A process for preparation of synergistic herbal preparation of

extracts picrorhiza kurroa and glycyrrhiza glabra having

medicinal value

Abstract

A synergistic herbal preparation of extracts of Picrorhiza Kurroa Royle or its close species of the
same family such as Picrorhiza Scrophulariflora Pennel and Neopicrorhiza Scrophulariiflora,
and Glycyrrhiza Glabra having medicinal values and Antiviral and Immune boosting activity
against DNA virus -bacteriophage pi AT 300 μg/ml and plaque reduction assay

Description

A PROCESS FOR PREPARATION OF SYNERGESTIC HERBAL PREPARATION OF

EXTRACTS OF PICRORHIZA KURROA AND GLLYCYRRHIZA GLABRA

HAVING MEDICINAL VALUES

The present invention relates to an process for preparation of synergestic herbal preparation of
extracts of Picrorhiza Kurroa Royle or its close species of the same family such as Picrorhiza
Scrophulariflora Pennel and Neopicrorhiza Scrophulariiflora and Gllycyrrhiza Glabra having
medicinal values and Antiviral and Immune boosting activity against DNA virus-bacteriophage
p1 AT 300 pg/ml by plaque reduction assay by stabilizing picrorhiza kurroa from oxidising there
by formulating it with other plant extracts which can be of use for treating organ specific to
many disorders/diseases caused due to microbes, bacteria, viruses and maybe due to
contamination of water, air or food of any kind.

Background:

To treat human disorders and diseases by using various plant parts traditionally many theories
have been published from ages, without exactly knowing the mechanism or site of action of these
plant parts or the extracts.

Plant based medicines are typically well tolerated, with less severe side effects as well as a
smaller range of side effects, with improved patient tolerance, while providing sufficient
efficacy. Yet this medication has neither been fully accepted nor used by common man due to its
non-acceptable formulation and dosage forms. In recent years research has been conducted to
understand and refine the process of extraction and formulating it in more patient acceptable
manner such as a ready to use formulation such as tablets, capsules, syrups, injectables etc.,

Any plant part or its extracts have been widely used in traditional medicine since the ages,
almost all the known plants or its extracts have been identified for their specific functionality to
treat many disorders. In general the entire plant parts are used for extraction or parts of plants are
dried under shade and powdered and given to patients for treating. In recent times many
methodologies have been adopted and tried for extracting site specific such as leaves, stems roots
etc. for extracting the alkaloids/glycosides/lipids using various processes. But all the plant
extracts or lipids need to be extracted under controlled conditions because of their unstable or
fast reaction nature say for they may be highly oxidising- deoxidising in nature of and hence
there is a need to stabilize the extracts specially the lipid extracts. A stabilized composition
which even dose not loose its medicinal value and at the same should not create any adverse
effects when consumed as medicine or any other form intended for prophylactic or treatment of
any disorder/disease condition.

A number of attempts have been made to extract picrorhiza kurroa for treatment of diseases. For
example, as prior art nos WO2011027364, WO2011024 96, WO2009098702, EP1837027,
WO2007093897, EP1637153, R0121767, CN1500798, US2003044512, EP0890360 and
US6136316 Indian applications 0225/DEL/2009, 2095/MUM/2006, 2141/DEIJ1997,
1330/DEL/2004, 823/DEL/1996, 0623/DEL/1996 and Indian patent nos 177155 and 178866.
However all these attemps have been made wherein the extract of picrorhiza kurroa has been
combined with other plants and not Glycyrrhiza Glabra (which belong to Fabaceae Family).
Furthermore in none of the said attempts the extract of picrorhiza kurroa are combined with oil
soluble vitamin in a developed procedure to make the extarct highly stable and at the same time
noh-toxic/compatible/enhanced for medicinal activity.

Particular reference is made here of prior art under no WO200107062 which also comprises of a
synergistic composition comprising the extract of Glycyrrhiza glabra and Picrorhiza kurroa.
However the said patent is limited to the treatment of Liver and Liver associated aliments
whereas the present invention if in respect of treating organ specific to many disorders/diseases
caused due to microbes, bacteria, and viruses and maybe due to contamination of water, air or
food of any kind. Also in prior art no WO200107062 the Composition ration is 2-1 :1-3 whereas
ingredeinets in the present invention are Mixed in a different ratio. While extraction process is
done in prior art no WO200107062 independently and the obtained extracts is mixed in said ratio
in the present invention extraction process is done together. While comparing the manufacturing
process the said in prior art no WO200107062 is different as it involves water in the process in
the present invention water mixing is not involved. Also in the prior art no WO200107062 Lipids
are discarded, Lipids are the important components in the present invention. Finally and most
importantly in prior art no WO200107062 no Stablization to prevent Oxidation of the product is
mentioned in the present invention stabilization or deoxidation process is carried on to prevent
oxidation by adding oil soluble vitamins.

