Claims:Claims:
1. The present technology was described the effective and cost cutting single step process of extraction and purification of Methyl-ß-orsellinate from lichen Rocella montagnei.
2. As claimed in claim 1, collection of Rocella montagnei was done from hilly area using sterile polyethylene bag and was stored at -80ºC.
3. As claimed in claim 2, collected sample was subjected to three rounds of cold extraction using methanol as solvents. Then crude extract was obtained using rotary evaporator (Superfit, India).
4. As claimed in claim 3, crude extract was subjected to colum chromatography using solica gel (60-120 mesh). The fractions were obtained using hexane and ethyl acetate as an successive replacement of hexane by ethyl acetate by 2.5%.
5. As claimed in claim 4, the fractions were analysed using TLC using 90% chloroform and 10% methanol. The plate was observed and it was found that the first group was a pure compound after hexane wash.
6. As claimed in claim 5, the pure compound was characetrized and analysed as Methyl-ß-orsellinate. Further, the pure compound was showed good antibacterial activity against clinical pathogens (E. Coli, K.pnemoniae, Proteus vulgaris and Staphylococcus epidermidis). , Description:Description of the present patent work
Objectives:
• Extraction of lichen Rocella montagnei compounds using methanol and by adopting cold extraction procedure
• Isolation and purification of Methyl-ß-orsellinate from the lichen Rocella montagnei through a column chromatographic set up.
• Recrystallization of the fractions containing the compound methyl orsellinate to get a pure crystal.
• Confirmation of purity of the isolated compound through Thin Layer Chromatography (TLC), NMR and GC-MS.
Background of prior art:
A variety of chromatographic procedures have been followed to purify the valuable secondary metabolites of lichens. Out of the various methodologies adopted, column chromatography proves to be an effective method to isolate the secondary metabolites of lichens in bulk quantities. Among the secondary metabolites, phenolic compounds have major roles in several allelopathic processes, protection of plants against several herbivores and also in the response of plants to various environmental changes. Various compounds of the lichen can be isolated by using different solvent systems. The type and quantity of the compound to be isolated will depend on the type of solvent system used. This is because of the principle of polarity-based separation. So, optimising the solvent system is very important and this is species specific too. In this study, an approach has been made to optimise the solvent system for Rocella montagnei lichen. Usually the solvent system may include one solvent of desired polarity or may be a series of solvents ranging from low polar to high polar. Here, two solvent systems were adopted in a sequential manner to isolate the compounds from the crude extract of the lichen under study. One system was Hexane : Ethyl acetate and the other system was Chloroform : Methanol. By varying the concentration of each of the solvent, the polarity can be varied step-by-step so that all the compounds of the crude extract will be extracted, each in one particular polarity.
Advantage over conventional methods:
A wide range of solvent systems and chromatographic techniques have been used previously for the purification of the compound in this study. But, all the techniques had one or more disadvantages which also had a problem of the production cost.
Table 1. Purification of methyl orselllinate by conventional methods
Conventional Method Disadvantage
C18-functionalized silica gel flash column chromatography with Methanol and water as solvent system Very less yield of 0.031% of thalli and 0.28% of crude extract
n-Hexane and Acetone solvent system Rechromatography has to be done to get the desired compound
Dichloromethane and ethyl acetate solvent system Less yield of 0.014% of thalli and 0.26% of crude extract and also an additional alcoholysis step
Two-directional thin layer chromatography (TLC) with the solvent systems benzene-dioxane-acetic acid 90:24:4 (1st direction) and hexane-ether-formic acid (2nd direction) Only traces of the compound were obtained
Series of chemical modifications of Methyl-3-orsinol Tedious process involving lots of chemicals

