FIELD OF THE INVENTION
The present invention relates to Amla (Phyllanthus emblica) composition. More particularly, to dried Amla with high Vitamin-C content.
BACKGROUND OF THE INVENTION
Amla, also called as Indian gooseberry (Phyllanthus emblica), is a tree belonging to the Euphorbiaceae family, native to the Indian coastal areas, and plains. Its edible fruit is extensively used in both dried and fresh forms in ancient Ayurvedic medicine. The myriad beneficial health effects, such as the promotion of longevity, strengthening the lungs, fighting chronic lung problems as well as upper respiratory infections are attributed to this fruit.
Amla is one of the most frequently used ingredients in Ayurveda, the ancient Indian system of medicine, for the treatment of several disorders such as common cold, scurvy, cancer and heart diseases. It is believed that the major constituent in Amla which is responsible for these beneficial effects is Vitamin-C. Vitamin-C, also referred to as L-Ascorbic Acid, is an important nutrient for humans. Although it is mainly prevalent in the form of L-Ascorbic Acid, other forms, such as, L-xylo-Ascorbic Acid or L-threo-hex-2-enoic acid y-lactone are also found. Humans cannot synthesize Vitamin-C in their bodies and have to obtain it from natural sources, such as fruits and vegetables. Vitamin-C is not only an antioxidant, anti-inflammatory and anti-mutant, but also a very effective free-radical scavenger.
Vitamin-C possesses antioxidant property, as it is an electron donor and a reducing agent. “By donating its electrons, it prevents other compounds from being oxidized. However, by the very nature of this reaction, Vitamin-C itself is oxidized in the process.” Padayatty S J, et al., “Vitamin-C as an antioxidant: evaluation of its role in disease prevention,” J Am Coll Nutr. 2003 February; 22(1):18-35, 19. Researchers in the last few decades have established the beneficial role of Amla extract as a biological antioxidant. Its high antioxidant power is attributed to the presence of Vitamin-C.
The stability of Vitamin-C varies markedly as a function of external factors, such as pH, heat etc. Vitamin-C is an unstable compound which decomposes easily under extreme conditions (Fennema, 1977; Lee & Coates, 199). Usually, Vitamin-C present in Amla gets oxidised during the drying process.
Degradation of Vitamin-C proceeds via both aerobic and anaerobic pathways (Huelin, 1953; Johnson et al.; 1995) and depends upon several factors, such as oxygen, heat, light (Robertson & Samaniego, 1986), storage temperature and storage time (Fellers, 1988; Gordon & Samniego-Esguerra, 1990). In aqueous solution Vitamin-C is a strong reducing agent. By giving up two electrons, it is oxidized to dehydro Vitamin-C by molecular oxygen, in the presence of metal ions as catalyst. Dehydro Vitamin-C is further oxidized by molecular oxygen to compounds that are no longer physiologically active. The rate of aerobic oxidation is pH dependant and is accelerated under alkaline conditions, i.e., at high pH. The acid catalyzed anaerobic oxidation of Vitamin-C results in the formation of Furfural as the major product. However, degradative oxidation occurs slowly under anaerobic and non-aqueous conditions. Exposure of an aqueous solution of Vitamin-C to ultraviolet radiation results in photochemical oxidation under both aerobic and anaerobic conditions. Aerobic oxidation of Vitamin-C occurs mainly during the processing of citrus juices (Huelin, 1953), whereas anaerobic degradation occurs mainly during storage, (Johnson et al.; 1995; Lee & Nagy, 1988a; Solomon et al.; 1995) which is particularly observed in thermally preserved citrus juices. It was reported that several reactive products are formed on degradation of vitamin C (Eskin, 1990; Huelin, Coggiola, Sidhu & Kennett, 1971) which react with amino acids to form brown pigments (Clegg, 1964; Larisch et al.; 1998). Hence, there exists a need in the art to develop a unique process that prevents aerobic and anaerobic degradation of Vitamin-C during the processing and drying of Amla.
SUMMARY OF THE INVENTION
The present invention provides a process to prepare dry salted Amla with a high Vitamin-C content.
In accordance with an embodiment of the invention, there is provided a process for preparing a dry salted Amla, comprising the steps of: (a) chopping a Amla into a plurality of small pieces; (b) mixing vacuum salt with the small pieces to form a salted mixture; and (c) drying the salted mixture in the absence of sunlight to obtain the dry salted Amla.
In accordance with another embodiment of the invention, the salted mixture is dried at a temperature of < 40 °C.
In accordance with another embodiment of the invention, the percent loss in weight on drying of the dry salted amla is not more than 8.0% w/w.
In accordance with another embodiment of the invention, the dry salted Amla has a vitamin-C content of at least 0.7% w/w.

