FORM 2
THE PATENT ACT 1970
(39 OF 1970)
&
The patents rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rale 13)
1. TITLE OF THE INVENTION-
Novel process to extract Lupeol and Saponin from selected plants
2. APPLICANT (S):
a. Name : Prof Dr. Deokule Subhash S.
b. Nationality : Indian
c. Address : Department of Botany, Savitribai Phule Pune University,
Ganeshkhind Road, Pane-411007, Maharashtra, India
a. Name : Dr. Joseph Tessy
b. Nationality : Indian
c. Address : Department of Botany, Savitribai Phule Pune University,
Ganeshkhind Road, Pune-411007, Maharashtra, India
a. Name : Dr. Mokat Digambar N.
b. Nationality : Indian
c. Address : Department of Botany, Savitribai Phule Pune University,
Ganeshkhind Road, Pune-411007, Maharashtra. India
a. Name : Ms. Raut Sharayu G.
b. Nationality : Indian
c. Address : Flat no. 701, Heramb Residency apartment, Behind Ekalavya
College, Bhujbal township, Kothrud, Pune - 411 038, Maharashtra, India.
a. Name : Dr. Jagtap Surest D.
b. Nationality : Indian
c. Address : Herbal Medicine
Interactive Research School for Health Affairs (IRSHA),
Bharati Vidyapeeth University, Pune Satara Road, Pune - 411 043.
Maharashtra, India.

3. PREAMBLE TO THE DESCRIPTION

PROVISIONAL COMPLETE
NOT APPLICABLE The following specification describes the invention.
4. Description
Field of the Invention
This invention relates to a process to enhance the yield of Lupol and Saponin having anti-dysenteric activity from Barringtonia actuangula (L.) Gaesta. Naregamia alata W & Arn. Aspect of the invention is through test for Saponin, High Performance Thin Layer Chromatography (HPTLC), and High Performance Liquid Chromatography (HPLC).
Background of the invention:
Barringtonia acutangula (L.) Gaertn.:
It is known as Indian oak This comes under the family Baningttoniaceae It is seen in deciduous and evergreen forest, of India mostly along the banks of rivers, streams. This plant is seen in Kasargode, Kottayam, Thrissur, Kollam, Alappuzha, Palakkad and Malapuram Districts of Kerala (Sasidharan, 2004). This is a medium sized glabrous tree 10-15m in height with pale grey slender young branches with rough dark simple and alternate leaves and bears beautiful flowers (Pandey & Chadha, 1996).
The juice of the leaves is a remedy for diarrhoea. The baric has medicinal properties and it is used as an astringent. It is administered during diarrhoea and blennorrhoea and as a febrifuge in malaria (Jain, 1991).
Pal et al. (1991 & 1994) reported the presence of tritefpenoid glucoside and saponins in
Barringtonia acutangula. Other constituent reported from the plants are, barringtonic acid,
Acutangulosides A-F, Monodesmosidk Saponins, Mine triterpene saponins, acutanguloside A-F,
acutanguloside D-F methyl esters (5a-7a), single triterpene aglycone (1), three monodesmosidic
glucuronide saponins of barringtogenol C namely barringtosides A, B and C, barringtoside A, 3-o-
beta-D-xylopyranosyi (l=>3)-[beta-D-galactopyranosyl (l->2)]-beta-D-glucuronopyranosyl
barringtogenol C; brringtoside B, 3-o-beta-D-xylopranosy (1-3)-] beta-D-galactopyranosyl (l->2)]-
b'eta-D- glucuronopyranosyl barringtogenol C; barringtoside B, 3-o-beta-D-xylopyranosyl (l->3)-]
beta-D-galactopyranosyl (l->2)]-beta-D-glucuronopyranosyl-21-o-tigloyl-28-o-isobutyryl
barringtogenol C; barringtoside C, 3-o-alpha-L-arabinopyranosy (l->3)-[beta- D-galactopyranosyl (1->2)]-beta-D-glucuronopyranosyl barringtogenol C. Saponin present in plant is responsible for anti-diarrhoeal activity (Anantaraman and Pillai, 1956; Barua and Chakraborti, 1964, 1965; Barua and Dutta, 1968; Barua et al, 1972, 1963; Braun and Mouchacea, 2000; Chakraborti and Barua, 1963, Mills et al, 1994).
Naregamia alata W, & Arn,:
It belongs to family Meliaceae. The plant is a small, under shrub upto 30 cm in height. This is the monotypic genus native of India. It is found to be growing in Western Ghats, from north to south ascending up to 900m. It is commonly found throughout South India in all districts of Kerala. It is endemic to peninsula India (Hooker,, 1872)..
The whole plant is medicinally important It is-acrid, sweet, cooling, aromatic, alexeteric, vulnerary, emetic, cholagogue, expectorant, depurative and antipyretic. The root contains an alkaloid called naregamia, wax, gum, asparagines, starch etc It is pungent and aromatic but not having any characteristic taste. The plant-is being used in_indigenous. System_of.medicine.in_the_treatment of