Another patents under no 1019/CHE/2010 is worth mentioning here wherein the cited patent
does not have any claims on the method of manufacturing/extracting process of the combinations
of Glycyrrhiza glabra and Picrorhiza kurroa and the process of stabilizing the obtained molecule
using Oil soluble vitamin. The patent claims mentioned under no 1019/CHE/2010 only speaks on
the extracted molecules functionality, its medicinal values and how it has been proved to have
medicinal properties using various laboratory method's such as culture plate techniques and
animal studies. The patent does not speak on delivery system/route of administration and only
says broadly on family of the plant PICRORHIZA KURROA than directly mentioning about the
plant name. The cited patent under nos 101'9/C HE/2010 do not have or do not claim any point
on lipophilic extraction process and stabilization process. Object of the invention:

In the present invention active constituents of the plant picrorhiza kurroa (which belong to
scrophulariaceae family) which are iridoid glycoside picrosides I, II, III and kutkoside, known
collectively as kutkin are extracted for its lipids along with its glycosides for its medicinal
properties (indications and medicinal values are been elaborated). This lipid is very reactive as it
gets oxidised rapidly once the solvent such as n-hexane or ethanol is removed from the extract
and hence there is a huge demand or need for developing a methodology for stabilising the
compound which will prevent the oxidation process.

In the present invention a methodology is developed wherein an oil soluble vitamin such as
Vitamin E or Vitamin A is added in a developed procedure. This mixture is highly stable and at
the same time non-toxic/compatible/enhanced for medicinal activity. This stebilised extract is
then formulated in a suitable delivery system such as soft gel capsule or a capsule or a tablet
form for convenient usage.

The invention is further proved to be effective for a stabilized extract combination picrorhiza
kurroa (which belong to scrophulariaceae family) and Glycyrrhiza Glabra (which belong to
Fabaceae Family) for Synergistic medicinal activity of combinational lipophilic extracts of both
plants. Apart from its "existing and proven medicinal values (indications have been elaborated) it
has Antiviral and Immune boosting activity (detailed study report has been established).

Detailed Description of Invention. Picrorhiza which has been traditionally used to treat worms,
constipation, low fever, scorpion sting, asthma and ailments affecting the liver such as
hepatoprotective, anticholestatic, antioxidant, and immune-modulating activity and Glycyrrhiza
Glabra which has the compound glycyrrhizic acid, found in liquorice, is routinely used
throughout Japan for the treatment and control of chronic viral hepatitis, and there is a possible
transaminase-lowering effect. Hepatoprotective mechanisms have been demonstrated in mice.
Recent studies indicate that glycyrrhizic acid disrupts latent Kaposi sarcoma (as also
demonstrated with other herpesvirus infections in the active stage), exhibiting a strong anti-viral
effect.

As combined preparation of both Picrorhiza Kurroa and Gllycyrrhiza Glabra has a synergistic
activity for its medicinal values mentioned above individually and apart from the above
mentioned medicinal values it has Antiviral and Immune boosting activity and a detailed study
report for the same has been established in laboratory. It can be stated that during the described
test and under the experimental conditions reported, from the plaque reduction assay it is clear
that the extracts has shown antiviral activity against DNA virus-bacteriophage p1 AT 300 g/ml
which is minimal inhibitory concentration for 0.37 MOI (multiplicity of Infection) value.
Spectrophometric analysis and haemocytometer analysis also supports the plaque reduction
assay. Both the methods have got significant correlated results with plaque reduction.
Individually both the plant description is Picrorhiza kurroa Names

Indian Name :Kutki & Kuru

Botanical Name Picrorhiza kurroa Other Names :Katuka, Kuru & Kadu

It is a small perennial herb from the Scrophulariaceae family. The rhizome of Picrorhiza has
been traditionally used to treat worms, constipation, low fever, scorpion sting, asthma and
ailments affecting the liver. Current research on Picrorhiza kurroa has focused on its
hepatoprotective, anticholestatic, antioxidant, and immune-modulating activity.

