The present invention generally relates to the field of Chemistry and Phytochemicals. More
specifically it relates to a method of preparation of high content conessine extract from
Holarrhena antidysenterica bark and the resulting extract.
5
BACKGROUND OF THE INVENTION
Holarrhena antidysenterica is a medicinal plant abundantly found in India especially in
Himalayan ranges. According to the classical Ayurvedic literature, the plant holds extreme
economic importance. It is exported in the form seed powder, bark powder, kutajakwatha,
10 Kutaja Prapati Vati and as herbal dietary supplement. Seeds of HA are mainly used as an antidiabetic remedy. Medicinal preparations from Holarrhena antidysenterica (kutaj) bark have
been used extensively in treatment of gastric disorders such as amoebic dysentery from time
immemorial. The bark of the plant is rich in alkaloids and few of the key ingredients show
potent amoebicidal effect. Within this, Conessine, one of the major steroidal alkaloid reported
15 from kutaj bark is responsible for amoebicidal action. This is a major constituent of Holarrhena
antidysenterica, responsible for numerous pharmacological activities.
Problems with known methods: There is limited literature available related to conessine
content in Holarrhena antidysenterica stem bark extracts so far. It is known that the extracts
20 are prepared from different solvents, reported to contain 0.3-0.5% of conessine content. In
addition, the commercial extracts provided by herbal industries lack standardization parameters
related to conessine content. Other alternate classical methodologies of extraction of alkaloids
are available which use acid-base treatment but the process is complicated, involves number of
expensive reagents and multiple steps. In addition, the conessine content by alkaloids
25 extraction technique has not been reported so far. Moreover the yield of the total extract is less
since only alkaloids containing fraction is separated. Therefore these available processes are
not viable for better production of conessine.
Importance of present invention: The prime advantage of the present invention is that it is a
30 simple, efficient and commercially viable and cost-effective method of preparing Holarrhena
antidysenterica extract from its bark with high concentration of conessine content as well
as higher yield, when compared with the other available classical methodology of isolation of
alkaloids.
3
Before going to the related prior art and the detail description of the invention, definitions of
few scientific terminologies are discussed below.
Chromatography –Chromatography is a laboratory process for the separation of a mixture.
5 When the mixture is dissolved in a fluid called the mobile phase, which carries it through a
structure holding another material called the stationary phase. The various constituents of the
mixture travel at different speeds, causing them to separate. The separation is based on
differential partitioning between the mobile and stationary phases. A Chromatogram is the
visual output of the chromatograph. In the case of an optimal separation, different peaks or
10 patterns on the chromatogram correspond to different components of the separated mixture
(https://en.wikipedia.org/wiki/Chromatography).
HPTLC analysis - High-performance thin-layer chromatography (HPTLC) is an
enhanced form of thin-layer chromatography(TLC). A number of enhancements can be made
to the basic method of thin-layer chromatography to automate the different steps, to increase
15 the resolution achieved and to allow more accurate quantitative measurements. Automation is
useful to overcome the uncertainty in droplet size and position when the sample is applied to
the TLC plate by hand. HPLC works on the principle that some molecules take longer than
others to pass through a chromatography column. This depends on the affinity of the molecule
with the mobile phase (liquid or gas) and the stationary phase (solid or liquid)
20 (https://en.wikipedia.org/wiki/High-performance_thin-layer_chromatography)
Rf value -The Retention Value (Rf value) is defined as the ratio of the distance moved by the
solute and the distance moved by the solvent along the paper, where both distances are
measured from the common Origin or Application Baseline, that is the point where the
sample is initially spotted on the paper.
25 (http://www.marz-kreations.com/Chemistry/Chromatography/Dyes/RF-Values.html)
Rf Value = Distance from Baseline travelled by Solute/Distance from Baseline travelled by
Solvent
PRIOR ART PATENTS AND PUBLICATIONS
30
Patent Application No. CN102153614A discloses “Method for preparing effective monomer of
total alkaloid extract of Holarrhena antidysenterica and application thereof”
4
The invention relates to a method for preparing an effective monomer of a total alkaloid extract
of Holarrhena antidysenterica and application thereof. The method comprises the following
steps of: (1) refluxing and extracting Holarrhena antidysenterica by using 90 percent ethanol
to obtain an ethanol extract; (2) dissolving the ethanol extract in water, regulating the pH value
