DESC:FIELD OF THE INVENTION

The present invention relates to an herbal formulation used for the management of neurologic and cardiovascular disorders using the fermented dry powder of sea buckthorn pulp. More specifically, the present invention describes a method for extracting a plurality of biological markers from the sea buckthorn pulp liquid.
BACKGROUND OF THE INVENTION

There are more than 600 known disorders that afflict the nervous system. Nervous system disorders have varied etiologies, but ultimately all have devastating effects on the individuals suffering from them and cardiovascular disease also is a major contributor to patient illness and mortality. It also is a primary driver of health care expenditure, costing more than 326 billion each year in India. Hypertension, or high blood pressure, or other neuro-hormonal disorders, is estimated to affect over 50 million people in India alone.
A number of drug treatments have been proposed for the management of neurological disorders, and other cardiovascular disorders. These include anaesthetics, anticonvulsants, antiemetic’s, antiparkinsonian agents, CNS stimulants, muscle relaxants, narcotic analgesics (pain relievers), nonnarcotic analgesics (such as acetaminophen and NSAIDs), and sedatives, and other medicament.
Hippophae rhamnoides also known as vinegar willow and blackthorn belongs to the genus Hippophae, which is widely distributed in the northwest region of China. It is a traditional herbal medicine used by Tibetan medicine practitioners in China. At present, Sea-buckthorn is allowed to be used in food, medicine, and other industries as a medicine and food. It has a wide range of application prospects.
Several patent applications are present that disclosed the use of Hippophae rhamnoides and their applications. A few of them are listed below

KR101685438B1 discloses a composition for treating and preventing physical stress-related diseases containing vitamin tree extract as an active ingredient.

US2005214394A1 discloses a methods and compositions for prevention and therapy of cancer using a therapeutically effective amount of an extract of Hippophae rhamnoides (sea-buckthorn) leaves, berries, and seeds are provided. Novel uses of these compositions in different stages of cancer therapy are disclosed. Novel compositions comprising Hippophae rhamnoides extracts that preferentially inhibit COX-2 over COX-1 are provided. Compositions comprising therapeutically effective amounts of at least one chemotherapeutic agent in addition to Hippophae rhamnoides are provided.

The need for the present invention arises as none of the existing inventions have been characterized for an effective and approach related to a variety of neurologic and cardiovascular disorders. The present invention provides an insight into the underlying problems at the molecular level and provides an effective approach that scientifically elucidates the mechanism of action, hence the present invention has an edge over the conventional remedies mentioned in the prior art.

OBJECTIVE OF THE INVENTION

The main objective of the present invention is the management of neurologic and cardiovascular disorders using the fermented dry powder of sea buckthorn pulp.
Yet another objective of the present invention is to provide a herbal formulation that helps in slows down the early cognitive decline and neurologic disorders.
Yet another objective of the present invention is the increased therapeutic efficacy of the biomarkers, which are identified using the fermented dry powder of sea buckthorn pulp.
Yet another objective of the present invention is that the present invention helps increase the general body resistance against physical and mental stress.

Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided herein below, in which various embodiments of the disclosed invention are illustrated by way of example.

SUMMARY OF THE INVENTION

The present invention describes a method for extracting a plurality of biological markers from the sea buckthorn pulp liquid. The method comprising of a sea buckthorn pulp liquid in the concentration range of 100 to 300mg. The sea buckthorn pulp liquid is fermented in a bioreactor and is freeze-dried at 0 to -80? to obtain a dry powder. This dry powder is further analyzed to obtain and identify the plurality of biological markers in the assay composition. Herein, the plurality of biomarkers comprises a first marker, a second marker, a third, and a fourth marker. In an embodiment of the present invention, the markers are selected from but not limited to Quercetin, Omega Fatty Acid, Vitamin C, Folic Acid, Vitamin K, and Lactic acid. In another aspect of the invention, the markers are measured in the assay composition using a multiplex assay format. In an embodiment of the present invention, the method discloses an herbal formulation for the management of neurologic and cardiovascular disorders using the fermented dry powder of sea buckthorn pulp. In yet another aspect of the invention, the herbal formulation is using an at least one pharmaceutically accepted excipient, wherein the at least one pharmaceutically accepted excipient is selected from a group of minerals, vitamins, salt, fillers, binders, and combinations thereof. In an aspect of the present invention, the biomarkers are extracted using different techniques and are disclosed in the description of the present invention. In yet another aspect of the invention, the herbal formulation is a phytopharmaceutical product.
The main advantage of the present invention is the management of neurologic and cardiovascular disorders using the fermented dry powder of sea buckthorn pulp.
Yet another advantage of the present invention is that the present invention slows down the early cognitive decline and neurologic disorders.

