DESC:FIELD OF THE INVENTION
The present invention provides a composition comprising therapeutically effective amounts of extracts of Nyctanthes arbortristis and Andrographis paniculata for treating inflammatory conditions.
BACKGROUND OF THE INVENTION
Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants, and is a protective response involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, and initiate tissue repair.
Inflammation can be either short-lived (acute) or long-lasting (chronic). Acute inflammation goes away within hours or days. Chronic inflammation can last months or years, even after the first trigger is gone. Symptoms of inflammation include redness, a swollen joint that may be warm to the touch, joint pain, joint stiffness, a joint that doesn’t work as well as it should, and the like. Some types of arthritis are a result of inflammation, such as rheumatoid arthritis, psoriatic arthritis, gouty arthritis. Other painful conditions of the joints and musculoskeletal systems like osteoarthritis, fibromyalgia, muscular low back pain, muscular neck pain, and the like, also arise due to inflammation. Yet another inflammatory condition that is severely debilitating is ankylosing spondylitis.
Rheumatoid arthritis, or RA, is an autoimmune and inflammatory disease, which means that the immune system attacks healthy cells in our body by mistake, causing inflammation (painful swelling) in the affected parts of the body. The inflammation associated with rheumatoid arthritis can damage other parts of the body as well. While new types of medications have improved treatment options dramatically, severe rheumatoid arthritis can still cause physical disabilities.
Conventionally there are three general classes of drugs commonly used in the treatment of rheumatoid arthritis: non-steroidal anti-inflammatory agents (NSAIDs), corticosteroids, and disease modifying anti-rheumatic drugs (DMARDs). NSAIDs and corticosteroids have a short onset of action while DMARDs can take several weeks or months to demonstrate a clinical effect. The initial mode of treatment of rheumatoid arthritis includes a combination of DMARD tablets and other medications. These medicines ease the symptoms of the condition and slow down its progression. In some cases, the patient may be advised to use painkillers, such as paracetamol or a combination of paracetamol and codeine, to relieve the pain associated with rheumatoid arthritis. In addition to, or instead of, the painkillers mentioned above, a non-steroidal anti-inflammatory drug (NSAID)are prescribed. This may be a traditional NSAID, such as ibuprofen, naproxen or diclofenac and a COX-2 inhibitor, such as celecoxib or etoricoxib. These medicines can help relieve pain while also reducing inflammation in the joints. It must be noted that taking NSAIDs can increase the risk of serious stomach problems, such as internal bleeding. DMARDs work by blocking the effects of the chemicals released when the immune system attacks joints, which could otherwise cause further damage to nearby bones, tendons, ligaments and cartilage. The DMARDs that may be used include: Methotrexate, leflunomide, hydroxychloroquine, sulfasalazine. However, this treatment leads to many side effects such as fatigue, loss of appetite, a sore mouth, diarrhoea, headaches, hair loss (methotrexate), dizziness, joint aches and pains, itching or mild rash.
There is, therefore, a need for a treatment which uses natural ingredients with no side effects or minimal side effects, as the treatment may go on for longer duration due to the chronic nature of the disease.
Burgos R A et al., 2009 has investigated the efficacy of Andrographis paniculate for the relief of rheumatoid arthritis symptoms. Fenfen Li et al., 2018 discloses a combination of andrographolide (from Andrographis paniculate) and methotrexate combined therapy for treatment of arthritis. WO2018065836A1 discloses a topical anti-arthritis and anti-paralytic herbal oil-based composition comprising Nyctanthes arbortristis, but it does not disclose any oral composition in combination with Andrographis paniculate. CN105031516A discloses a Chinese herbal composition for treating erysipelas, an inflammatory condition of the skin using 40 parts of Rheum officinale, 18 parts of golden cypress, 16 parts of Curcuma longa, 14 parts of Nyctanthes arbortristis, 12 parts of Arisaema heterophyllum, 16 parts of Polygonum cuspidatum, 14 parts of Scutellaria baicalensis, 12 parts of Tetrapanax papyrifer pollen, 10 parts of cape jasmine, 15 parts of Gueldenstaedtia diversifolia,25 parts of cupressin, 18 parts of Morinda angustifolia,8 parts of asiatic bilberry and 80 parts of Radice trichosanthis. However, none of the prior arts disclose an oral anti-inflammatory composition containing Andrographis paniculate and Nyctanthes arbortristis.
OBJECT OF THE INVENTION
Some of the objects of the present disclosure are listed herein below.
