limitations on the inventive concept embodied herein.
EXAMPLES: Example-l
Dashmularishta is one the most complex formulations mentioned in ancient Ayurvedic literature. The formulation contains 67 ingredients in all as described in Bhaishajya Ratnavali (Ambikadutta Shastri, Kaviraj (1987): in Hindi translation & commentary to Bhaishajya Ratnavali, ed. by Rajeshwaradutta Shastri, Chaukhambha Sanskrit Sansthan, Varanasi, 8th edition, p-796.) including the source of inoculum viz. Woodfordia fruticosa and Jaggery used as nutrient in the medium. The prepared medium contains 40% initial sugar and a range of spices being topped during fermentation. This blend of spicy ingredients inhibits the rapid growth of fermenting organisms.
In one experiment, 250 ml of Dashmularishta medium was distributed into Seven sets of Erelynemayor flasks and three types of culture systems were run in parallel. In the first set of flasks, the Woodfordia fruticosa flowers were used as source of inoculum; following the traditional method of fermentation. The second set was inoculated with a well-known MTCC 178 strain of Saccharomyces cereviceae. In another set, the Baker's Yeast was inoculated. In rest of 4 sets, DRF-UDS - 004/Wf, DRF-UDS - 16/Wf, DRF-UDS - 017/Wf individually, and a combination of all three strains were inoculated. All these sets were incubated under controlled conditions allowing a complex anaerobic fermentation.
It was observed that, all the flasks in Set - 1, containing W. fruticosa flowers as source of inoculum were contaminated by fungi on day 4. MTCC 178 strain produced alcohol rapidly during initial phase, though it was slowed down later. On day 2 the concentration of alcohol reached 6.56%. The set containing Baker's yeast as inoculum was found to attain 8.71% alcohol levels by the end of day-3. When fermentation continued, the set has shown a final level of 11.53% alcohol. As against these results, the new cultures and their combination have shown a distinct pattern of alcohol generation. On day 2 the alcohol was found to range between 1.98 to 3.22%. This pace continued in a fairly, uniform manner till the end of day 8.
This experiment clearly indicates that, the cultures of yeast isolated by the invention work in a uniform pace and attain the requisite levels not too slow and too fast. This pattern of biosynthesis is more desirable in an Ayurvedic formulation like, Dashmularishta.
Example - 2
In one experiment, 250 ml of Dashmularishta medium was distributed into Erelynemayor flasks and these flasks were inoculated with DRF-UDS - 005/M1. All these flasks were incubated under controlled conditions allowing a complex anaerobic fermentation.
It was observed that, the rate of fermentation was steady and consistent and attain 6.3 % alcohol level by the end of day-4. When fermentation continued, the medium has shown a final level of 10.13% alcohol.
This experiment clearly indicates that, the cultures of yeast isolated by the invention work in a uniform pace and attain the requisite levels not too slow and too fast. This pattern of biosynthesis is more desirable in an Ayurvedic formulation like, Dashmularishta.
Further experiments were conducted with only three cultures isolated from Woodfordia
fruticosa viz. DRF-UDS - 004/Wf, DRF-UDS - 016/Wf and DRF-UDS - 017/Wf and their
combinations.
Example-3
In another experiment on Dashmularishta, the batch volume was increased to 3.5 liters net, using a modern; microprocessor based bench-top fermentor (Bioflo-3000 - with a gross
volume of 5 liters). When Dashmularishta was produced in this bioreactor going by a purely traditional process, it was observed that:
a) The powered dry flowers of Woodfordia fruticosa occupy 20% of fermentor capacity resulting in a reduced volume of out-put.
b) The fermentation process was completed after 21 days of controlled conditions of incubation.
At next stage, the extract of Dashmularishta was prepared including the flowers of W. fruticosa and the Baker's yeast was used in lieu of flowers. This time, the batch size could be increased to the full capacity of bioreactor used in the experiment. However, the fermentation process was completed within 52 hours, making the process too rapid.
Contrary to these two methods, processing of Dashmularishta could be completed on day 9, when the medium was inoculated with any one of the three cultures viz. DRF-UDS - 004/Wf, DRF-UDS - 016/Wf, DRF - 017AVf or the combinations thereof. This example illustrates that, the new cultures identified by the invention carryout the process not very slow and not too rapid but do it, in a medium duration of time frame. Further, the new process enables to make use of available fermentor capacity, to the full-extent as against the traditional process. Such feasibility comes from a fact that, W. fruticosa flowers required by the formula are utilized during extraction phase.