Yet another advantage of the present invention is the increased therapeutic efficacy of the biomarkers, which are identified using the fermented dry powder of sea buckthorn pulp.
Yet another advantage of the present invention is that the present invention helps increase the general body resistance against physical and mental stress.
Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided herein below, in which various embodiments of the disclosed invention are illustrated by way of example

DETAILED DESCRIPTION OF THE INVENTION

Definition

The term “a” or “an”, as used herein, is defined as one. The term “plurality”, as used herein, is defined as two as or more than one. The term “another”, as used herein, is defined as at least a second or more. The terms “including” and/or “having”, as used herein, are defined as comprising (i.e., open language).
The term “comprising” is not intended to limit the present invention with such terminology rather is used in a wider sense. Any invention using the term comprising could be separated into one or more claims using “consisting” or “consisting of”. The term “comprising” may be used interchangeably with the terms “having” or “containing”.
Reference in this document to “one embodiment”, “certain embodiments”, “an embodiment”, “another embodiment”, and “yet another embodiment” or similar terms, throughout the document means that a specific feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of such phrases in various places, this specification throughout are not necessarily all referring to the same embodiment. Furthermore, the specific features, structures, or

characteristics are combined in any suitable manner in one or more embodiments without limitation.
The term “or” as used herein is to be interpreted as inclusive or meaning any one or more combinations. Therefore, “A, B or C” means any of the following: “A; B; C; A and B; A and C; B and C; A, B and C”. An exception to this definition will occur only when a combination of elements, functions, steps, or acts are in mutually exclusive, inherently.
As used herein, the term "one or more" generally refers to, but is not limited to, singular as well as the plural form of the term.
The drawings featured in the figures are to illustrate certain convenient embodiments of the present invention and are not to be considered as a limitation to that. Term "means" preceding a present participle of operation indicates the desired function for which there is one or more embodiments, i.e., one or more methods, for achieving the desired function and that one skilled in the art could select from these or their equivalent in view of the disclosure herein and use of the term "means" is not intended to be limiting.
The present invention describes a method for extracting a plurality of biological markers from the sea buckthorn pulp liquid. The method comprising of a sea buckthorn pulp liquid in the concentration range of 100 to 300mg. The sea buckthorn pulp liquid is fermented in a bioreactor and is freeze-dried at 0 to -80? to obtain a dry powder. This dry powder is further analyzed to obtain and identify the plurality of biological markers in the assay composition. Herein, the plurality of biomarkers comprises a first marker, a second marker, a third, and a fourth marker.
In an embodiment of the present invention, the markers are selected from but not limited to Quercetin, Omega Fatty Acid, Vitamin C, Folic Acid, Vitamin K, and Lactic acid.

In another aspect of the invention, the markers are measured in the assay composition using a multiplex assay format. In yet another aspect of the invention, the herbal formulation is a phytopharmaceutical product.

In an embodiment of the present invention, the method discloses an herbal formulation for the management of neurologic and cardiovascular disorders using the fermented dry powder of sea buckthorn pulp.
In yet another aspect of the invention, the herbal formulation is using an atleast one pharmaceutically accepted excipient, wherein the at least one pharmaceutically accepted excipient is selected from a group of minerals, vitamins, salt, fillers, binders, and combinations thereof.

The experimental results were conducted to evaluate the Neuroprotective efficacy of Herbal Based Standardized Extracts of Seabuckthron on Rat Model of Alzheimer Type of Dementia of test compound.

1. Materials and methods Test Article:
Product Name: Fermented Sea buckthorn Pulp Powder
Physical Appearance: Brown /yellowish colored Coarse powder
Odor: Characteristic
Mfg. Date: Jan 21
Exp. Date: Dec 22

2. Study Design:
2.1 Grouping and dosage of animals
In the stereotaxic apparatus and the skull was exposed by a midline sagittal incision on the scalp. Two holes were drilled through the skull for placement of injection cannulae into the lateral cerebral ventricles using the following coordinates: 0.8 mm posterior to bregma; 1.4 mm lateral to sagittal suture; and 3.6 mm ventral from the surface of the brain. A single dose of gentamicin (40 mg/kg, i.p.) was injected following surgery, and a daily application of antiseptic powder (Neosporin®) was done to prevent sepsis.