5 to be between 1 and 2 by using concentrated hydrochloric acid, and filtering; (3) regulating the
pH value of filtrate obtained after the filtration to be between 9 and 11 by using stronger
ammonia water or aqueous solution of sodium hydroxide, and extracting by using an organic
solvent to obtain the total alkaloid extract of Holarrhena antidysenterica; and (4) performing
MCI-gel column chromatography, methanol aqueous solution gradient elution, and thin-layer
10 chromatography on the total alkaloid extract, wherein obtained effective monomer ingredients
comprise conessine, isoconessimine, conessimin and conimin.
The invention discloses application of the total alkaloid extract of the Holarrhena
antidysenterica in preparing medicaments for preventing and treating Alzheimer disease or
medicaments for improving intelligence, and application of the effective monomer ingredients
15 of the total alkaloid extract of Holarrhena antidysenterica in preparing the medicaments for
preventing and treating Alzheimer disease or the medicaments for improving intelligence.
In this patent inventors used 90% of Ethanol as an ingredient of extraction, whereas in the
present invention Methanol has been used.
Patent Application No. US20100040568A1discloses “Steroidal compounds as melanogenesis
20 modifiers and uses thereof”
The invention relates to a steroidal compounds of formula I, for example, conessine, and the
use of such compounds and compositions thereof to modulate (e.g., inhibit) melanogenesis and
pigmentation. The invention also provided plant extracts containing a compound of formula I,
for example, conessine and the use of such a plant extract to modulate (e.g., inhibit)
25 melanogenesis and pigmentation. The compound or plant extract may be prepared as
pharmaceutical and cosmetic compositions and may be used for the prevention and treatment
of conditions that are related to aberrant melanogenesis activity.
In this patent, the detailed conessine extraction procedure and its yield have not been
discussed.
30
Journals/books
N. Kumar, B. Singh, P. Bhandari, A. P. Gupta, V. K.Kaul, 2007. Steroidal Alkaloids from
Holarrhena antidysenterica(L.) Wall. Chem. Pharm. Bull. 55(6), 912—914.
5
The paper discloses about the chemical investigations on the stem bark of Holarrhena
antidysenterica resulted in the isolation of a new steroidal alkaloid designated as
holadysenterine (1), together with three known steroidal alkaloids, conessine (2),
isoconessimine (3) and kurchessine (4). Their structures were elucidated on the basis of 1D5 and 2D-NMR techniques and high-resolution mass spectrometry.
The technique of isolation of conessine is different from the present invention.
K. K. Bhutani, R. M. Vaid, M. Ali, R. Kapoor, S. R. Soodan, D. Kumar, 1990. Steroidal
Alkaloids from Holarrhena antidysenterica. Phytochemistry, 29 (3), 969-972.
The paper discloses three new steroidal alkaloids, regholarrhenine D, E and F, along with the
10 known alkaloid kurchessine, were isolated from the stem bark of Holarrhena antidysenterica.
The structures were elucidated on the basis of spectral and chemical evidence. It was shown
that the first alkaloid possesses the endo N-OH function in lieu of the endo N-Me function in
conessine. The second alkaloid is the C-7 stereoisomer of the previously isolated alkaloid
kurcholessine. The third alkaloid was established as the 18-hydroxymethyl analogue of
15 kurchessine.
The above invention does not discuss about the high yield extraction of conessine.
R. K. Dwivedi, R.K. Sharma, 1990. Quantitative estimation of Holarrhena antidysenterica
bark total Alkaloids in crude drugs and In the Body Fluids of man and rat. Journal of
20 Ethnopharmacology, 30, 75-89.
The paper discloses a turbidimetric method was developed for the quantitative estimation of
the total alkaloids of kutaj bark in crude medicinal preparations and in the body fluids of man
and rat. The alkaloids were colloidally precipitated with Dragendorff’s reagent as complex salts
of potassium iodobismuthate in extremely dilute solutions. The finely sub- divided orange25 brown precipitate gave a coloured, clear homogeneous suspension in the presence of gum
Arabic. Optical density of such suspensions changed linearly with the change in alkaloid
concentration, when prepared within the standardized experimental conditions that included
control of ion concentration and temperature of the reaction mixture. Observations revealed the
reversible nature of the alkaloid-reagent reaction. Crude medicinal preparations from three
30 different pharmaceutical sources contained varying concentrations of the alkaloids. Complete
recovery of the alkaloids was possible from plasma and urine, while significant amounts of the
alkaloids were lost to blood cells and faecal contents in man and rat.
6
The above invention does not discuss about the high yield extraction of conessine.
S. Sinha, A. Sharma, P. H. Reddy, B. Rathi, N.V.S.R.K. Prasad, A.Vashishtha, 2013.
Evaluation of phytochemical and pharmacological aspects of Holarrhena antidysenterica
(wall.): A comprehensive review. Journal of pharmacy research, 6, 488-492.