Example- 4
Ashokarishta is one among the five top-selling Ayurvedic formulations falling under Asava-Arishta range. Described in Bhaishajya Ratnavali (Ambikadutta Shastri, Kaviraj, 1987: in Hindi translation & commentary to Bhaishajya Ratnavali, ed. by Rajeshwaradutta Shastri, Chaukhambha Sanskrit Sansthan, Varanasi, 8th edition, p-722.) the formulation contains 15 ingredients, including Jaggery and the flowers of Woodfordia fruticosa - the source of fermenting organism.
In a series of experiments with this formulation at 4 liters volume size, using the Bioflo-3000 bench-top bioreactor, it was observed that, 2 out 5 batches were contaminated due to fungi from Woodfordia fruticosa. In rest of three batches, the requisite alcohol levels could be
attained on day 16 . In these qualified batches, the batch yield was reduced due to the bulky nature of flower powder occupying voluminous space in the fermentor.
In the next stage of experiments, the flowers of Woodfordia fruticosa were used during extraction phase. The fermentation of this modified medium was carried-out, using the yeast strains isolated during invention, DRF-UDS - 004/Wf, DRF-UDS - 016/Wf and DRF-UDS -017/Wf individually and a combination thereof in a concentration ranging between 1-5% (v/v). During this series of experiments, no batch was contaminated. The required levels of alcohol could be attained on day 8. Since, the use of powdered flowers could be avoided, the fermentor's capacity could be fully utilized.
The above example suggests that, the yeast cultures identified during the invention work effectively in the medium of Ashokarishta as well, providing all advantages of new process to it's full-extent.
Example- 5
In another experiment, 250 ml of Ashokarishta medium was distributed into Seven sets of Erelynemayor flasks and seven types of culture systems were run in parallel. In the first set of flasks, the Woodfordia fruticosa flowers were used as source of inoculum; following the traditional method of fermentation. The second set was inoculated with a well-known MTCC 178 strain of Saccharomyces cereviceae. In another set, the Baker's Yeast was inoculated. In rest of 4 sets, DRF-UDS - 004/Wf, DRF-UDS - 16/Wf, DRF-UDS - 017/Wf individually, and a combination of all three strains were inoculated. All these sets were incubated under similar, controlled conditions allowing a complex anaerobic fermentation.
It was observed that, all the 2 of 3 flasks in Set - 1, containing W. fruticosa flowers as source of inoculum were contaminated by fungi on 2nd day. In the rest, the alcohol content reached 10.16% after 16 days. MTCC 178 strain produced alcohol beyond 7% v/v as on day 2nd and above 11% on day 4th. The set containing Baker's yeast as inoculum was found to attain 9.38% alcohol levels by the end of day-1. As against this rapid generation of alcohol, the new cultures and their combination could complete the process of Ashokarishta on day 8 with 9.27% of alcohol content in all the member flasks.
This experiment clearly indicates that, the cultures of yeast isolated by the invention behave distinctly. They produce alcohol in a slow and consistent manner unlike the Yeast strains used for baking or brewing purposes. On the other hand, the traditional way of using W. fruticosa is prone to a high risk of fungal contamination. Contrary to them, the new process identified by the invention helps to overcome all the critical issues governing the process of Ashokarishta.
Example- 6
Lohasava is another member of Asava-Arishta group. This formulation contains Iron and is used in treatment of anaemia. Besides Iron, it contains 13 other ingredients including Jaggery and Woodfordia fruticosa - the source of fermenting organisms.
In one experiment, 250 ml of Lohasava medium was distributed into Seven sets of Erelynemayor flasks and seven types of culture systems were run in parallel. In the first set of flasks, the Woodfordia fruticosa flowers were used as source of inoculum; following the traditional method of fermentation. The second set was inoculated with a well-known MTCC 178 strain of Saccharomyces cereviceae. In another set, the Baker's Yeast was inoculated. In rest of 4 sets, DRF-UDS - 004/Wf, DRF-UDS - 16/Wf, DRF-UDS - 017/Wf individually, and a combination of all three strains were inoculated. All these sets were incubated under similar, controlled conditions allowing a complex anaerobic fermentation.