Method: Freshly prepared Aß1-42 peptide solution at a nominal dose of 5µM/kg into the lateral cerebral ventricles using the previously reported coordinates. Alzheimer’s disease (AD) was induced in wistar rats by intracerebroventricular administration of Amyloid Beta (ICV-Aß1-42) bilaterally injected into the cerebral ventricles. Behavioral assessment for abnormalities in learning and memory was monitored by Morris water maze and passive avoidance task.

The test dosages (One dose 600 mg/kg) was selected along with control group. The test compound was administered orally using an oral gavage tube. The test compound was suspended in water with 0.5% carboxymethyl cellulose prior to administration. Animals in the control group were handled in an identical manner to the test group subjects. The control group received the same vehicle used for administering the test compound in test groups.

3. ADMINISTRATION OF THE TEST ARTICLE
After one week of acclimatization in the animals of disease group and treatment group brain surgery has been done by using the stereotaxic apparatus to induce the Alzheimer disease in animals. The brain surgery took two weeks to heal the wound after the recovery from brain surgery the animals are subjected for treatment by using the test article. The animals were divided into two groups each group, control and treatment. The animals were dosed daily once with the test article for 15 days. Test formulations was administered by oral gavage to each rat of treatments groups of all dose groups animals using the using a suitably graduated disposable syringe. The volume of administration to individual rat was adjusted according to its body weight that was recorded just before dosing.

4. OBSERVATIONS
Behavioral Parameters: Behavioral Assessment for Cognitive Function. Single animal behavioral analysis might not be able to cover the entire features of memory and learning. Hence, performed a series of behavioral studies like Morris water maze (MWM) and passive avoidance analysis. Since the introduction of MWM in 1984, it has been widely reported in rodents for behavioral analysis. Moreover, short-term memory loss was determined by passive avoidance analysis using retention latency time.

Morris water maze (MWM) Test:In our study, a circular water tank with the dimensions of 120 cm diameter and 60 cm height was selected for performing MWM analysis. The tank was filled with water (27 ± 1 °C) until a depth of 40 cm and was further divided into four equal quadrants like southwest, southeast, northwest, and northeast. In one of the selected quadrants, a hidden platform (10 cm × 5 cm) was positioned 2 cm beneath the water surface, and it has been located in the same spot during the entire experimentation. Each rat’s swimming was tracked and recorded using video camera linked to a computer system. Prior to start of training, animals were permitted to freely swim in the tank for about 60 s by removing the hidden platform. Further, animals that were provided training have a ceiling time of about 60 s containing four trials per session (once from each quadrant) for 4 days. Once the animals climb onto the hidden platform, they are allowed to remain there itself for 30 s before the beginning of next session. If the animal is unable to reach the platform in the experimental time of 60 s, it was gently located over the hidden platform and allowed to stay here for the same time interval. Moreover, the time required to identify the hidden platform was recorded as latency in seconds by utilizing ANY-maze video tracking device (Stoelting Co., USA).

Passive Avoidance Test:
In brief, the device consists of two chambers; one is illuminated, and the other one is a dark chamber. However, they were divided by a guillotine door and have a shock scrambler grid floor in both of the chambers (Inco Ambala, India). In the acquisition trial, each animal was positioned in the illuminated chamber, and later at 60 s the separating door was unlocked. The time spent to enter the dark compartment was recorded as initial latency, and if any animal takes above 60 s, it was excepted for further procedure. Once the animal enters the dark chamber, the separating door was locked and supplied an electrical foot shock with a capacity of 50 V over the grid floor for about 3 s. Retention latency was determined after 24 h in a similar manner, but the electrical foot shock was not applied and the latency time was measured up to 300 s. Short retention latency time indicates lesser retention memory. The apparatus was properly cleaned and dried between trails with dilute (70% v/v) alcohol.

Biochemical Parameters:
The neuro-inflammatory markers TNF-a and Amyloid beta were estimated using standard ELISA kits as per manufacturer’s instructions. Level of oxidative stress markers like malonyldialdehyde (MDA), Catalase, reduced glutathione (GSH) and superoxide dismutase (SOD) was also analyzed as per following protocols.