5 The paper discloses that the medicinal plants are generating an ever-increasing amount of
interest due to the effectiveness, low cost and minimal side-effects associated with drugs
derived from them. Holarrhena antidysenterica, belonging to the family Apocynaceae, is
commended for the medicinal applications of its stem bark, leaves and seeds in Ayurveda.
During the past century, studies on the phytochemical and pharmacological nature of the plant
10 have yielded important results regarding the chemical constituents present and have also
verified the traditionally claimed properties associated with the plant viz. analgesic,
antibacterial, anti-diarrhoeal, anti-amoebic, anti-inflammatory and anti-haemorrhoidal
activities. Moreover, recently some other properties have also been discovered viz.
antimalarial, anti-diabetic, anti-oxidant, anti-urolithic, anti-mutagenic, CNS-stimulating,
15 Angiotensin-converting-enzyme inhibitory and acetylcholinesterase inhibitory activity. This
review discusses the findings of studies on the aforementioned properties of the plant in detail
and 68 alkaloids isolated from various parts of plant to justify its widespread use in the
treatment of a variety of diseases and suggests future lines of research.
The above invention does not discuss about the high yield extraction of conessine.
20 R.K. Thappa, K. Tikk U, B. P. Saxena, R.M. Vaid, K.K. Bhutani, 1989.Conessine as a
larval growth inhibitor, sterilant, and antifeedant from Holarrhena antidysenterica wall.
Insect Sci. Applic., 10 (2), 149-155.
The paper discloses about Conessine, the steroidal alkaloid of Holarrhena antidysenterica
Wall, possesses a wide range of activities against four insect species viz. Aedes aegypti,
25 Dysdercus koenigii, Spodoptera litura and Pieris brassicae. In D. koenigii the compound
inhibits the egg hatching of treated adults and nymphs. In Ae. aegypti the larval developmental
periods are extended, resulting in a high mortality rate. Such effects are produced at very low
dosages of 0.5 to l0 ppm. Antifeedant activity is observed against larvae of S. litura and P.
brassicae at concentrations of 0.005 to 0.2 % of conessine.
30 The above invention does not discuss about the method of extraction of high quality
conessine.
7
K. Das, R. Dang, 2017. Influence of Demographic Location and Solvent Extraction on
Pharmacognostical Assessment and Identification of Conessine Content in Different
Parts of Holarrhena antidysentrica through HPTLC Analysis. Indian Journal of
Pharmaceutical Education and Research, 51(3).
5 The present patent application is aimed at comparative pharmacognostical studies in terms of
macroscopic and quantitative microscopy on different solvent (chloroform, methanol and
water) extracted leaves, stem and root parts of HA, procured from the Bangalore soil zone,
Karnataka, India. In this paper, the methanol stem bark extract showed highest percentage of
yield of 14.80 ± 0.02* followed by a root methanol extract (12.60 ± 0.21). But HPTLC figure
10 showed that the maximum amount of active constituents, i.e., conessine content observed
with the methanol extract of bark (0.51%), followed by a methanolic root extract (0.48%)
and leaf chloroform extract (0.31%) whereas the leaf methanol extract showed conessine
content 0.05%.
The above paper is close to the present invention. Although the method used in the above
15 publication shows the ~12% yield of extraction, but conessine content is 0.51%, whereas the
present invention shows 2-3% conessine content in the extraction. Therefore the current
method has an extreme importance in medicinal field.
From the above, it is clear that there is no disclosure in the prior art regarding this highly potent
20 method of preparing high content conessine described in this present invention.
SUMMARY OF THE INVENTION
In the present invention, a unique simple, efficient and commercially viable method of
preparing Holarrhena antidysenterica extract from its bark with high concentration of
25 conessine has been developed. The process shows much higher overall yield (10-12%)
compared to other conventional alkaloid extraction methods as well as this simplified process
requires only 2-3 steps. In this process Methanol and 5% Diethyl amine have been added in
coarsely ground Holarrhena antidysenterica bark to make a thick paste which is high in
conessine content. The process requires less number of reagents, steps, energy to complete the
30 extraction and almost negligible pollution is generated throughout the process.
8
BRIEF DESCRIPTION OF THE DIAGRAMS
Figure 1: Linearity curve for conessine
Figure 2: Chromatogram overlay of standard conessine and H. antidysenterica extract
5 Figure 3: Spectral scan match of standard conessine and H. antidysenterica extract
OBJECTS OF THE PRESENT INVENTION
It is an object of the present invention to disclose an extract from the bark of Holarrhena
10 antidysenterica which has high concentration of conessine that is 2-3% and has a higher yield
(10-12%).
Another object of the present invention is to disclose a unique simple, efficient and
commercially viable method of preparing Holarrhena antidysenterica extract from its bark.