The solvent system used in the present study is very effective in the isolation of Methyl-ß-orsellinate in two aspects, which are as follows
• A high yield of the compound is obtained
• The desired compound is obtained directly in the first step without the need of any further modification
Summary of the invention:
The cold extraction procedure was followed to extract the compounds from Rocella montagnei lichen. About 21 g of the lichen thalli were coarse powdered. Then it was subjected to cold extraction procedure by using 150 ml of methanol for 1 cycle. 1stcycle was left for 3 days at 100 rpm under room temperature. About 3 cycles were performed to extract all the compounds from the lichen followed by use of solvents such as acetone and water to confirm that the lichen is now devoid of valuable compounds.
All the three methanol cycle extraction were mixed and was subjected to a preliminary Thin Layer Chromatography procedure to optimise the solvent systems. Hexane & Ethyl acetate and Chloroform & Methanol were found to be the optimised solvent systems for performing column chromatography of the lichen compounds for further purification. The methanolic extract was subjected to rotary evaporation to dry the extracted compound mixture which weighed about 5 g. The dried powder was mixed with silica gel (60-120 mesh) and kept for a couple of days. Before loading into the column, this mix was heated at 90º C for 10 minutes. The dry column was prepared such that the ratio of the extract to that of silica gel is 1:10. First, the column is given a hexane wash to remove the impurities. The polarity of the solvent system is gradually increasedon successive replacement of hexane by ethyl acetate by 2.5%.
The solvent ratio was as follows: 100% hexane, 97.5% hexane+2.5% ethyl acetate, 95% hexane+5% ethyl acetate and so on until 100% ethyl acetate was reached. Now, when about 23 fractions were collected, the solvent was changed to 100% chloroform and the polarity was increased by adding methanol until the system contained 20% methanol. A total of 33 fractions were obtained using the optimized using the two different mentioned solvent systems. Finally, the column was washed with 100% methanol. All the collected fractions were allowed to evaporate and the fractions which were left with similar appearing crystals were grouped. Finally, 7 groups were clubbed and finalised for further purification using TLC and repeated solvent washes. The 7 groups are as follows:
Group 1: Fractions 4 to 8
Group 2: Fractions 23 & around
Group 3: Fractions 24 & 25
Group 4: Fraction 26 & around
Group 5: Fraction 33 & around
Group 6: Initial 100% methanol fraction
Group 7: Final 100% methanol fraction
All the 7 fractions were subjected to Thin Layer Chromatography. Acetone and hexane systems were not suitable as they couldn’t separate the compounds in each group. Polarity was increased and TLC was performed using 90% chloroform and 10% methanol. The plate was observed and it was found that the first group was a pure compound after hexane wash. The purified compound of Methyl-ß-orsellinate was identified using standard compound. Groups 3, 4, 5 & 6 were mixed as they had similar multiple spots and made to run along with group 2. All the showed multiple spots with similar ones among them. Groups 2, 3 & 4 and groups 5 & 6 were mixed together and were subjected to rechromatography procedure. Antimicrobial activity was observed for purified Methyl-ß-orsellinate against clinical pathogens and was found to be more significant level compared to standard drug.

Inventive step:
The red-circled step of the above flowchart is the inventive step in this study. The use of a novel solvent system, Hexane:Ethyl acetate and Chloroform:Methanol facilitated the high yield of Methyl-ß-orsellinate.
Results:
The usage of a novel system of solvents, Hexane:Ethyl acetate and Chloroform:Methanol in column chromatography for purifying the compounds of Rocella montagnei lichen produced a very high yield of Methyl-ß-orsellinate compound which is 8.36% of the dry thalli weight and 35.1% of the crude extract obtained. The percentage yield of the pure compound by the present method is very significant and effective cost cutting technology.
Table 2. Purification and yield % of methyl orsellinate from different lichen species
Technique followed Lichen species Initial amount taken (g) Yield obtained (%)
Lichen thalli Crude extract lichen thalli crude extract
Hexane and ethyl acetate as solvent system Rocella montagnei 21 5 8.36 35.1
Methanol and water as solvent system Umbilicaria antarctica 10 1.1 0.031 0.28
Dichloromethane and ethyl acetate solvent system Pseudo cyphellarianudata 118 6.08 0.014 0.26

Table 3. Antimicrobial activity of purified methyl orsellinate from R. montagnei
Name of the Bacteria / Fungi Crude methanolic extract of R. montagnei lichen
Zone of inhibition (mm±SE) Fraction subjected to Initial purification
Zone of inhibition (mm±SE) Final purified compound (Methyl orsellinate)
Zone of inhibition (mm±SE) Chlorom-phenicol (30mcg)
(mm±SE) Nystatin (30mcg)
(mm±SE)
For 50 µg For 100 µg For 50 µg For 100 µg For 50 µg For 100 µg
Bacteria
Escherichia coli 12.2±0.072 16.1±0.05 7.1±0.061 15.7±0.06 -ND- 13±0.145 26±0.21
Klebsiella pneumoniae -ND- 14.2±0.12 -ND- 13.9±0.11 -ND- 13.4±0.18 20±0.13
Proteus vulgaris -ND- 13.9±0.14 -ND- 14.5±0.05 -ND- 15.3±0.05 20±0.20
Staphylococcus epidermidis 10.3±0.1 23.4±0.08 11.8±0.13 10.7±0.02 14.1±0.141 -ND- 20±0.18
Fungi
Candida albicans 9.4±0.6 8.3±0.072 -ND- 6.8±0.077 -ND- -ND- 23±0.34
Candida krusei 8.6±0.8 18±0.068 -ND- 14.3±0.09 -ND- 8.2±0.079 21±0.41