Estimation of MDA levels: The quantitative measurement of MDA, lipid peroxidation end product was done using the method described by Wills (Wills, 1966). In brief, 0.1 ml of brain homogenate was heated at 95°C for 1 h with 0.1 ml of 8.1% sodium dodecyl sulphate (SDS), 0.75 ml 20% glacial acetic acid, 0.75 ml of 0.8% thiobarbituric acid (TBA) and 0.3 ml of distilled water. The supernatant was collected by centrifugation at 10000 rpm for 10 min. The amount of MDA in the supernatant was estimated spectrometrically at 532 nm using Tetramethoxypropane as standard. (Epoch microplate spectrophotometer, Biotek, Winooski, US). The values are represented as a nanomole of MDA per milligram of protein.

Estimation of SOD: The estimation of SOD is performed based on autoxidation epinephrine at pH 10.4. Briefly, the supernatant of brain homogenate was mixed with 0.8ml of 50 mM glycine buffer (pH 10.4) and 0.02 ml of (-)-epinephrine was added. After 5 min the absorbance was measured at 480 nm. (UV-1800 Spectrophotometer, Shimadzu, Japan). The activity of SOD was represented as U/milligram of protein.

Estimation of GSH levels: The reduced glutathione in brain homogenate was estimated by Ellman method. In brief, an equal volume of sulphosalicylic acid (5%) and homogenate were mixed and cold digested for 1 h at 4 °C. Further, the supernatant was collected after centrifuging at 10000rpm for 10min at 4 °C. The 50µl of supernatant was mixed with 450µl of phosphate buer and 1.5 ml of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) in 0.1 M phosphate buer, (pH 8.0). After incubating the above reaction mixture for 10 min at 37 °C, the absorbance was measuredimmediately at 412 nm using a spectrophotometer. (Epoch microplate spectrophotometer, Biotek,Winooski, US). Results were represented as micromole per milligram protein.

Estimation of Catalase: CAT is a hemeprotein, localized in the peroxisomes or the microperoxisomes. This enzyme catalyses the decomposition of H2O2 to water and oxygen and thus protecting the cell from oxidative damage by H2O2 and OH. CAT is a key component of the antioxidant defense system. Inhibition of these protective mechanisms results in enhanced sensitivity to free radical-induced cellular damage.0.1 ml of supernatant was added to cuvette containing 1.9 ml of 50 mM phosphate buffer (pH 7.0) Reaction was started by the addition of 1.0 ml of freshly prepared 30 mMH2O2.The rate of decomposition of H202 was measured spectrophotometrically from changes in absorbance at 240 nm using a spectrophotometer. (Epoch microplate spectrophotometer, Biotek,Winooski, US).

5. RESULTS
Test Result (Control vs treated)
Passive Avoidance Task Non-Significant
Morris Water Maze Non-Significant
Lipid Peroxidation (MDA) Decreasing trend
Superoxide dismutase (SOD) Decreasing
Catalase Decreasing
Glutathione Reductase (GSH) Decreasing

6. CONCLUSION
The extracts drug did not show any significant neuroprotective effect when experiment was performed on the animal models. The biochemical parameters are evaluated to conclude the results where the levels of oxidative markers such as GSH, MDA, SOD and catalase were decreased in the treatment group when compared to the control group but not significant statistically. The assessment of the behavioral parameters were also non-significant when performed using MWM and Passive Avoidance task task to evaluate the learning and memory features of animals. As, the study was performed on used animals as a pilot study and the number of animals used in the study were significantly less because of which it is very difficult to conclude any significant result. Hence, this study needs to be reconducted with appropriate number of newly acquired animals in order to remove any confounding variables from the study.

Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided herein, in which various embodiments of the disclosed present invention are illustrated by way of example and appropriate reference to accompanying drawings. Those skilled in the art to which the present invention pertains may make modifications resulting in other embodiments employing principles of the present invention without departing from its spirit or characteristics, particularly upon considering the foregoing teachings. Accordingly, the described embodiments are to be considered in all respects only as illustrative, and not restrictive, and the scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description or drawings. Consequently, while the present invention has been described with reference to particular embodiments, modifications of structure, sequence, materials and the like apparent to those skilled in the art still fall within the scope of the invention as claimed by the applicant.