The primary object of the present disclosure is to provide a composition for treating inflammatory conditions.
Another object of the present disclosure is to provide a composition that does not suffer from the limitations of known compositions based on synthetic compounds, which cause several side effects, including fatigue, diarrhoea, headaches, joint aches and the like.
Yet further object of the present disclosure is to provide a composition comprising only natural ingredients.
Still further object of the present disclosure is to provide a composition for treatment and management of inflammatory diseases such as rheumatoid arthritis.
SUMMARY OF THE INVENTION
In an aspect of the present disclosure, there is provided a composition for treating inflammatory conditions, the said composition comprising (i) about 80 mg to about 450mg of an extract of Andrographis paniculata, (ii) about 400mg to about 2000mg of an extract of Nyctanthes arbortristis, and (iii) pharmaceutically acceptable carriers. The extract is usually an aqueous, alcoholic or hydroalcoholic extract of Nyctanthes arbortristis and Andrographis paniculata.
In another aspect of the present disclosure, the pharmaceutically acceptable carriers are selected from the group comprising diluents, disintegrants, binders, lubricants, solvents, cosolvents, isotonicity agents, chelating agents, buffers, pH-adjusting agents, solubilizers, stabilizers, dispersion agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, and flavouring agents.
In yet another aspect of the present disclosure, the composition is in the form of a dosage form selected from the group comprising capsules, tablets, granules, powders filled in sachet, oral liquid solutions, oral liquid suspensions, syrups, lozenges, solid dispersion, oral films and dry suspensions for reconstitution. In a preferred aspect of the present disclosure, the composition is in the form of a tablet. In another embodiment, the composition is in the form of a capsule. The tablet and capsules may be prepared by processes conventional in the art. They act as a convenient means to administer the combination of the extracts of the present disclosure to the patients.
In still another aspect of the present disclosure the composition is used for treating rheumatoid arthritis and is administered to a subject such that the composition comprises about 80 mg to about 450 mg of Andrographis paniculata and about 400 mg/ to about 2000 mg of Nyctanthes arbortristis .
DETAILED DESCRIPTION OF THE INVENTION
Andrographis paniculata (AP) is an important medicinal plant and widely used around the world. It belongs to the family Acanthaceae. AP is used as a traditional herbal medicine in Bangladesh, China, Hong Kong, India, Pakistan, Philippines, Malaysia, Indonesia, and Thailand and is ethnobotanically used for the treatment of snake bite, bug bite, diabetes, dysentery, fever, and malaria. In the Unani and Ayurvedic medicines, AP is one of the commonly used medicinal plants. In recent times, commercial preparations of this plant extract are also used in certain countries. The aerial parts of AP have been described for its numerous uses in the extraction of phytoconstituents; however, leaves, stems, roots, and whole plants have also been reported for phytochemicals with pharmacological activities.
Taxonomic Hierarchy: Taxonomic hierarchy of AP is as follows:
Domain: Eukaryota,
Kingdom: Plantae,
Subkingdom: Tracheobionta,
Superdivision: Spermatophyta,
Division: Angiosperma,
Class: Dicotyledonae,
Subclass: Gamopetalae,
Series: Bicarpellatae,
Order: Personales,
Family: Acanthaceae,
Subfamily: Acanthoideae,
Tribe: Justiciae,
Subtribe: Andrographideae,
Genus: Andrographis,
Species: A. paniculata
Andrographis paniculata contains andrographolide which is chemically described as (S,E)-4-Hydroxy-3-(2-((1R,4aS,5R,6R,8aS)-6-hydroxy-5-(hydroxymethyl) -5,8a-dimethyl-2-methylenedecahydronaphthalen-1-yl)ethylidene)dihydrofuran-2(3H)-one (Formula I).

Formula I
Nyctanthes arbortristis (NAT) Linn, belonging to the family Oleaceae, is known to emit pleasant and strong fragrance during the whole night, hence popularly called “Harsinghar” in Hindi and “Night Jasmine” in English. The juice of its leaves and decoction are commonly used in traditional system of medicine to treat rheumatism, arthritis, and inflammatory disorders.