BRIEF DESCRIPTION OF DRAWINGS:
The invention can be described in the terms of the following figures where-
Figure 1A Mass spectrum of standard Vitamin-C solution showing main precursor ion at m/z 175.15 in the negative mode.
Figure 1B Daughter scan mass spectra of standard Vitamin-C solution showing major daughter ions at m/z 114.91 & 87.05.
Figure 2A Mass spectrum of Vitamin-C in sample (Batch 1) showing main precursor ion at m/z 175.15 in the negative mode.
Figure 2B Daughter scan mass spectrum of Vitamin-C in sample (Batch 1) showing major daughter ions at m/z 114.98 & 86.99.
Figure 3A Mass spectrum of Vitamin-C in sample (Batch 2) showing main precursor ion at m/z 175.15 in the negative mode.
Figure 3B Daughter scan mass spectrum of Vitamin-C in sample (Batch 2) showing major daughter ions at m/z 114.91 & 87.05.
Figure 4A Mass spectrum of Vitamin-C in sample (Batch 3) showing main precursor ion at m/z 175.15 in the negative mode.
Figure 4B Daughter scan mass spectra of Vitamin-C in sample (Batch 3) showing major daughter ions at m/z 114.91 & 87.05.
Figure 5A Mass spectra of sample (Batch 4) in the negative mode.
Figure 5B Daughter scan mass spectra of sample (Batch 4).
Figure 6A Mass spectra of sample (Batch 5) in the negative mode.
Figure 6B Daughter scan mass spectra of sample (Batch 5).
DETAILED DESCRIPTION OF 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 provides a dry salted Amla composition, which has higher Vitamin-C content compared to conventionally dried ones, i.e. the ones dried in the presence of sunlight. The present invention further discloses a process for preparing said composition.
The fresh green Amla are washed and chopped into small pieces. The small pieces of Amla are mixed with vacuum salt and dried at a temperature of < 40 °C in the absence of sunlight. Drying is continued till the percent loss on drying is not more than 8%.
The term “Vacuum salt” used herein refers to rock salt that has been refined. The rock salt is dissolved in clean water to form a salt solution. This brine is purified from contaminants with special chemicals and thereafter the water is evaporated under vacuum. The salt thus obtained is > 99.9% pure sodium chloride.
The present invention is more particularly described in the following non-limiting examples that are intended as illustrations only since numerous modifications and variations within the scope of the present invention will be apparent to a skilled artisan. 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 made available from the chemical suppliers.

Example 1.
Process for preparing dry salted Amla
14.00 kg of fresh green Amla were washed in fresh water. Further processing of washed Amla was carried out in the absence of sunlight. Amla was chopped with a stainless steel knife into small pieces having length, breadth and thickness of approximately 11 mm. The weight of Amla pieces, obtained after chopping, was 10.300 kg. 1.700 kg Vacuum salt, i.e. 17.50% by weight of Amla pieces, was mixed with the Amla pieces and the mixture was dried in a tray drier at a temperature in the range from 35 to 40 °C, in the absence of sunlight. Drying was continued till a percent loss on drying of not more than 8% was achieved. The dry salted Amla thus obtained were packed in a controlled environment (< 25% relative humidity) in air tight containers/packages.