It was observed that, all the flasks in Set - 1, containing W. fruticosa flowers as source of inoculum were contaminated by fungi on day 2nd. MTCC 178 strain produced alcohol 14.12%v/v as on day 2nd and when the experiment was continued further, the alcohol content reached 15.88% on day 8th. The set containing Baker's yeast as inoculum was found to attain 8.79% alcohol levels by 2nd day and on day-8 it reached 14.01%. As against this rapid generation of alcohol, the new cultures and their combination could rise alcohol content slowly and consistently to attain the levels ranging between 8.74% to 10.81% as on 8* day.
It must be noted that, the maximum alcohol content recommended for Asava-Arishta should not exceed 12% v/v in the end product.
This experiment clearly indicates that, the traditional way of using W. fruticosa is prone to a high risk of fungal contamination. Available yeast culture produce alcohol at a steeply rising
manner. On the other hand, cultures of yeast isolated by the invention behave distinctly. They produce alcohol in a slow and consistent manner, which is more desirable in Ayurvedic formulations. This very advantage of the invention helps to overcome all the critical issues governing the process of Lohasava.
Example- 7
Drakshasava is another formulation falling in the category. As per Yogaratnakara10 (Laskhmipati Shastri (1988): in Vidyotini Hindi commentary to Yogaratnakara, ed. by Shastri, B.S., Chaukhambha Sanskrti Sansthan, 4th ed., p-288.), it is prepared from 17 ingredients including Sugar and Woodfordia fruticosa used for topping during fermentation.
In one experiment, Drakshasava medium was prepared as per the formula. As modification, the flowers of Woodfordia fruticosa were also added during the extraction phase. Jaggery was dissolved and Honey was added as per the textual formula in the medium. This medium
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was dispensed into 4 sets of Erelynmayor flasks.
The fermenting organisms isolated during invention viz. DRF-UDS - 004/Wf, DRF-UDS -16/Wf, DRF-UDS - 017/Wf were inoculated individually and the combination thereof. The alcohol content in all these sets was observed to rise constantly from day - 2 onwards and reached 10.92% on day 7th. This process duration is much less, as compared to the traditional process, which takes 13 days to attain the 10.5% alcohol level in the finished product.
Example- 8
Among Asava-Arishta formulations, Kumaryasava is the member addressing liver ailments. It contains Kumari - the Aloe barbadensis as main ingredients. The formulation uses 41 other herbs besides Jaggery, honey and the flowers of Woodfordia fruticosa as the source of fermenting organism. Thus, the formulation is highly complex in nature with 45 ingredients.
In one experiment, the extract of Kumaryasava prepared as per textual formula excepting that, the flowers of W. fruticosa were combined during extraction phase. A culture medium was prepared by adding honey and Jaggery as per the textual formulation and it was dispensed into 5 sets of Erylenemayor flasks. In the first set, Baker's yeast was inoculated as fermenting organism and in the rest of them, the new cultures viz. DRF-UDS - 004/Wf,
DRF-UDS -16/Wf, DRF-UDS - 017/Wf were inoculated individually and the combination thereof.
In the first set, 10.87% alcohol levels could be attained by 2nd day, showing the rapid nature of fermenting organism. On the other hand, in rest of tubes the alcohol content was noted to be ranging between 1.94% and 2.53% on day-2 and between 9.8% and 10.75% on day 8th.
This example also substantiates the earlier observations that, the new cultures viz. DRF-UDS - 004/Wf, DRF-UDS -16/Wf, DRF-UDS - 017/Wf and their combinations behave distinctly, in the medium of an Asava-Arishta nature. Such behaviour of the organisms is advantages considering the specificity of process required for the range.
Example-9
The above examples demonstrate the efficiency of the process in traditional/ generic Ayurvedic formulations - for which, the formula was laid down ages ago. In order to examine the dynamics of alcohol generation in proprietary combinations, few more experiments were carried-out.
In one of such experiments, a combination of Gurmar leaves (Gymnema sylvestre), Methi seeds (Trigonella foenwngrecum), Vijayasar heartwood (Pterocarpus marsupium), Jamun seeds (Eugenia jambolana), Karela seeds (Momordica cherantia) and Dhataki (Woodfordia fruticosa) was taken and extract was obtained.