various ailments such as in acute dysentery, wounds, ulcers, vitiate conditions of pitta and vata, halitosis, cough, asthma, bronchitis, splenomegaly, scabies, purities, dyspepsia, catarrh, anaemia, malarial fevers, rheumatism and skin itching (Nadkarni, 1927, Jain, 1991). Decoction of the stem and leaves has been used in treatment of dysentery. Naregamia has recently been tried in Madras to treat acute dysentery and also as an emetic and expectorant. Roots are used as emetic, cholagogue, expectorant, and in the treatment of acute dysentery and chronic bronchitis as reported by Dymock et al. (1890), Ambasta (1986) and Agarwal (1997). Naregamia is a wonderful drag in the case of dysentery, treated by a decoction of the bark in rice water. Lupol and Saponin present in plants are having anti-dysentric activity from Barringioma acutangula and Naregamia alaia.
Saponin:
Saponins are a class of chemical compounds found in particular abundance in various plant species. More specifically, they are amphipathic glycosides grouped phenomenologically by the soap¬like foaming they produce when shaken in aqueous solutions, and structurally by having one or more hydrophilic glycoside moieties combined with a lipophilic triterpese derivative. Saponins have historically been understood to be plant-derived, but they have also been isolated from marine organisms such as sea cucumber. Saponins are indeed found in many plants. Most saponins, which readily dissolve in water, are poisonous to fish. Therefore, in ethnobotany, they are primarily known for their use by indigenous people in obtaining aquatic food sources.
Lupeol:
Lupeoi is a pharmacologically active triterpenoidL It has several potential medicinal properties. Lupeol is found in a variety of plants, including mango, Acacia visco mdAbrtmia vittosa. It is also found in dandelion coffee. Lupeol is produced by several organisms from squalene epoxide. Dammarane and baccharane skeletons are formed as intermediates The reactions are catalyzed by the enzyme lupeol synthase. Lupeol has a complex pharmacology, displaying antiprotozoal, antimicrobial, anti-inflammatory, w&ifajmm and chemppreventive properties.
Summary of the invention:
This invention relates to a new process to enhance the yield of Lupol and Saponin from Barringtonia acutemgula and Naregamia alaia. Aspect of the invention is through test for Saponin, High Performance Thin Layer Chromatography (HPTLC).
In phytochemistry, plants were analyzed by qualitative tests for the presence of saponin. The phytochemical investigations were fiirther supported by HPTLC analysis using solvent system consisted of toluene: acetone : acetic acid (GAA) in ratio of 8.9 : 0.9 : 0.2. The active principle lupeol appeared as blue band on visual observation after derivatization in the HPTLC analysis at an Rf value of 0.42. Quantitative analysis was achieved using standard lupeoi The amount of lupeoi present in Barrintonia acutangula 0.05% and Naregamia alaia lupeol is absent.
In case of saponin the solvent system used was ethylacetate: etfaanol (96%) : water : ammonia (25%) in ratio (6.5 : 2.5 ; 9 ; 1) respectively. The standard saponin was spotted 0.1 pg/pl. The derivatization was carried out by using anisaldehyde -sulfuric acid reagent and later heating it at 100°C for 10 minutes. Active principle saponin appeared in bluish band on visual observation after derivatization in the HPTLC analysis at an Rf value 0.06. Quantitative analysis was achieved using standard saponin.