One more object is to disclose a method which requires less number of reagents and fewer steps
15 and lesser energy to complete the extraction and almost negligible pollution is generated
throughout the process.
DETAILED DESCRIPTION OF THE INVENTION
20 The present invention discloses a method of preparation of high content conessine extract from
Holarrhena antidysenterica bark and the resulting extract. The said extract made from
Holarrhena antidysenterica bark which possesses very high conessine content (2-3%). The
method of extraction results in higher yield. The steps of extraction include grinding of the
bark of Holarrhena antidysenterica and adding 5% diethylamine solution. Finally the solution
25 is filtered and concentrated under negative pressure to get the brownish paste which is actually
the final extract containing very high conessine content (2-3%).
The process is described by way of following example.
Example:
1. 10 Kg of Holarrhena antidysenterica bark was procured from Raipur, Chhattisgarh.
30 2. The bark was coarsely grinded and passed through 20 mesh sieve size.
3. 100 litre of 5% diethylamine solution was prepared in methanol.
9
4. The plant material was transferred in extractor and 100 litre of 5% diethylamine solution
was added.
5. Extraction process of the mixture was performed for 4-5 hrs. in extractor between 55-60
°C temperatures.
5 6. The solution was taken filtered and concentrated under negative pressure to get the final
extract in the form of brownish paste.
7. Finally the HPTLC analysis was performed to know the quantification of conessine
content.
Material Required for Extraction and Quantification:
10
1. Extraction unit: The stainless steel extractor is being used for preparation of Holarrhena
antidysenterica extract with high concentration of conessine.
2. Required Reagents: Methanol, Diethylamine
3. Mobile phase for HPTLC for Quantification: The ratio Toluene: Ethyl acetate: Diethyl
15 amine is 6: 2.5: 1
4. Rf value: 0.5-0.6
5. Visulalizing Agent: Dragondorff reagent
Flowchart: The Comparative result using different methods
20
Following the methodology described before, here is the flowchart showing the conessine
content extracted from different methods. In the first case, when only Methanol is added to the
10
coarsely powdered Holarrhena antidysenterica bark to make brownish thick paste, the result
shows only 0.4-0.8% of conessine content. In the third case, it is shown that 10% Diethyl amine
was added to the Methanol and the received conessine content is 0.8-1.2%.
5 After few combinations, the highest conessine content (2-3%) was received, when 5% Diethyl
amine was added with Methanol to make the brownish paste.
The process records the highest conessine content received till date. The higher conessine
content is shown by the linearity curve (Figure 1). Secondly, the quantification of conessine
10 content in H. antidysenterica bark extract was established by HPTLC analysis (Figure 2).The
developed methodology was authenticated by chromatogram overlay of standard with sample
and spectral scan match (Figure 3) at 520nm. Analysis confirms the multifold increase (400-
600 percent/ 4-6 times) in conessine content by the methodology developed when compared
with the classical extraction methodology.
15
Novelty, Inventive Steps and Industrial Application of the invention
NOVELTY
20 The present invention discloses a method of preparation of high content conessine extract from
Holarrhena antidysenterica bark and the resulting extract. The method of preparation of the
same is unique, simple, efficient and commercially viable. The extract made from this
simplified process possess much higher overall yield (10-12%) and high conessine content
(2-3%). None of the other already disclosed methods show the same yield or conessine
25 content. Hence the present invention is novel.
INVENTIVE STEP
The present invention discloses a simplified process for preparing Holarrhena antidysenterica
extract with much higher content (2-3%) of conessine. Technical advancement lies in using
30 Methanol and adding 5% Diethyl amine in coarsely ground Holarrhena antidysenterica bark
to make a thick paste which is high in conessine content. Economical advantage lies in the
fact that since the content of conessine is very high, the method becomes commercially viable.
The process requires less number of reagents, steps, energy and almost negligible pollution is
11
generated throughout the process thus it duly qualifies the two criteria of Inventiveness very
effectively.
INDUSTRIAL APPLICATION
5 Conessine is one of the major constituents of Holarrhena antidysenterica responsible for
numerous pharmacological activities. This medicinal plant is used extensively in treatment of
gastric disorders such as amoebic dysentery. The process of present invention shows much
higher overall yield (10-12%) compared to other conventional alkaloid extraction methods as
well as this simplified process requires only 2-3 steps and very few reagents to complete the
10 extraction.
The process can easily be adopted for industrial scale development as it is very simple and
much faster and with much higher yield.