Jaggery was dissolved in the extract and a blend of powdered Pippali (Piper longum), Elaichi (Elettaria cardamomum), and Javitri (Myristica fragrans), greater cardamom (Ammonium subulatum) was used as spicy topping during the process. The herbal formulation of this experiment is designed to address diabetes.
This medium was dispensed in 5 sets of Erelynemayor flasks and DRF-UDS - 004/Wf, DRF-
UDS -16/Wf, DRF-UDS - 017/Wf were inoculated individually and the combination thereof
respectively. In the last set, Baker's yeast was used as inoculum. When alcohol contents
were monitored in this experiment on a day-today basis, it was noted that, the alcohol
generation was completed by baker's yeast on day-3, rest of cultures took 9 days. These
observations clearly hint at the utility of the new process in a proprietary herbal formulation.
Example -10
In yet another experiment, the extract was prepared from a proprietary, anti-arthritic combination. The formulation contained Rasna (Vanda roxburghiana), Mustak (Cyperus rotundus), Karkatshringi (Pistacia integerrima), Yastimadhu (Glycerrhiza glabra), Vriddhadaru (Argyria speciosa), Eranda (Ricinus communis) and Dhataki (Woodfordia fruticosa). Jaggery was dissolved in this herbal extract and a blend of powdered Sunthi (Zingiber officinale), Ajwain (Trachyspermum ammi), Pippali (Piper longum), Elaichi (Elettaria cardamomum) was used as Prakshep.
This medium was dispensed in 5 sets of Erelynemayor flasks and DRF-UDS - 004/Wf, DRF-UDS -16/Wf, DRF-UDS - 017/Wf were inoculated individually and the combination thereof respectively. In the last set, Baker's yeast was used as inoculum. When alcohol contents were monitored in this experiment on a day-today basis, it was noted that, the alcohol generation was completed by baker's yeast on day-2, rest of cultures took 7 days. These observations clearly hint at the utility of the new process in a proprietary herbal formulation.
Example -11
In yet another experiment, a combination of Kantakari (Solanum xanthocarpum), Shireesha (Albizzia lebbeck), Goskhura (Tribulus terrestris), Karkatshringi (Pistacia integerrima), Vasa (Adathoda vasica) and Dhataki (Woodfordia fruticosa) was taken and extract was obtained.
Jaggery was dissolved in the extract and a blend of powdered Elaichi (Elettaria cardamomum), Lavang (Syzygium aromaticum), Pippali (Piper longum), and Nagakeshar (Mesua ferrea) was used as spicy topping during the fermentation.
This medium was dispensed in 5 sets of Erelynemayor flasks and DRF-UDS - 004/Wf, DRF-UDS -16/Wf, DRF-UDS - 017/Wf were inoculated individually and the combination thereof respectively. In the last set, Baker's yeast was used as inoculum. When alcohol contents were monitored in this experiment on a day-today basis, it was noted that, the alcohol generation was completed by baker's yeast on day-3, rest of cultures took 9 days. These observations clearly hint at the utility of the new process in a proprietary herbal formulation.
All the above experiments revealed that the new cultures isolated from the study have shown great potential for the generation of different Asava/Arishtas. The fermentation duration is about 7 days in all the experiments and the alcohol content is less than 11 % v/v. Fermentation parameters have shown the potentiality of these culture in different conditions. However the crux of any new process lies in the efficacy of end product. In order to assess their pharmacological activity several different samples were prepared and they were tested for their pharmacological activity.
These investigations are illustrated in the foregoing examples.
The summary of these pharmacological studies were explained as follows:
Example -12
Anti-inflammatory Activity of Dashmularishta:
Six different samples were prepared using various cultures. The identification of the sample is as follows based on the culture used for the preparation of the sample.
Sample 'A': DRF-UDS - 004/Wf Sample 'B': DRF-UDS - 016/Wf Sample 'C: DRF-UDS - 017/Wf Sample 'D': Mixed culture of the above three Sample rE': Baker's Yeast Sample 'F: Traditional method
Study Protocol: The samples were evaluated using an acute inflammation model induced by Carrageenan and formalin. The samples were also evaluated using Freund's Complete Adjuvant induced chronic inflammation models in rats.