CLAIMS:1. A method for extracting a plurality of biological markers from the sea buckthorn pulp liquid, the method comprising of:
a sea buckthorn pulp liquid in the concentration range 100 to 300mg, the sea buckthorn pulp liquid is fermented in a bioreactor, the fermented sea buckthorn pulp liquid is freeze-dried at 0 to - 80? to obtain a dry powder,
the dry powder is further analyzed to obtain and identify the plurality of biological markers in the assay composition.
wherein, the plurality of biomarkers comprises a first marker, a second marker, a third, and a fourth marker.
2. The method as claimed in claim 1, wherein, the markers are selected from but not limited to Quercetin, Omega Fatty Acid, Vitamin C, and Folic Acid.
3. The method as claimed in claim 1, wherein the marker is measured in the assay composition using a multiplex assay format.
4. The method as claimed in claim 1, wherein the method discloses an herbal formulation for the management of neurologic and cardiovascular disorders using the fermented dry powder of sea buckthorn pulp, wherein, the herbal formulation is a phytopharmaceutical product .
5. The herbal formulation as claimed in claim 4, wherein the herbal formulation is using an atleast one pharmaceutically accepted excipient, wherein the atleast one pharmaceutically accepted excipient is selected from a group of minerals, vitamins, salt, fillers, binders, and combinations thereof.
6. The markers as claimed in claim 1, wherein, the first marker, Quercetin is extracted using the following steps:
200mg of sea buckthorn is treated with 10mL Dimethyl sulphoxide and 0.1N Methanolic Hydrochloric Acid (10:90), resulting in a solution,

the above solution is sonicated for 20min with intermediate shaking and vortex further for 15 min,
the above solution is then filtered with a 045µ Nylon filter.
7. The markers as claimed in claim 1, wherein, the second marker, omega fatty acid is extracted using the following steps:
a sea buckthorn pulp liquid of 250 mg is treated with 100mg pyragallol, 4 mL ethanol, 10mL of 14% Hydrochloric Acid into 250 round bottom flask,
Shake and reflux the treated liquid for 60min at 80? on waterbath, after cooling the solution, add 25mL diethyl ether and 25mL petroleum ether and vortex for 5min,
transfer the treated liquid into a centrifuge tube and centrifuge for 5min at 1000rpm,
Separate the organic upper layer from the treated liquid and evaporate on waterbath at 90 ?. Then add 2mL chloroform and 2mL diethyl ether, Shake and evaporate it,
Rinse the beaker by adding 2mL Methanolic boron trifluoride and 1mL toluene and transfer into GC-HS Vial. Rinse the same beaker with 1mL methanol. Seal the vial and mix it. Kept in the oven at 105? for 45 min.
Cool and treated this organic solution with 5mL 20% sodium sulfate solution and then transfer the whole content of GC-HS vial into a centrifuge tube containing 2mL n-Hexane. Vortex for 5min and then Centrifuge for 5min at 3000rpm. The upper layer of the treated liquid is used to study omega fatty acid.
8. The markers as claimed in claim 1, wherein, the third marker, Vitamin C is extracted using the following steps:
a sea buckthorn pulp liquid in the concentration range of 100 to 200mg is treated with 5mL acetonitrile and water in the ratio of (40:60),

the treated liquid is sonicated for 30min with intermediate shaking, and is made up to 10mL with acetonitrile and sonicate further for 15 min,
the treated liquid is then filtered with a 045µ Nylon filter to get the desired product.
9. The markers as claimed in claim 1, wherein, the fourth marker, folic acid is extracted using the following steps:
Transfer about 1.0 g of a sea buckthorn liquid into 50 ml volumetric flask, add 5 ml 0.05 N Sodium Hydroxide Solution. Sonicate for 20 minutes with continuous vigorously shaking at below 10 ? of sonicator bath temperature and then add 15 ml water slowly through the wall of the volumetric flask (i.e. Drop wise to avoid foaming), Sonicate for 10 Minutes with intermittent shaking at below 10 ? of sonicator bath temperature and Adding 5 ml methanol slowly and mix carefully to avoid the foaming. Then makeup to volume slowly up to the mark with methanol. Transfer the content of the volumetric flask into a centrifuge tube and centrifuge the tube content at 3000 RPM for 10 minutes. Filter it to get the desired product.