WE CLAIM:

1.A method of preparation of high content conessine extract from Holarrhena antidysenterica bark WHEREIN the same consists of following steps:
- Grinding of Holarrhena antidysenterica bark and sieving it;
- Adding 5% diethylamine solution prepared in methanol into the powdered Holarrhena antidysenterica kept in extractor;
- Performing the extraction process of the mixture for 4-5 hrs. in extractor between 55-60 °C temperatures; and
- Filtering and concentrating the solution under negative pressure to get the brownish paste.

2. The method of preparation of high content conessine extract from Holarrhena antidysenterica bark as claimed in claim 1 WHEREIN the yield of the extract is 10-12%.

3. The extract obtained by the process as claimed in claim 1 WHEREIN the same has conessine content of 2-3%.

, Description:FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(SEE SECTION 10 AND RULE 13)

METHOD OF PREPARING HIGH CONTENT CONESSINE EXTRACT FROM HOLARRHENA ANTIDYSENTERICA BARK AND THE RESULTING EXTRACT

AYURVET LIMITED

Village - Katha, Post Office – Baddi,
District –Solan, Pincode – 173205,
Himachal Pradesh, India
Email: krk@ayurvet.in
Contact: 09218593483

The following specification particularly describes the invention and the manner in which it is to be performed
FIELD OF INVENTION
The present invention generally relates to the field of Chemistry and Phytochemicals. More specifically it relates to a method of preparation of high content conessine extract from Holarrhena antidysenterica bark and the resulting extract.

BACKGROUND OF THE INVENTION
Holarrhena antidysenterica is a medicinal plant abundantly found in India especially in Himalayan ranges. According to the classical Ayurvedic literature, the plant holds extreme economic importance. It is exported in the form seed powder, bark powder, kutajakwatha, Kutaja Prapati Vati and as herbal dietary supplement. Seeds of HA are mainly used as an anti-diabetic remedy. Medicinal preparations from Holarrhena antidysenterica (kutaj) bark have been used extensively in treatment of gastric disorders such as amoebic dysentery from time immemorial. The bark of the plant is rich in alkaloids and few of the key ingredients show potent amoebicidal effect. Within this, Conessine, one of the major steroidal alkaloid reported from kutaj bark is responsible for amoebicidal action. This is a major constituent of Holarrhena antidysenterica, responsible for numerous pharmacological activities.

Problems with known methods: There is limited literature available related to conessine content in Holarrhena antidysenterica stem bark extracts so far. It is known that the extracts are prepared from different solvents, reported to contain 0.3-0.5% of conessine content. In addition, the commercial extracts provided by herbal industries lack standardization parameters related to conessine content. Other alternate classical methodologies of extraction of alkaloids are available which use acid-base treatment but the process is complicated, involves number of expensive reagents and multiple steps. In addition, the conessine content by alkaloids extraction technique has not been reported so far. Moreover the yield of the total extract is less since only alkaloids containing fraction is separated. Therefore these available processes are not viable for better production of conessine.

Importance of present invention: The prime advantage of the present invention is that it is a simple, efficient and commercially viable and cost-effective method of preparing Holarrhena antidysenterica extract from its bark with high concentration of conessine content as well as higher yield, when compared with the other available classical methodology of isolation of alkaloids.
Before going to the related prior art and the detail description of the invention, definitions of few scientific terminologies are discussed below.

Chromatography –Chromatography is a laboratory process for the separation of a mixture. When the mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases. A Chromatogram is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture
(https://en.wikipedia.org/wiki/Chromatography).
HPTLC analysis - High-performance thin-layer chromatography (HPTLC) is an enhanced form of thin-layer chromatography(TLC). A number of enhancements can be made to the basic method of thin-layer chromatography to automate the different steps, to increase the resolution achieved and to allow more accurate quantitative measurements. Automation is useful to overcome the uncertainty in droplet size and position when the sample is applied to the TLC plate by hand. HPLC works on the principle that some molecules take longer than others to pass through a chromatography column. This depends on the affinity of the molecule with the mobile phase (liquid or gas) and the stationary phase (solid or liquid) (https://en.wikipedia.org/wiki/High-performance_thin-layer_chromatography)
Rf value -The Retention Value (Rf value) is defined as the ratio of the distance moved by the solute and the distance moved by the solvent along the paper, where both distances are measured from the common Origin or Application Baseline, that is the point where the sample is initially spotted on the paper.
(http://www.marz-kreations.com/Chemistry/Chromatography/Dyes/RF-Values.html)
Rf Value = Distance from Baseline travelled by Solute/Distance from Baseline travelled by Solvent