Carrageenan Induced Acute Inflammation Model: 40 Wistar rats of either sex weighing 150 -200 g were kept under standard laboratory conditions in the departmental animal house. A balanced pellet diet and water were provided ad libitum. They were divided into 8 groups containing 5 animals each. The first group served as control receiving only water in lieu of medication.
Animals in rest of 6 groups received a pre-treatment with one of the six samples of Dashmularishta at a dose of 7 ml/kg of body weight for one week. On day 8th, 0.1 ml of 1% Carrageenan solution was injected sub-cutaneously into plantar region of left hind paw. Immediately after this injection, one more dose of Dashmularishta sample was administered in the test groups while; the control received water - in lieu of the drug.
The volume of left hind paw immediately before the administration of phlogistic agent and after 3 hours for each animal using a Mercury Filled Plethysmograph.
The 8th group didn't receive the phlebotic agent and hence served as a normal control. The volume of paw in this group was taken as standard, normal volume of paw for comparison purposes.
Formalin Induced Acute Inflammation Model: The study protocol was similar as above excepting that, Formalin was used phlebotic agent to induce oedema in the paw. The volume of paw was measured after 4 hours of injecting formalin.
After completion of these test procedures, the means volumes of paw oedema were calculated for each group and the data was analyzed using Unpaired, one way Student's " t" test. P<0.05 was considered significant.
Results of Acute Inflammation Study: Of the Six samples used in the study, 3 samples viz. Sample A, C and Sample D were found to be significantly effective in controlling the inflammation caused by carrageenan. In case of formalin induced inflammation, none of the samples was found to be effective.
Freund's Adjuvant Induced Chronic Inflammation Model: Experimental animals were distributed at random, into 7 groups containing 6 each. The first group was marked for control purposes and therefore, received water in lieu of study samples. Animals in rest of 6 groups were assigned to pre- treatment with one of the 6 samples of Dashmularishta at a dose of 7 ml/kg. b. w. in two divided doses for a period of 15 days.
On day 16 , Freund's Adjuvant was injected into left hind paw - after measuring the initial volume of paw. Treatment with Dashmularishta was continued for 11 days after this injection.
The measurements of paws in every group were taken at 18 hours after injection and on days 2, 3, 5, 7, 9 and 11. After completion of these test procedures, the means volumes of paw oedema were calculated for each group and the data was analyzed using Unpaired, one way Student's " t" tests. P<0.05 was considered significant.
Results in Chronic Inflammation Model: Sample B was found to offer a highly significant protection against chronic inflammation induced by Freund's Adjuvant.
The above example amply demonstrates that, the invented process promotes the efficacy of Dashmularishta. This benefit is in addition to the other process advantages.
Example -13
Uterotonic Activity of Ashokarishta:
Six different samples were prepared using various cultures. The identification of the sample is as follows based on the culture used for the preparation of the sample.
Sample T: DRF-UDS - 004/Wf
Sample XT. DRF-UDS - 016/Wf
Sample 'HI': DRF-UDS - 017/Wf
Sample TV": Mixed culture of the above three
Sample "V": Baker's Yeast
Sample 'VI: Traditional method
Study Protocol: This study was done in two experimental sets-up, in comparison to standard utero-tonic substance, 5 - hydroxy-tryptamine.
Experiment - 1: Female rats weighing between 150 - 200 g were pretreated with stilbestrol at a dose of 1 mg/kg administered intra-peritoneally, 24 hours prior to the experiment. Then they were sacrificed and the uteri were isolated. One of the horns was suspended in water bath and the rhythmic contractions were abolished by using "de-Jalton's solution". When the
spontaneous contractions became regular, the responses of test samples and other agonists were recorded using a frontal lever isometrically connected to a polygraph. One of the test samples included a combination of 5 - hyroxytryptamine and Ashokarishta, to examine the potentiation effect of Ashokarishta on conventional uterotonic agents.
Experiment - 2: In the second set-up, the rats were pretreated with one of samples of Ashokarishta - at a dose of 1 ml per day for a period of 3 weeks before sacrifice. After sacrifice, rest of experiment was carried-out as described in experiment - 1.