Origin

Picrorhiza kurroa also known as kutki is found in the North-Western Himalayan region from
Kashmir to Kumaun and Garhwal regions in India and Nepal.

Chemical composition / key active constituents Kutkin, a bitter glycosidal principle, is reported.
Also isolated D-mannitol, vanillic acid and some steroids are present. Kutkin was later shown to
be a stable mixed crystal of two C-9 iridoid glycosides-Picroside I and Kutakosid, Apocynin has
been isolated from the plant. Picroside II has been isolated and shown to have hepatoprotective
activity. With the help of preparative HPLC, larger Quantities of picrosides have been isolated,
permitting precise structure identification and biological experiments.
R - Ή ¾' - CSiHKTiwyt , ricroswte I

Pharmacology

Alcoholic extract of the plant and kutkin possess hepatoprotective activity. Plant is a potent
immunostimulant of both cell mediated and humoral immunity and exhibits choleretic activity in
dogs. Picrorhiza kurroa is also benefical in the management of bronchial asthma.

Remedies For

Protects the liver against hepatotoxins, hepatoprotective properties, Potent antioxidant activity,
Modulates liver enzyme levels, anti-inflammatory action anti- allergy action.

Dosage

3-4 gm of drug is generally given as antiperiodic and 0.6-1.2 gm as bitter tonic. Typical adult
dosage is 400 to 1500 mg/day, with dosages up to 3.5 g/day sometimes being recommended for
fevers

Glycyrrhiza glabra

Names

Indian Name '.Liquorice

Botanical Name .Glycyrrhiza glabra (syn: Liquirita officinalis (L) Other Names :Lacrisse, sweet
licorice, licorice root

It was one of the most widely known medicines in ancient history, and records of its use include
Assyrian tablets of around 2000 BC and Chinese herbals of the same period. Theophrastos of
Lesbos, writing in the fourth century BC wrote that 'it has the property of quenching thirst if one
holds it in the mouth'. Dioscorides gave the plant its botanical name (Greek glukos = sweet, riza
= root). Its 3th century English name was Lycorys, a corruption of glycyrrhiza.. Liquorice
(Glycyrrhiza glabra) has long been used for both culinary and medical purposes. Used for
flavoring and sweetening candies and medical remedies, licorice also has potent effects of its
own, particularly for ulcers and adrenal insufficiencies. Whole. It is also used for asthmatic
coughs, as an antispasmodic and ulcer remedy, and to cool 'hot' conditions.

Origin

The roots are unearthed in the autumn of the fourth season. It is grown in India, Spain, Iran,
Russia, China & Italy. cYiemical composition / key active constituents '

The herb contains glycyrrhizin, glycyrrhetinic acid, flavonoids, asparagine, iso- flavonoids.and
chalcones.

Pharmacology Licorice contains the glycoside, glycyrrhizin which has a similar structure and
activity as the adrenal steroids. Licorice has an anti-inflammatory activity similar to cortisone
and has been found useful for arthritis and allergies. In addition licorice has been used for mild
Addison's disease and other adrenal insufficiencies, such as hypoglycemia. Licorice also acts like
the hormone, ACTH, causing sodium retention, potassium depletion, and water retention.

Excess consumption of licorice can lead to the classic symptoms of hypertension, with edema,
increased blood pressure, potassium loss, and muscular weakness. The Deglycyrrhizinated form
is most often used to avoid the hypertensive side effects of the glycyrrhetinic acid in whole
Licorice. Licorice and DGL have a mild laxative effect and can protect the intestinal lining by
increasing the production of mucus, thus alleviating heartburn and ulcers. Licorice and DGL also
have a demulcent action and have been used for coughs and other bronchial complaints.

Remedies For Glycyrrhiza is widely used in bronchial problem's such as catarrh, bronchitis, cold,
flu and coughs. It reduces irritation of the throat and yet has an expectorant action. It produces its
demulcent and expectorant effects. It is used in relieving stress. It is a potent healing agent for
tuberculosis, where its effects have been compared to hydrocortisone. Glycyrrhiza is also
effective in helping to reduce fevers (glycyrretinic acid has an effect like aspirin), and it may
have an antibacterial action as well. Its use in the treatment of chronic inflammations such as
arthritis and rheumatic diseases, Anti-inflammatory, chronic skin conditions, and autoimmune
diseases in general. Dosage

600 mg/day DGL extract or 1 , 000 mg of regular extract 1/2 hour before meals.