Taxonomic Hierarchy: Taxonomic hierarchy of NAT is as follows:
Domain- Eukaryota
Kingdom- Plantae
Phylum- Spermatophyta
Class- Magnoliopsida
Subclass- Asteridae
Order- Scrophulariales
Family- Oleaceae
Genus- Nyctanthes L
Species- Nyctanthes arbortristis

Nyctanthes arbortristis contains Beta-sitosterol (b-sitosterol), which is chemically described as (3S,8S,9S,10R,13R,14S,17R)-17-[(2R,5R)-5-ethyl-6-methylheptan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol (Formula II).

Formula II

While AP and NAT have been known individually for treating inflammation and various other ailments, there is no art that uses therapeutically effective amounts of a combination of the extracts of both AP and NAT for use in the treatment of inflammation or inflammatory conditions. The present invention provides a composition comprising therapeutically effective amounts of extracts of Nyctanthes arbortristis (NAT) and Andrographis paniculata (AP) for treating inflammatory conditions. Preferably, the invention provides a composition comprising therapeutically effective amounts of extracts of Nyctanthes arbortristis (NAT) and Andrographis paniculata (AP) for treating arthritis.
In an embodiment, the present disclosure provides a composition for treating inflammatory conditions, the said composition comprising (i) about 80mg to about 450mg of an extract of Andrographis paniculata, (ii) about 400 mg to about 2000 mg of an extract of Nyctanthes arbortristis, and (iii) pharmaceutically acceptable carriers.
In another embodiment of the present disclosure, the composition comprises about 84mg of an extract of Andrographis paniculata and about 420mg of an extract of Nyctanthes arbortristis.
In yet another embodiment of the present disclosure, the composition comprises about 420 mg of an extract of Andrographis paniculata and about 1680mg of an extract of Nyctanthes arbortristis.
In a preferred embodiment of the present disclosure, the composition comprises about 300mg of an extract of Andrographis paniculata and about 900mg of an extract of Nyctanthes arbortristis.
In a more preferred embodiment of the present disclosure, the composition comprises about 100mg of an extract of Andrographis paniculata and about 300mg of an extract of Nyctanthes arbortristis. A single tablet composition comprising 100mg AP and 300mg NAT may be administered one to three times a day. Thus, the tablets may be used to administer a total daily dose of 300mg of AP and 900mg of NAT, i.e. by administering three tablets daily. The whole plant, i.e. all parts of AP and NAT, were used. The parts were dried, pulverized and then used for extraction. Extraction procedure is generally of three types: Solid/liquid extraction, liquid/liquid extraction and acid/base extraction. Solvent extraction is the most prevalent type of extraction, followed by distillation extraction, pressing, and sublimation, all of which are based on extraction principles. The extraction procedure may be conventional or non-conventional. The major factors based on which the extraction procedure varies are the time taken for extraction, the temperature of extraction, the dimension of the tissue or its part to be extracted, the ratio of solvent and sample, and the pH of the solvent, among some others.
Solid/liquid extraction is used in traditional procedures. It is based on the principle that when a solid component comes in contact with a solvent (liquid), the soluble parts of the solid dissolve in the solvent, and this migration of the soluble ingredients into the solvent occurs along a concentration gradient. The rate of transfer is determined by the constituent concentrations and continues until equilibrium is attained. Different types of conventional techniques are maceration (cold and hot), digestion, infusion, elution (lixiviation), decoction, reflux extraction, tincture, percolation, steam distillation, hydrodistillation, soxhlet extraction, serial exhaustive extraction, and fermentation (aqueous-alcoholic extraction). These methods may be used for the extraction of AP and NAT to obtain extracts that can then be mixed in different ratios or amounts, to prepare a composition that is suitable for administration to patients.
In an embodiment of the present disclosure, the extract is an aqueous, alcoholic or hydroalcoholic extract of Nyctanthes arbortristis and Andrographis paniculata. The solvents that are suitable for obtaining the extracts of the plants mentioned above, include but are not limited to, water, methanol, ethanol, propanol, isopropanol, dimethyl formamide, dimethyl sulfoxide, dichloromethane methylene dichloride, acetone, hexane, diethyl ether and other solvents conventionally used in the extraction of herbal products. These solvents can be used alone or in admixture, in various quantities, as may be required. A skilled person is adept with the knowledge of assessing the suitable solvent needed for the extraction, and can use the same (See Abubakar et al, J Pharm Bioallied Sci. 2020 Jan-Mar; 12(1): 1–10). In preferred embodiments of the present invention, the solvent used is selected from water, alcohol or mixtures thereof. In highly preferred embodiments, methanol, ethanol and dichloromethane may be used as the solvents for extraction of Nyctanthes arbortristis and Andrographis paniculata. The individual extracts thus obtained may then be mixed in defined amounts to obtain the composition of the present invention.