Detailed description of the figures:
Figure 1: Banintonia acutangula Figure 2: Naregamia aiaia Figure 3: HPTLC of Saponin Figure 4: Saponin- Standard Figure 5: Saponin 3D Image Figure 6: HPTLC of Lupeo! Figure 7: Lupeol - Standard Figure 8: Lupeo! 3D Image
Detailed description of the invention:
Detailed experimental studies
The leaves of Barringtonia acutangula (Figure 1) and Naregamia alata (Figure 2) collected from different parts of Kerala, India and identified by BSI of India western circle. Voucher specimens have been deposited in the Botanical Survey of India (BSI) Western Circle Pune, India, and also in the department of Botany Umversity of Pune. The leaves were shade dried and pulverized. The pulverized plant materials were extracted with ethanol. These extracts were initially screened for antidysenteric.
Test for saponins
Sections were placed directly in 1 drop of concentrated H2SO4 on a slide, which gives a characteristic sequence of colour reactions, beginning immediately with yellow, changing to red within 30 minutes and finally becoming violet or blue green in a short time.
To determine localization of the saponin, sections were put in saturated Barium hydroxide
solution for about 24 hrs- Sections ware washed with calcium chloride then placed in Potassium dichromate. Yellow colour indicated the presence of saponins.
High performance thin layer chromatography (HPTLC);
HPTLC technique was followed for the qualitative analysis and the confirmation of chemicals present
in the studied plants.
HPTLC is a versatile separation technique included various steps as given below:
1) Selection of HPTLC plates and adsorbent
2) Sample preparation
3) Application of sample
4) Development (separation)
5) Detection including post-chromatographic derivatization
6) Quantification
7) Documentation
1) Selection of HPTLC! plates and adsorbent:
For preparative TLC work, plates with adsorbent thickness of 1.0 - 2.0 mm are available in addition to chemically modified layers- Aluminum sheet (0.1 mm thick) sheets as support offer the same advantage as polyester support but with increased temperature resistance. However, with eluent containing high concentration of mineral acids or concentrated ammonia one may find problem, as they will chemically attack aluminum. Aluminum sheets are otherwise compatible with organic 'Solvents and organic acids such as formic acid and acetic acid.

Plate size: The pre-coated TLC/ HPTLC plates in size of 20 x 20 cm with aluminum or polyester support are procured mainly for economic reasons. Before handling the pre-coated plates for any experimental work, it is important to note direction of the application of adsorbent as chromatographic developments have to be performed in that direction only. Pre-washing of pre-coated plates with large surface area absorb not only water vapours and the impurities from atmosphere but other volatile substances often condense particularly after the packing has been opened and exposed to laboratory. To avoid any possible interference, due to impurities with the chromatographic separation particularly in case of quantitative work, pre-washing of plates were done".
Activation of pre-coated plates: Plates exposed to high humidity or kept on hand for long time may have to be activated by placing in oven at 110- 120° C for 30 minutes prior to sample spotting.
2) Sample Preparation:
Dissolved dosage form with complete recovery of intact compound(s) of interest and minimum of matrix with a suitable concentration of analyie(s) for direct application on the HPTLC plate. Besides, maximizing the yield of analyte(s) in the selected solvent stability of analytes during extraction and analysis must be considered and ensured. Therefore, the choice of a sellable solvent for a given analysis is very important. For normal phase chromatography using silica gel pre-coated plates (more than 80-90% HPTLC analysis is done using silica gel as adsorbent) solvent for dissolving the sample should be non-polar and volatile as far as possible. It is preferable to keep the solvent as simple as possible and quantity employed is limited to ensure complete extraction of analytes and minimum of extraneous components. Sample and reference substances should be dissolved in the same solvent to ensure comparable distribution at starting zones,
3) Application of sample:
Applied 1-10 pi volume for TLC and 0.5-5 for HPTLC keeping the size of starting zone(s) down to minimum; 2-4 mm (TLC) and 0.5-1 mm (HPTLC) in the concentration range of 0.1 -1 µg/µl for TLC/ HPTLC. However, volume and concentration primarily depend on the component under analysis and their sensitivity to various detection techniques.
4) Development (Mobile phase):
Mobile phase was selected by taking into consideration chemical properties of analytes and the adsorbent layer. Pre-conditioning (Chamber saturation):
Drying cupboard or hot plates are employed. Hot pistes with regulated range of temperature i.e. 50-190° C + 2° C are extensively being employed for heating the chromatogram.
5) Detection and visualization:
As soon as the development process is complete the plate is removed from the chamber and dried to remove the mobile phase completely. The zones can be located by various physical chemical biological-physiological methods. There is apparently no difficulty in detecting colored substances or colorless substances in short wave ultra-violet (UV) regioa (254 nm) or with intrinsic fluorescence such as riboflavin quinine sulphate.