Determination of Vitamin-C content in dried salted Amla:
A rapid simple and sensitive method based on liquid chromatography and tandem mass spectrometry (LC/MS/MS) with an electrospray ionization (ESI) source for the simultaneous analysis of fourteen water-soluble vitamins (B1, B2, two B3 vitamers, B5, five B6 vitamers, B8, B9, B12 and C) in various food matrices, i.e. maize flour, green and golden kiwi and tomato pulp, was reported in Gentili, et al., Rapid Commun Mass Spectrom, 2008, 22, 2029-43. Likewise, a HPLC method for detection of lipophilic Vitamin-C derivatives was reported in Tai, A. et al., J Chromatogr B, 2006, 840, 38-43. Using the HPLC method for determining the Vitamin-C content in Amla, afforded inaccurate results, due to the similar retention times of Vitamin-C and other compounds present in Amla. Hence, the present invention uses a combination of chromatographic techniques and mass spectroscopy to accurately quantify the Vitamin-C content in Amla.
As used herein, the term “chromatography” refers to the separation of a mixture of chemical entities. The mixture of chemical entities is carried over a stationary liquid or solid phase by a liquid or gaseous mobile phase. The mixture is separated into its component due to the preferential adsorption of a particular chemical entity over the stationary phase.
As used herein, the term “liquid chromatography” refers to the separation of a mixture of chemical entities dissolved in a liquid as the liquid moves through a column of finely divided stationary phase. The components dissolved in the liquid phase are separated due to the preferential adsorption of certain components over others, on the solid phase.
As used herein, the term “high performance liquid chromatography” or “HPLC” refers to liquid chromatography, wherein the separation of the different components of a mixture is increased by forcing the mobile phase under high pressure through a stationary phase.
Prior to mass spectrometry, it is advisable to enrich the Vitamin-C relative to other components in the sample. HPLC is used to enrich the sample in Vitamin-C. A person skilled in the art may select HPLC columns and instruments compatible with Vitamin-C. The chromatographic column consists of a stationary phase to facilitate the separation of the chemical entities. The stationary phase is bonded to alkyl groups, such as C-4, C-8, or C-18, where 4, 8 and 18 represent the number of carbon atoms in a saturated hydrocarbon chain. Such chemically bonded stationary phases are prepared by reacting an organosilane with silica gel support material in a suitable solvent. Solvent elution modalities, such as a gradient mode or an isocratic mode may be used. As used herein, the term “gradient mode” refers to eluting the components of mixture by using a mobile phase having constant composition. As used herein, the term “gradient mode” refers to eluting the components of a mixture, with a mobile phase having varying composition.
The sample is prepared by dissolving a powder, prepared by crushing the dried Amla composition, in an acetic acid solution. Such a sample is called a test solution. The concentration of acetic acid solution is 0.1%. The concentration of Vitamin-C in the test solution is compared with that of Vitamin-C in the standard solution. The standard solution is prepared by dissolving known quantity of reference standard Vitamin-C in acetic acid solution.
As used herein, the term “mass spectrometry” refers to techniques of detecting and identifying compounds based on their mass/charge (m/z) ratio. A mass spectrometer generally includes an ionizer and an ion detector. The analyte may be ionized to a positive ion or a negative ion. The analyte, i.e. Vitamin-C is ionized to a negatively charged ion. As used herein, the term “ionizing” refers to the process of generating an analyte ion having net charge. A negative ion is one having a net negative electric charge of one or more electron units. As used herein, the term “negative ion mode” refers to the mode of operating a mass spectrometer in which negative ions are detected.
As used herein, the term “electrospray ionization” or ESI refers to methods in which a solution, carrying an analyte, is passed through a capillary. When a high positive or negative potential is applied across the capillary, the solution is drawn into the shape of a cone. On reaching a certain threshold voltage limit, jets of fluid, in the form of small droplets, are emitted from this cone. This string of droplets is allowed to flow through an evaporation chamber, wherein solvent evaporation takes place, leading to a reduction in the size of the droplets. As the droplets become smaller and smaller, the repulsion between the like charges increases until such a point wherein they explosively release individual ions and neutral molecules. These charged ions help in identifying the analyte molecules based on their mass to charge ratio (m/z). Vitamin-C is ionized to negatively charged ion using ESI.
After the sample has been ionized, the negatively charged ions of Vitamin-C may be analyzed to determine their mass-to-charge ratio. The mass-to-charge ratio may be determined using quadrupole analyzers, ion traps analyzers, magnetic and electric sector analyzers and time-of-flight analyzers.
The resolution of mass spectrometry can be increased by using “tandem mass spectrometry or “ms/ms”. In “tandem mass spectroscopy”, the ion of interest, which is generally the precursor ion, is selected from among several interfering ions and is further fragmented into daughter ions by collision with an inert gas. The precursor ions gain kinetic energy by colliding with the inert gas and subsequently dissociate by a process referred to as “unimolecular decomposition”. It should be noted that sufficient energy must be imparted to the precursor ions so that the bonds in it break due to increase in vibrational energy. As used herein, the term “precursor ion” refers to the negative ion formed by ionizing the analyte, i.e. Vitamin-C.