Combined Lipid extract Mixture of the said two plants has not been established anywhere in
literature search

Method of Extraction The extraction process involves

Collection of dried underground parts of the plant Glycyrrhiza Glabra which belong to Fabaceae
Family & Picrorhiza Kurroa Royle or its close species of the same family such as Picrorhiza
Scrophulariflora Pennel and Neopicrorhiza Scrophulariiflora of Scrophulariaceae Family. These
underground parts are cleaned thoroughly to ensure that the parts are free from impurities such as
Mud, dust etc.

These root/underground parts are individually pulverized in to small pieces of dimension from 1
micron thick to 5 mm.

The obtained mixture is mixed in equal to approximately equal portion (1 :1) [ any composition
ranging from 15 % to 80 % Picrorhiza Kurroa Royle and 85 % to 20 % Glycyrrhiza Glabra and
vice versa ratio and mixed using Octagonal blender/conical blender for obtaining
uniform/homogeneous mixture.

This mixture is taken and mixed with solvents such as n-pentane, n-hexane, n- propanol, ethanol,
methanol, pyridine, acetic acid, isooctane, cyclohexane, cyclopentane, carbon tetrachloride,
toluene, diethyl ether chloroform. This is mixed with proportion in a range of 10-90% of
homogenous mixture with 90-10% of solvents e.g.1. If 10 grams of homogenous mixture is taken
it is mixed with 40 grams solvent. e.g. 2. If 10 grams of homogenous mixture is taken it can be
mixed with 90 grams of solvent e.g.3. If 10 grams of homogenous mixture is taken it can be
mixed with 60 grams of solvent.

The above obtained mixture containing homogenous mixture of underground plants parts and
solvent is charged in to a reactor which can be of glass, or of medical grade stainless steel or any
related material which can with stand the temperature and does not react with the compound, it is
heated with uniform temperature of 25°- 150 0 C for 1-6 hours with continuous stirring at 5-60
rpm speed.

The obtained solution is filtered with filter cloth of 0.5-5 μ for twice or if required thrice
depending up on the removal of suspended particulate matter.

The obtained solution is rich in extract of the underground plant parts and selected solvent for
example n-hexane.

This mixture is loaded in a flat bottom container which provides maximum surface area and I
exposed to Nitrogen gas or any inert gas which is passing from a 3 - 5 μ heap filter with a mild
air blow. With this blow the excess solvent present in the solution which is obtained is
evaporated.

To this product which is remaining in the dish Oil soluble vitamin more specific vitamin such as
Vitamin E is added in 5 to 95 % of the mixture obtained from the above reaction with 95 to 5 %
of oil soluble vitamin such as Vitamin E, for example 1. For every 1 grams of mixture 10 grams
of Vitamin E is added

2. For every 90 grams of mixture 10 grams of Vitamin E is added

3. For every 50 grams of mixture 50 grams of Vitamin E is added

4. For every 40 grams of mixture 10 grams of vitamin E is added. After adding the above
mixture is stirred continuously 5-30 minutes without heating.

The mixture is again loaded in a flat bottom container which provides maximum surface area and
I exposed to Nitrogen gas which is passing from a 3 - 5 μ heap filter with a mild blow. With this
blow the excess solvent present in the solution which is obtained is evaporated.

The final mixture is pure mixture of Glycyrrhiza Glabra & Picrorhiza kurroa extracts in a highly
stabilised form which is stored in air tight container in a cool and dry condition more preferably
at 20-25 degree centigrade. A part of the solution is sent for testing for sterility and purity and
stability. The mixture obtained has a new molecular formula of C24 H26 014 (IR spectrum
Exhibited Bands fig enclosed) and a molecular weight of 538.45484.

Method of Formulation/ Delivery system

The above mixture which passes the purity and sterility test is ready for formulation.

The mixture can be dispensed/formulated as Soft gelatine capsule by mixing the suitable dosage
in any inert Pharma/medical grade oil such as sesame oil or olive oil)

Film/Uncoated/sugar-coated tablets by adding the therapeutic content of the product with

suitable binders and base material and is converted in to tablet method using dry granulation
method Made in to pellets and given in Capsular form by adding suitable therapeutic range of
product and is made in granular form using suitable base materials and filled in to hard gelatine
capsules.

Made in to granules and given in Syrup base by making the materials in to granules and mixed
with sugar syrup form Can be administered directly as injectable as lipholosed form.