Results: Uterotonic effect of Ashokarishta Per-se: With sample B there was a spontaneous contraction observed. Rest of the samples had no effect on uterine contraction patterns at any of the dose levels.
All the samples exhibited a potentiating effect when combined with 1 mg of 5 -hydroxytryptamine. This effect was much marked with Sample 'IT.
5 samples were found to exhibit marked utero-tonic activity when used for pretreatment. Of them, sample 'IT was found to exhibit a marked activity.
The above results indicate that, Sample 'II' exhibits more potent utero-toinc activity as compared to others.
The above example amply demonstrates that, the invented process promotes the efficacy of Ashokarishta. This benefit is in addition to the other process advantages.
Example -14
Hepatoprotective Activity of Kumaryasava:
Six different samples were prepared using various cultures. The identification of the samples
are as follows based on the culture used for the preparation of the sample.
Sample 'A*: DRF-UDS - 004/Wf Sample 'B': DRF-UDS - 016/Wf Sample 'C: DRF-UDS - 017/Wf Sample 'D': Mixed culture of the above three
Sample E': Baker's Yeast Sample 'F: Traditional method
Study Protocol: The hepatoprotective role of Kumaryasava was evaluated in two different experimental sets-up.
Experiment - 1: 56 Male Wistar Adult rats weighing 250 - 300 grams were divided into 8 groups comprising 7 animals each. All the animals were maintained under standard laboratory conditions as per the guidelines CPCSEA. They were allowed a standard rat chaw pellet diet and distilled water ad libitum.
A sample of blood was drawn from retro-orbital plexus, on day 0 - before, they were assigned to any treatment.
Of these eight groups, first group received only distilled water at a dose of 10 ml/kg of b.w. through oro-gastric incubation for a period of 7 days in a row. No hepatotoxic drug was administered for this group and hence, it served as a normal, untreated control. Second group received a similar treatment with distilled water but at the end of 7 days; they also received a dose of hepatotoxic drug to induce liver damage. Animals assigned to Group - 3 to Group - 8 were treated by one of samples of Kumaryasava (labeled as Sample A, Sample B, Sample C etc.) at a dose of 0.54 ml/200 gm of body weight for a period of 7 days.
On day 8th, a sample of blood was drawn to measure the liver functions. Later, galactosamine was administered through i.p. route, at a single dose of 800-mg/kg body weight to each of animals assigned to Group - 2 to Group - 8. The animals in first group were not given this hepatotoxic drug.
On day 9th, another sample of blood was drawn and later the animal was sacrificed and the sample of liver was collected for histo-pathology examination. The liver tissue was also used for estimation of Liver glycogen. The blood samples drawn at 3 time intervals (day 0, day 8th and on day 9th) were analyzed for LFT which included estimation of Serum Bilirubin, Alkaline phosphatase & SGPT.
Experiment - 2: A similar experimental protocol system was followed for second set of experiments up to day 7th. On day 8th, a sample of blood was drawn from retro-orbital plexus. Afterwards, another hepatotoxin viz. thioacetamide was administered at a dose of 600 mg/kg b.w. through intra-peritoneal route.
On day 9th, another sample of blood was drawn for LFT.
Later, the animals were anaesthetized by administration of thiopentene sodium (40 mg/kg b.w.) and the abdomen was opened in layers. The bile duct was identified and the bile was collected for one hour, using a scalp-vein set. After this, the animal was sacrificed and the liver was collected for histo-pathology examination.
The blood samples were evaluated for LFT comprising Serum bilirubin, Alkaline phosphatase and SGPT.
Analysis of Results: For the individual groups, the data was pooled and mean (± SD) was calculated for parametric data. Analysis was done using Student's paired Y test. Unpaired Y test was done to compare the values of different drug groups. The same method was followed for both sets of experiments.
Results: Of the 6 samples, sample 'A' showed better protection if, increase in levels of SGPT and alkaline phosphatase is taken into consideration. This sample was also found to preserve the liver glycogen content as compared to other samples. On the other hand, all the samples were found to prevent the rise in SGPT or alkaline phosphatase
However, these results are yet to be confirmed from histo-pathological studies on liver tissues which, are in progress.