PRIOR ART PATENTS AND PUBLICATIONS

Patent Application No. CN102153614A discloses “Method for preparing effective monomer of total alkaloid extract of Holarrhena antidysenterica and application thereof”
The invention relates to a method for preparing an effective monomer of a total alkaloid extract of Holarrhena antidysenterica and application thereof. The method comprises the following steps of: (1) refluxing and extracting Holarrhena antidysenterica by using 90 percent ethanol to obtain an ethanol extract; (2) dissolving the ethanol extract in water, regulating the pH value to be between 1 and 2 by using concentrated hydrochloric acid, and filtering; (3) regulating the pH value of filtrate obtained after the filtration to be between 9 and 11 by using stronger ammonia water or aqueous solution of sodium hydroxide, and extracting by using an organic solvent to obtain the total alkaloid extract of Holarrhena antidysenterica; and (4) performing MCI-gel column chromatography, methanol aqueous solution gradient elution, and thin-layer chromatography on the total alkaloid extract, wherein obtained effective monomer ingredients comprise conessine, isoconessimine, conessimin and conimin.
The invention discloses application of the total alkaloid extract of the Holarrhena antidysenterica in preparing medicaments for preventing and treating Alzheimer disease or medicaments for improving intelligence, and application of the effective monomer ingredients of the total alkaloid extract of Holarrhena antidysenterica in preparing the medicaments for preventing and treating Alzheimer disease or the medicaments for improving intelligence.
In this patent inventors used 90% of Ethanol as an ingredient of extraction, whereas in the present invention Methanol has been used.
Patent Application No. US20100040568A1discloses “Steroidal compounds as melanogenesis modifiers and uses thereof”
The invention relates to a steroidal compounds of formula I, for example, conessine, and the use of such compounds and compositions thereof to modulate (e.g., inhibit) melanogenesis and pigmentation. The invention also provided plant extracts containing a compound of formula I, for example, conessine and the use of such a plant extract to modulate (e.g., inhibit) melanogenesis and pigmentation. The compound or plant extract may be prepared as pharmaceutical and cosmetic compositions and may be used for the prevention and treatment of conditions that are related to aberrant melanogenesis activity.
In this patent, the detailed conessine extraction procedure and its yield have not been discussed.

Journals/books
N. Kumar, B. Singh, P. Bhandari, A. P. Gupta, V. K.Kaul, 2007. Steroidal Alkaloids from Holarrhena antidysenterica(L.) Wall. Chem. Pharm. Bull. 55(6), 912—914.
The paper discloses about the chemical investigations on the stem bark of Holarrhena antidysenterica resulted in the isolation of a new steroidal alkaloid designated as holadysenterine (1), together with three known steroidal alkaloids, conessine (2), isoconessimine (3) and kurchessine (4). Their structures were elucidated on the basis of 1D- and 2D-NMR techniques and high-resolution mass spectrometry.
The technique of isolation of conessine is different from the present invention.
K. K. Bhutani, R. M. Vaid, M. Ali, R. Kapoor, S. R. Soodan, D. Kumar, 1990. Steroidal Alkaloids from Holarrhena antidysenterica. Phytochemistry, 29 (3), 969-972.
The paper discloses three new steroidal alkaloids, regholarrhenine D, E and F, along with the known alkaloid kurchessine, were isolated from the stem bark of Holarrhena antidysenterica. The structures were elucidated on the basis of spectral and chemical evidence. It was shown that the first alkaloid possesses the endo N-OH function in lieu of the endo N-Me function in conessine. The second alkaloid is the C-7 stereoisomer of the previously isolated alkaloid kurcholessine. The third alkaloid was established as the 18-hydroxymethyl analogue of kurchessine.
The above invention does not discuss about the high yield extraction of conessine.