Therapeutic Value : The prepared formulation by any of the above rhentioned delivery system
has shown the following medicinal values

The molecule has Glutathione producing activity

Can have positive effect in treating both RNA and DNA based viral diseases and , their related
complications

Can have Antimalarial and Antiprotozoal activity.

The molecule has immune regulatory and anticancer and hepato protective activity.

The molecule has proven medicinal value in treating Dengue, Yellow fever.

The molecule can be of of use in treating Gastroenteritis, Influenza , Leukemia, Rabies.

The molecule has a proven ability in treating Thalassemia

Claims

Claims:
1. A synergestic herbal preparation of extracts of Picrorhiza Kurroa Royle or its close species of
the same family such as Picrorhiza Scrophulariflora Pennel and Neopicrorhiza Scrophulariiflora
and Gllycyrrhiza Glabra having medicinal values and Antiviral and Immune boosting activity
against DNA virus-bacteriophage p1 AT 300 pg/ml and plaque reduction assay.
2. A synergestic herbal preparation as claimed in claim 1 wherein active constituents of the plant
picrorhiza kurroa are extracted for its lipids along with its glycosides wherein an oil soluble
vitamin is added in a developed procedure to make the preparation stable and non-
toxic/compatible/enhanced for medicinal activity.
3. A synergestic herbal preparation as claimed in any one of claims 1 and 2 which is formulated
in a suitable delivery system such as soft gel capsule or a capsule or a tablet form for convenient
usage.
4. A synergestic herbal preparation as claimed in any one of claims 1 , 2 and 3 wherein the
extraction process involves:
Collection of dried underground parts of the plant Glycyrrhiza Glabra & Picrorhiza kurroa;
Cleaning of the underground parts to ensure that the parts are free from impurities such as Mud,
dust etc.;
Individual pulverization of the underground parts in to small pieces of dimension from 1 micron
thick to 5 mm;
Mixing of the obtained mixture to approximately equal portion (1 :1) [ any composition ranging
from 15 % to 80 % Picrorhiza Kurroa Royle and 85 % to 20 % Glycyrrhiza Glabra and vice
versa ratio and mixed using Octagonal blender/conical blender for obtaining
uniform/homogeneous mixture;
Mixing of the mixture with solvents such as n-pentane, n-hexane, n-propanol, ethanol, methanol,
pyridine, acetic acid, isooctane, cyclohexane, cyclopentane, carbon tetrachloride, toluene, diethyl
ether chloroform. This is mixed with proportion in a range of 10-90% of homogenous mixture
with 90- 10% of solvents;
Charging of above obtained mixture containing homogenous mixture of underground plants parts
and solvent into a reactor which can be of glass, or of medical grade stainless steel or any related
material which can with stand the temperature and does not react with the compound, it is heated
with uniform temperature of 25°- 150 0 C for 1-6 hours with continuous stirring at 5- 60 rpm
speed;
Filtering the obtained solution with filter cloth of 0.5-5 μ for twice or if required thrice
depending up on the removal of suspended particulate matter;
Loading the mixture in a flat bottom container which provides maximum surface area and
exposing to Nitrogen gas or any inert gas which is passing from a 3 - 5 μ heap filter with a mild
air blow;
Adding an oil soluble vitamin in 5 to 95 % of the mixture obtained from the above reaction with
95 to 5 % of oil soluble vitamin;
Stirring the above mixture continuously 5-30 minutes without heating;
Loading the mixture in a flat bottom container which provides maximum surface area and
exposing to Nitrogen gas which is passing from a 3 - 5 μ heap filter with a mild blow;
Storing mixture in air tight container in a cool and dry condition more preferably at 20-25 degree
centigrade.
5. A synergestic herbal preparation as claimed in any one of claims 1 to 4 wherein the mixture
can be dispensed/formulated as: Soft gelatine capsule by mixing ' the suitable dosage in any inert
Pharma/medical grade oil such as sesame oil or olive oil);
FJIm/Uncoated/sugar-coated tablets by adding the therapeutic content of the product with
suitable binders and base material and is converted in to tablet method using dry granulation
method;
Pellets and given in Capsular form by adding suitable therapeutic range of product and is made
in granular form using suitable base materials and filled in to hard gelatine capsules;
Granules and given in Syrup base by making the materials in to granules and mixed with sugar
syrup form;
Administered directly as injectable as lipholosed form.