Example -15
Haematinic Effects of Lauhasava Samples:
Six different samples were prepared using various cultures. The identification of the samples are as follows based on the culture used for the preparation of the sample.

Sample 'A': DRF-UDS - 004/Wf Sample 'B*: DRF-UDS - 016AVf Sample 'C: DRF-UDS - 017/Wf Sample 'D': Mixed culture of the above three Sample "E': Baker's Yeast Sample 'F: Traditional method
Study Protocol: The haematinic profile of each sample of Lauhasava was evaluated in experimentally induced anaemia under two distinct situations (a) when the animal receives a normal diet after inducing anaemia and (b) when animal continues to receive iron deficient diet even during the period of treatment for anaemia. Its haematinic profile was also compared to administration of Ferrous sulfate.
Experimental Set-up: 170 Wistar strain female rats (weighing 120 - 180 grams) by a) were used in the study at the on-set of experiment. To induce anaemia, two distinct methods were employed simultaneously. Firstly, all the se animals were fed on 500 ml of Milk per day through feeding bottles. This diet was maintained for 3 weeks in a row - with a liberal access to water ad libitum. Secondly, a controlled bloodletting was done by a puncture of retro-orbital plexus at a rate of 1 ml per day per rat. To ensure the induction of anaemia, weekly estimations of Hb content was carried out on the withdrawn blood sample.
By this method, a 50% drop was seen in normal value of 13.88 g/dL at the end of three weeks in almost all the animals. After this phase, the anaemic animals were divided at random into 8 groups consisting of 20 animals each.
The first group was assigned to administration of distilled water at a dose of 1 ml per day for 21 days after induction of anaemia. The second group received treatment with Ferrous sulfate syrup at a dose of 0.9 gm/100 g of b.w. in two divided doses for 21 days.
Animals in rest of six groups received one of the six samples of Lauhasava at a dose of 0.27 ml/100 g b.w. in two divided doses for 21 days after induction of anaemia.
For the purposes of dietary regimen, each of these 8 groups was subdivided into two subgroups consisting of 10 animals each. The first of these subgroups received a standard rat
pellet diet and water ad libitum throughout the treatment period of 21 days. The second subgroup continued to receive an Iron deficient diet comprising of 500ml milk per day, fed through feeding bottle.
Blood samples were collected from animals in different treatment groups on day 0, 3, 7, 14 and 21 to monitor the progress in Hb levels. The % age increase in Hb, RBC counts and other haematological parameters were arrived at and the results were compared to by using Student's paired and unpaired 't' tests.
Results: After induction of anaemia, the basal level of Hb% dropped from 11.2 - 16.1 gm % to 4.4 - 9.9 gm. The following pointers review the results after initiation of post anaemic treatment.
♦ I subgroup "A" of animals which received normal diet along with assigned treatment, there was no change in Hb% day 3, post therapy. This was uniform observation across all treatment groups.
♦ In group 1 a, which received distilled water as its medication and a normal diet, the Hb5 was not restored to normalcy as on day 14th. However, on day 21st, there was a near normalcy in this group.
♦ In group 2 a, which received Ferrous sulfate treatment the Hb% was seen to be on rise from day 7th onwards. All the Lauhasava samples were also noted to improve the blood picture right from day - 7th. However, the best kind of haematological response was seen in the animals, which were assigned to Sample - F. The Hb% improved in this particular group improved consistently well from day 7th onwards.
♦ Such results were uniformly consistent with reference to all other haematological parameters.
♦ Sample 'C was found to be least active in this particular subgroup.
♦ The animals in subgroup B of different treatment groups received only iron deficient diet during post anaemic treatment period. It is expected that, the recovery from anaemia will be somewhat, slow in relation to subgroup - A.
♦ The Hb% was seen to improve in the animals receiving ferrous sulfate or Lauhasava samples from day 14th onwards, excepting the animals receiving treatment with sample F. On day 21st, it was noted that, treatment with Lauhasava samples marked as A, B & D was even better to treatment.with ferrous sulfate.
♦ Going by these results, Sample prepared by traditional method does not have a value in absence of normal dietary iron - while it is an effective formulation to treat experimental anaemia.
The overall impression is that, Sample 'A1, Sample 'B1, Sample T)1 and the Sample 'F are more effective than the rest of the two.