R. K. Dwivedi, R.K. Sharma, 1990. Quantitative estimation of Holarrhena antidysenterica bark total Alkaloids in crude drugs and In the Body Fluids of man and rat. Journal of Ethnopharmacology, 30, 75-89.
The paper discloses a turbidimetric method was developed for the quantitative estimation of the total alkaloids of kutaj bark in crude medicinal preparations and in the body fluids of man and rat. The alkaloids were colloidally precipitated with Dragendorff’s reagent as complex salts of potassium iodobismuthate in extremely dilute solutions. The finely sub- divided orange-brown precipitate gave a coloured, clear homogeneous suspension in the presence of gum Arabic. Optical density of such suspensions changed linearly with the change in alkaloid concentration, when prepared within the standardized experimental conditions that included control of ion concentration and temperature of the reaction mixture. Observations revealed the reversible nature of the alkaloid-reagent reaction. Crude medicinal preparations from three different pharmaceutical sources contained varying concentrations of the alkaloids. Complete recovery of the alkaloids was possible from plasma and urine, while significant amounts of the alkaloids were lost to blood cells and faecal contents in man and rat.
The above invention does not discuss about the high yield extraction of conessine.
S. Sinha, A. Sharma, P. H. Reddy, B. Rathi, N.V.S.R.K. Prasad, A.Vashishtha, 2013. Evaluation of phytochemical and pharmacological aspects of Holarrhena antidysenterica (wall.): A comprehensive review. Journal of pharmacy research, 6, 488-492.
The paper discloses that the medicinal plants are generating an ever-increasing amount of interest due to the effectiveness, low cost and minimal side-effects associated with drugs derived from them. Holarrhena antidysenterica, belonging to the family Apocynaceae, is commended for the medicinal applications of its stem bark, leaves and seeds in Ayurveda. During the past century, studies on the phytochemical and pharmacological nature of the plant have yielded important results regarding the chemical constituents present and have also verified the traditionally claimed properties associated with the plant viz. analgesic, antibacterial, anti-diarrhoeal, anti-amoebic, anti-inflammatory and anti-haemorrhoidal activities. Moreover, recently some other properties have also been discovered viz. antimalarial, anti-diabetic, anti-oxidant, anti-urolithic, anti-mutagenic, CNS-stimulating, Angiotensin-converting-enzyme inhibitory and acetylcholinesterase inhibitory activity. This review discusses the findings of studies on the aforementioned properties of the plant in detail and 68 alkaloids isolated from various parts of plant to justify its widespread use in the treatment of a variety of diseases and suggests future lines of research.
The above invention does not discuss about the high yield extraction of conessine.
R.K. Thappa, K. Tikk U, B. P. Saxena, R.M. Vaid, K.K. Bhutani, 1989.Conessine as a larval growth inhibitor, sterilant, and antifeedant from Holarrhena antidysenterica wall.
Insect Sci. Applic., 10 (2), 149-155.
The paper discloses about Conessine, the steroidal alkaloid of Holarrhena antidysenterica Wall, possesses a wide range of activities against four insect species viz. Aedes aegypti, Dysdercus koenigii, Spodoptera litura and Pieris brassicae. In D. koenigii the compound inhibits the egg hatching of treated adults and nymphs. In Ae. aegypti the larval developmental periods are extended, resulting in a high mortality rate. Such effects are produced at very low dosages of 0.5 to l0 ppm. Antifeedant activity is observed against larvae of S. litura and P. brassicae at concentrations of 0.005 to 0.2 % of conessine.
The above invention does not discuss about the method of extraction of high quality conessine.
K. Das, R. Dang, 2017. Influence of Demographic Location and Solvent Extraction on Pharmacognostical Assessment and Identification of Conessine Content in Different Parts of Holarrhena antidysentrica through HPTLC Analysis. Indian Journal of Pharmaceutical Education and Research, 51(3).
The present patent application is aimed at comparative pharmacognostical studies in terms of macroscopic and quantitative microscopy on different solvent (chloroform, methanol and water) extracted leaves, stem and root parts of HA, procured from the Bangalore soil zone, Karnataka, India. In this paper, the methanol stem bark extract showed highest percentage of yield of 14.80 ± 0.02* followed by a root methanol extract (12.60 ± 0.21). But HPTLC figure showed that the maximum amount of active constituents, i.e., conessine content observed with the methanol extract of bark (0.51%), followed by a methanolic root extract (0.48%) and leaf chloroform extract (0.31%) whereas the leaf methanol extract showed conessine content 0.05%.
The above paper is close to the present invention. Although the method used in the above publication shows the ~12% yield of extraction, but conessine content is 0.51%, whereas the present invention shows 2-3% conessine content in the extraction. Therefore the current method has an extreme importance in medicinal field.

From the above, it is clear that there is no disclosure in the prior art regarding this highly potent method of preparing high content conessine described in this present invention.

SUMMARY OF THE INVENTION
In the present invention, a unique simple, efficient and commercially viable method of preparing Holarrhena antidysenterica extract from its bark with high concentration of conessine has been developed. The process shows much higher overall yield (10-12%) compared to other conventional alkaloid extraction methods as well as this simplified process requires only 2-3 steps. In this process Methanol and 5% Diethyl amine have been added in coarsely ground Holarrhena antidysenterica bark to make a thick paste which is high in conessine content. The process requires less number of reagents, steps, energy to complete the extraction and almost negligible pollution is generated throughout the process.