6) Quantitative:
Spraying and dipping techniques are used for applying detection reagents. However, ir addition to other reasons as enumerated below dipping followed by evaporation Is essential both for precision and repeatability in ultimate quantitative analysis.
7) Documentation:
The use of application scheme and labeling every single chromatogram can avoid mistake ir respect of order of application. It is preferable to apply each sample and reference solution twice by following data - pair method. A lead pencil can be used to write on the chromatoplate. The plate should never be marked below the starting point, as layer is likely to get damaged affecting chromatographic distribution of the substances under analysis which may ultimately lead to error in scanning. The best way to label the chromatoplate is to mark above the level of solvent point, immediately after development Is completed, the solvent point should be marked both on left and right hand edges of the plate, this facilitated calculation of Rf values. The practice of cutting a scratch across the whole layer is no longer In use. The type of plate, chamber system, composition of mobile phase, running time and detection method should all be recorded. HPTLC protocol format given in the text may be adopted for recording all the relevant data.
Results:
Phytochemical test:
In phytochemistry, plants were analyzed by qualitative tests for the presence of various chemical constituents saponin. The phytochemicai Investigations were further supported by HPTLC analysis.
High Performance Thin Layer Chromatography (HPTLC):
In the case of lupeol analysis along with the mehanolic extract, standard lupeol was also spotted 0.1 µg/µl (0..µg/µl on the plate conc). The solvent system consisted of toluene: acetone : acetic acid (GAA) in ratio of 8.9 : 0.8 : 0.3. Derivatization was carried out by dipping the plate in anisaldehyde -sulfuric acid reagent and later heating it at 100°C for 10 minutes. The developed plate scanned at 580 nm using TLC scanner 3 and associate integration software (wincais 1.4.2 version). In case of saponin the solvent system used was ethylacetate : ethanol (96%) ; water: ammonia (25%) in ratio (6.8 : 2.7 : 9 : 1) respectively. The standard saponin was spotted 0.1 µg/µl. The derivatization was carried out by using anisaldehyde -sulfuric acid reagent and later heating it at 100°C for 10 minutes (Wagner and Bladt, 1996). The developed plates were scanned at 540nm using TLC scanner 3 and associate integration software (wincats 1.4.2 version).
Analytical studies ( Lupeol):
(Figure: 3, 4 and 5)
The active principle hipeol appeared as blue band on visual observation after derivatization in the HPTLC analysis at an Rf value of 0.42. Quantitative analysis was achieved using standard lupeol. The amount of lupeol present in Barrintonia acutangula 0.05% and Naregamia alata lupeol is absent.
Analytical studies { Saponin):
(Figure: 6,7 and 8)
The active principle saponin appeared in bluish band on visual observation after derivatization in the HPTLC analysis at an Rf value 0.06. Quantitative analysis was achieved using standard saponin. The amount of Saponin present m Barrintoma acutangula 0.4 % and amount of saponin present in Naregamia alata 1.20 % on dry wait basis respectively.