WE CLAIM:
1) A novel yeast strain having accession No DRF-UDS 004/wf and having the
following characteristics:
a) being sugar resistant,
b) capable of producing alcohol to the extent of about 7 to llv/v% continuously and consistently in a herbal extraction medium,
c) capable of overcoming resistance conferred by the herbal and spicy ingredients present in the medium,
d) capable of attaining biomass of about 1 to 4g/l, and
e) exhibiting the following bio-chemical properties:
i) urease test - positive,
ii) utilization of fructose test - negative,
iii) starch hydrolysis test - negative,
iv) xylose hydrolysis test - negative.
2) A yeast strain as claimed in claim 1 wherein the biomass attainable by the strain is about 1.7g/l, preferably 1.73 g/1.
3) A yeast strain as claimed in claim 1 wherein the alcohol produced by the strain is about 9v/v%, preferably 9.580 v/v%.
4) A novel yeast strain having accession No DRF-UDS 016/wf and having the following characteristics:

a) being sugar resistant,
b) capable of producing alcohol to the extent of about 7 to 11.5 v/v% continuously and consistently in a herbal extraction medium,
c) overcoming resistance conferred by the herbal and spicy ingredients in the medium,
d) capable of attaining biomass of about 1 to 4g/l, and
e) exhibiting the following bio-chemical properties:
i) urease test - negative,
ii) utilization of fructose test - positive,
iii) starch hydrolysis test - positive, xylose hydrolysis test - positive.
5) A yeast strain as claimed in claim 4 wherein the biomass attainable by the strain is about 1.5g/l, preferably 1.58 g/1.
6) A yeast strain as claimed in claim 4 wherein the alcohol produced by the strain is about 12 v/v%, preferably about 12.340 v/v%.
7) A novel yeast strain having accession No DRF-UDS 017/wf, and having the following characteristics:

a) being sugar resistant,
b) capable of producing alcohol to the extent of about 7 to 12 v/v% continuously and consistently in a herbal extraction medium,
c) overcoming resistance conferred by the herbal and spicy ingredients in the medium,
d) capable of attaining biomass of about 1 to 3.5 g/1, and
e) exhibiting the following bio-chemical properties:
i) urease test - negative,
ii) utilization of fructose test-positive,
iii) starch hydrolysis test - positive,
iv) xylose hydrolysis test - negative.
8) A yeast strain as claimed in claim 7 wherein the biomass attainable by the strain is about 1.6 g/1, preferably 1.65 g/1.
9) A yeast strain as claimed 7 wherein the alcohol produced by the strain is about 10.5 v/v%, preferably 10.520 v/v%.
10) A method for preparing yeast extract comprising the steps of:

a) preparing an aqueous medium comprising 10 to 40% sucrose derived from jaggery and minor ingredients such as hereindescribed,
b) inoculating the medium with a yeast comprising a strain as claimed in any of claims 1 to 3,
c) incubating the culture under aerobic conditions at a temperature in the range of 20-3 7°C for a period of 24 to 72 hours, and
d) maintaining the culture to obtain a liquid containing 5 to 30 mg of yeast biomass per ml.
11) A method as claimed in claim 10 wherein the yeast strains DRF-UDS 004/wf, DRF-UDS 017/wf and DRF-UDS 016/wf are isolated from Woodfordia fruticosa and Mudhuca latifolia.
12) A method as claimed in claim 10 wherein the minor ingredients are buffering agents, stabilizing and emulsifying agents.
13) A method as claimed in claim 10 wherein the culture is incubated at 25 to 32°C.
14) A method as claimed in claim 10 wherein the medium of step (a) is optionally sterilized at 100 to 130°C at a pressure of 15pi.
15) A method as claimed in claim 10 wherein the aerobic conditions comprise continuous aeration or injecting prefiltered air.
16) A method as claimed in claim 10 wherein the medium of step (a) contains cell destiny of yeast cultures ranging from 1 x 106 cfu per ml to 1 x 1010 cfu per ml after 48 hours of incubation.
17) Yeast strains bearing accession numbers DRF-UDS-004/wf, DRF-UDS-016/wf and DRF-UDS-017/wf substantially as hereindescribed and illustrated with reference to the examples.
18) A method for propagation of yeast substantially as hereindescribed and illustrated