BRIEF DESCRIPTION OF THE DIAGRAMS

Figure 1: Linearity curve for conessine
Figure 2: Chromatogram overlay of standard conessine and H. antidysenterica extract
Figure 3: Spectral scan match of standard conessine and H. antidysenterica extract

OBJECTS OF THE PRESENT INVENTION

It is an object of the present invention to disclose an extract from the bark of Holarrhena antidysenterica which has high concentration of conessine that is 2-3% and has a higher yield (10-12%).
Another object of the present invention is to disclose a unique simple, efficient and commercially viable method of preparing Holarrhena antidysenterica extract from its bark.
One more object is to disclose a method which requires less number of reagents and fewer steps and lesser energy to complete the extraction and almost negligible pollution is generated throughout the process.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses a method of preparation of high content conessine extract from Holarrhena antidysenterica bark and the resulting extract. The said extract made from Holarrhena antidysenterica bark which possesses very high conessine content (2-3%). The method of extraction results in higher yield. The steps of extraction include grinding of the bark of Holarrhena antidysenterica and adding 5% diethylamine solution. Finally the solution is filtered and concentrated under negative pressure to get the brownish paste which is actually the final extract containing very high conessine content (2-3%).
The process is described by way of following example.
Example:
1. 10 Kg of Holarrhena antidysenterica bark was procured from Raipur, Chhattisgarh.
2. The bark was coarsely grinded and passed through 20 mesh sieve size.
3. 100 litre of 5% diethylamine solution was prepared in methanol.
4. The plant material was transferred in extractor and 100 litre of 5% diethylamine solution was added.
5. Extraction process of the mixture was performed for 4-5 hrs. in extractor between 55-60 °C temperatures.
6. The solution was taken filtered and concentrated under negative pressure to get the final extract in the form of brownish paste.
7. Finally the HPTLC analysis was performed to know the quantification of conessine content.
Material Required for Extraction and Quantification:

1. Extraction unit: The stainless steel extractor is being used for preparation of Holarrhena antidysenterica extract with high concentration of conessine.
2. Required Reagents: Methanol, Diethylamine
3. Mobile phase for HPTLC for Quantification: The ratio Toluene: Ethyl acetate: Diethyl amine is 6: 2.5: 1
4. Rf value: 0.5-0.6
5. Visulalizing Agent: Dragondorff reagent

Flowchart: The Comparative result using different methods

Following the methodology described before, here is the flowchart showing the conessine content extracted from different methods. In the first case, when only Methanol is added to the coarsely powdered Holarrhena antidysenterica bark to make brownish thick paste, the result shows only 0.4-0.8% of conessine content. In the third case, it is shown that 10% Diethyl amine was added to the Methanol and the received conessine content is 0.8-1.2%.

After few combinations, the highest conessine content (2-3%) was received, when 5% Diethyl amine was added with Methanol to make the brownish paste.

The process records the highest conessine content received till date. The higher conessine content is shown by the linearity curve (Figure 1). Secondly, the quantification of conessine content in H. antidysenterica bark extract was established by HPTLC analysis (Figure 2).The developed methodology was authenticated by chromatogram overlay of standard with sample and spectral scan match (Figure 3) at 520nm. Analysis confirms the multifold increase (400-600 percent/ 4-6 times) in conessine content by the methodology developed when compared with the classical extraction methodology.

Novelty, Inventive Steps and Industrial Application of the invention

NOVELTY
The present invention discloses a method of preparation of high content conessine extract from Holarrhena antidysenterica bark and the resulting extract. The method of preparation of the same is unique, simple, efficient and commercially viable. The extract made from this simplified process possess much higher overall yield (10-12%) and high conessine content (2-3%). None of the other already disclosed methods show the same yield or conessine content. Hence the present invention is novel.

INVENTIVE STEP
The present invention discloses a simplified process for preparing Holarrhena antidysenterica extract with much higher content (2-3%) of conessine. Technical advancement lies in using Methanol and adding 5% Diethyl amine in coarsely ground Holarrhena antidysenterica bark to make a thick paste which is high in conessine content. Economical advantage lies in the fact that since the content of conessine is very high, the method becomes commercially viable. The process requires less number of reagents, steps, energy and almost negligible pollution is generated throughout the process thus it duly qualifies the two criteria of Inventiveness very effectively.

INDUSTRIAL APPLICATION
Conessine is one of the major constituents of Holarrhena antidysenterica responsible for numerous pharmacological activities. This medicinal plant is used extensively in treatment of gastric disorders such as amoebic dysentery. The process of present invention shows much higher overall yield (10-12%) compared to other conventional alkaloid extraction methods as well as this simplified process requires only 2-3 steps and very few reagents to complete the extraction.
The process can easily be adopted for industrial scale development as it is very simple and much faster and with much higher yield.