As used herein, the term “multiple reaction mode” or MRM refers to a method for the quantification of small molecules. The method employs two stages of mass filtering on a triple quadrupole mass spectrometer (Q1, Q2 and Q3). In the first stage, a precursor ion is selected in Q1 and is fragmented by collision with an inert gas in Q2. In the second stage, instead of obtaining a full scan ms/ms, wherein all the fragments generated in Q2 are detected by Q3, only those fragments characteristic of the precursor ion are analyzed in Q3. Such a targeted analysis of only the fragment ions of interest enhances the lower detection limit for the analyte.

Example 2
Estimation of Vitamin-C in Amla
Experimental Set-up
LC-MS/MS was carried out using a Micromass Quattro Micro triple-quad mass spectrometer (Waters/Micromass, UK) with the Waters 2695 high-performance liquid chromatography (HPLC) system consisting of a quaternary pump with a vacuum degasser, a thermostated column compartment and an auto-sampler. Amla samples extracts were passed through an Inertsil C18 150 × 4.6 mm, 5µ column from GL Sciences INC. (Japan). The mobile phase consisted of 0.1% methanolic glacial acetic acid & 0.1% glacial acetic acid in water (80:20, v/v). Vitamin-C was eluted using an isocratic mobile phase with a constant flow rate of 1 ml/min and the total LC run time was 5 min. Splitter in ratio of 1:1 used to split the LC flow and resultant eluant was introduced to the ESI-MS/MS system. The autosampler and column oven temperature were maintained at 8 °C and 45 °C, respectively. The injection volume was 10 µl. Negative ion electrospray ionization (ESI) was used to form deprotonated molecules [M-H] - at m/z 174.94 for Vitamin-C. The ESI source was maintained at 120 °C and chromatograms were acquired in the Multiple reaction monitoring (MRM) mode for the negative ion. The MRM transition of m/z 174.94-114.90 for Vitamin-C at collision energy 12 ev, was selected to obtain maximum sensitivity. For quantification, the parent molecule was fragmented into the daughter ions by collision with argon gas. The capillary and cone voltages were set at –2.80 kV and –22 V, respectively. Nitrogen gas was used as desolvation gas at a flow rate of 750 L/h and the desolvation temperature at 350°C. The extractor and RF lens voltage were set at 1 and 0.2 V, respectively. For the analyzer, the LM resolution 1 and HM resolution 1 were 12 V and ion energy 1 was 0.8 V; the entrance was 50 V and the exit was 50 V; the LM resolution 2 and HM resolution 2 were 12 V and ion energy 2 was 2 V; the multiplier voltage was 650 V. MRM was used with a dwell time of 0.2 s.
Sample solution preparation: 0.25 g of crushed powder of dry Amla was taken in a 25 mL volumetric flask. 15 mL of 0.1% glacial acetic acid in water was added into the volumetric flask and agitated for 20 minutes on a mechanical shaker. The volume was made up to 25 mL with 0.1% glacial acetic acid and the resulting solution was used as sample solution.
Standard solution preparation: 10.0 mg of Vitamin-C was taken in a 100 mL volumetric flask. 50 mL of 0.1% glacial acetic acid in water was added into the volumetric flask and agitated on a mechanical shaker to dissolve the content. The volume was made up to 100 mL with 0.1% glacial acetic acid to form a stock solution. 2 mL of the stock solution was further diluted to 20 mL with 0.1% glacial acetic acid and the resulting solution was used as a standard solution.