References:
Agarwal (1997), Drag plants of India (Vol. II), Kalyani Publishers: 79,517.
Ambasta (1986), The useful Plants of India. Publication and Information Directorate, Council of
Scientific and Industrial Research, New Delhi: 67-68,105, 176,178,193,393. Anaantaraman and Pillai (1956), Barringtogenol and Barringtogenol acid, two new triterpenoids
sapongenins from B. racemose. J.Chem.Soc. 4369-73. Barua and Chakraborti (1964), Triterpenoid XVffl. The constitution of Barringtogenol C;. Sci. and
Cul 30(7): 332-34. Barua and Chakraborti (1965), Triterpenoid XIV. The constitution of Barringtogenol C;-a new
Triterpenoid Sapogenin from Barringtonia acutangula Gaertn Tetrahedron, 21: 381-87. Barua and Dutta (1968), Treterpenoid-XXIX. The structureof Barringtogenol B- anew Triterpenoid
Sapogenin fromBarringtoma acutangula Gaertn, Tetrahedron, 24:1113. Barua et al (1972), Treterpenoid. XXXIX. The constitution of Brrinictoniacid-A new triterpene
acid from Barringtoma acutangula Gaertn, J. Indian Chem. Soc. 53:209-210. Barua et al. (1963), Triterpenoid —XIV: Studies on the constitution of Barringtogenol 'C' -A NEW
Triterpenoid Sapogenin from Barrinagtomia cautangula Gaertn J. Indian, Chem.Soc.
40(6):483-485. Braun and Mouchacea (2000), Cercosporoid hyphomyceies on Barringtonia spp- Sydowia. 52(2) :
73-77. Chakraborti and Barua (1963), Triterpenoid -XIV: The constitution of Barringtogeno 'D' -a new
triterpenoid sapogenin from Barrmagtoma cautangula Gaertn. Tetrahedron, 19: 1727-1732.
Dymock et aL (1997)> Pharmacographian indica, Bishen Singh Mahendra Pa! Singh Dehra Dun,
(Vol. II): 17,19,234-244. Hooker, (1872), Flora of British India, Bishen Singh Mahendra Pal Singh and M.P. Singh and
Periodical Experts, Delhi: (Vol.I): 542-543. Jain, (1991), Dictionary of Indian Folk Medicine and Ethnobotany, Deep Publishers : 33,35-36, 44,
76, 82. Mills et al. (1994), Acutangulosides A.F. Monodesmosidac Sapoaoins from the bark of Barringtonia
acutangula. Phytochemistry, 30, 35(5): 1315-8. Nadkarni, (1927), ELM. Nadukarni's Indian Materia mediea. Popularr Book Depot, Bombay, (3rd
edition), (Vol.1) : 176,199,273,452-453,474,842. Pal et al. (1991), Triterpenoid glucoside from Barringtonia acutangula, Phytochemistry, 35(5)
1315-1318. Pal et al. (1994), Sapomins from Barringtonia acutangula, Phytochemistry, 35(5) :1315-1318. Pandey & Chadha, (1996), Economic Botany, Vikas Publishing HousePvt. Ltd.: 117,138-139. Sasidharan (2004), Biodiversity Documentation for Kerala Part 6 Flowering plants, Kerala Forest
Research Institute, Peechi, Kerala. India: 1-3, 68, 90, 180, 247, 271.

5. Claims
We claim:
1. A method for extraction of Lupeol and Saponin using extraction, HPTLC from two different plant species.
2. Extraction method as in claim 1 is applicable for studied plants as shown below.
a) Barrintonia acutangula and
b) Naregamia alata

3. Extraction method present study as in claim 1, using plants as in claim 2 can be extracted using leaf powder in methanol as a solvent.
4. Method of extraction characterized in comprising the following process steps;
a. Leaf powder of plant material (passed through 80 mesh size sieve) as in claim 2
b. Leaf powder was extracted in methanol as solvent.
c. In case of Lupeol, the solvent system consisted of toluene: acetone : acetic acid (GAA) in
ratio of 8.9 : 0.8 : 0.3.
d. Derivatization was carried out by dipping the plate in anisaldehyde -sulfuric acid reagent
and later heating it at 100°C for 10 minutes.
e. In case of saponin the solvent system used was ethylacetate : ethanol (96%) : water :
ammonia (25%) in ratio (6.8 : 2.7 : 9 : 1) respectively.
f. The derivatization was carried out by using anisaldehyde -sulfuric acid reagent and later
heating it at 100°C for 10 minutes.
g. Yields were compared with those standard values of lupeol and saponin.
5. Method of extraction according to claim 4 characterized in that the method is the production of a solution form (methanol extract)
6. Method used for extraction as in claim 1, for the plant as in claim claim 2, using extraction method as in claim 3 & 4, is capable for increase the yield of lupeol and saponin.
7. According to any of the proceeding claims that exemplified, the said method shows increased enhanced yield and new sources for lupeol and saponin.