Calculation: - Content of Vitamin-C (in % w/w)

% w/w=(Area of Sample)/(Area of Standard)×(Weight of Standard)/(Weight of Sample)×(Dilution of Sample)/(Dilution ofStandard)×Purity of Standard

Areas of standard & sample represent the peak area calculated from the LC-MS in MRM (Multiple reaction Monitoring) mode. MRM mode was used for the quantification of Vitamin C. Peak area is calculated by the LC-MS system software on the basis of the relative abundance or intensity of that particular ion verses time. In figures 1 to 6, 175.15 represents the m/z ratio of main precursor ion (Parent ion) obtained from the mass spectra of standard & sample. Whereas, main daughter ions having m/z ratio 114.91 & 87.05 were obtained by further fragmentation of parent ion in Daughter scan mode, which is commonly used for the further confirmation of the particular compound.

Three batches of dry salted Amla, i.e. Batch 1, Batch 2 and Batch 3 were prepared according to the process given in Example 1. The process provided in Example 1 was replicated thrice to prepare batches 1, 2 and 3. Batch 4 and 5 were commercially available dried Amla samples, Batch 6 was prepared according to the process given in Example 1 and exposed to a temperature of 45 °C at a relative humidity of 75% for a period of 3 months in order to determine the shelf life of the product during storage, and Batch 7 was unprocessed fresh Amla. All the seven batches were analyzed according to the process described in Example 2 and the results are presented in Table 1.
Table 1. Vitamin-C content in Amla (% w/w)
Batch Remarks Vitamin-C (% w/w)
1 Prepared in absence of sunlight according to the process of Example 1 0.82
2 Prepared in absence of sunlight according to the process of Example 1 1.15
3 Prepared in absence of sunlight according to the process of Example 1 1.17
4 Commercially available sample Below detection limit
5 Commercially available sample Below detection limit
6 Prepared in absence of sunlight according to the process of Example 1 and exposed to a temperature of 45 °C and relative humidity of 75% for a period of 3 months 0.70
7 Unprocessed fresh Amla 0.61

While particular embodiments of the present invention are 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.

CLAIMS:
We Claim:
1. A process for preparing a dry salted Amla, the process comprising the steps of:
(a) chopping a Amla into a plurality of small pieces;
(b) mixing vacuum salt with the small pieces to form a salted mixture; and
(c) drying the salted mixture in the absence of sunlight to obtain the dry salted Amla.
2. The process for preparing dry salted Amla as claimed in claim 1, wherein the salted mixture is dried at a temperature of < 40 °C.
3. The process for preparing dry salted Amla as claimed in claims 1 and 2, wherein the percent loss in weight on drying of the dry salted Amla is not more than 8.0% w/w.
4. The process for preparing dry salted Amla as claimed in claims 1 to 3, wherein the dry salted Amla has a vitamin-C content of at least 0.7% w/w.
5. A dry salted Amla prepared by a process as claimed in claims 1 to 4.