ß-sitosterol content of Nyctanthes Abortristis and Andrographolide content of Andrographis paniculata are known to have anti-arthritic activity. These ingredients were extracted via fractionation from the plants of NAT and AP using conventional methods and formulated together into a composition.
Fractionation of extracts of AP and NAT is preferably carried out using n-butanol and petroleum ether. Other suitable solvents conventionally known in the art may also be used.
The aerial part of AP is most commonly used; its extracts contain diterpenoids, diterpene glycosides, lactones, flavonoids, and flavonoid glycosides. Among the various fractions of methanolic extract of Andrographis paniculata, the dichloromethane fraction has a major content of Andrographolide (ANG).
The present invention preferably uses the n-butanol fraction of the Nyctanthes abortristis, which contains the iridoid glycosides namely Arbortristoside A (major content), B and C. The petroleum ether fraction of Nyctanthes Abortristis leaves containing ß-sitosterol as the major content, and nyctanthic acid, is the preferred extract used in the compositions of the present invention.
The analysis of bioactive compounds present in the plant extracts is analysed through various techniques known in the art involving the applications of chromatographic techniques such as HPLC (High Performance Liquid Chromatography), TLC (Thin Layer Chromatography), HPTLC (High Performance Thin Layer Chromatography), OPLC (Optimum Performance Laminar Chromatography), GC (Gas Chromatography), PC (Paper Chromatography), CC (Column Chromatography) and it’s detection through Fourier Transform Infra-Red spectroscopy (FTIR), Nuclear Magnetic Resonance (NMR), and Mass Spectrometry (MS).
The compositions of the present invention are formulated for oral administration. The oral compositions of the present invention may be selected from, but are not limited to, tablets, capsules, granules, powders filled in sachet, oral liquid solutions, syrups, oral liquid suspensions, emulsions, lozenges, solid dispersion, oral films, dry suspensions for reconstitution and the like. In preferred embodiments of the present disclosure, the composition is in the form of a tablet.
The compositions may contain inert excipients that are conventionally used in oral formulations, but are not limited to, one or more agents selected from diluents, disintegrants, solvents, cosolvents, isotonicity agents, chelating agents, buffers, pH-adjusting agents, solubilizers, stabilizers, dispersion agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, flavouring agents, diluents and other known additives to provide an elegant presentation of the drug, i.e., a compound described herein or pharmaceutical composition thereof, or aid in the manufacturing of the pharmaceutical product.
Examples of suitable diluents that may be used in the compositions of the present invention include, but are not limited to, microcrystalline cellulose, dibasic calcium phosphate, lactose, talcum, calcium phosphate, magnesium carbonate, magnesium oxide, calcium sulphate, saccharose, monosaccharides, such as glucose, maltodextrin, dextrates, dextrin and combinations thereof.
Examples of suitable disintegrants that may be used in the compositions of the present invention include, but are not limited to, crosslinked polymers, starches and modified starches. Preferred disintegrants are crosslinked polyvinylpyrrolidone (crospovidone), crosslinked sodium carboxymethyl cellulose (croscarmellose sodium), corn starch and sodium starch glycolate and combinations thereof.
Examples of binders that may be used in the compositions of the present invention include, but are not limited to, maltose syrup powder, gum arabic, sodium alginate, propylene glycol alginate ester, hydrolyzed gelatin powder, fructose, carboxylvinyl polymer, hydrous silicon dioxide, agar powder, light anhydrous silicic acid, polyvinylpyrrolidone, copolydone, Polyvinyl acetate (PVA), cellulose acetate phthalate, dioctyl sodium sulfosuccinate, starch, cornstarch, tragacanth, lactose, concentrate glycerin, sucrose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose (HPC), hydroxypropylmethyl cellulose (HPMC), hydroxypropylmethyl cellulose phthalate, vinylpyrrolidone-vinyl acetate copolymer, glucose, pregelatinized starch, fumaric acid, povidone, polyvinyl alcohol, D-mannitol, and methyl cellulose and combinations thereof.
Examples of glidants that may be used in the compositions of the present invention include, but are not limited to, hydrous silicon dioxide, light anhydrous silicic acid, crystalline cellulose, synthetic aluminum silicate, titanium oxide, stearic acid, calcium stearate, magnesium stearate, calcium tertiary phosphate, talc and combinations thereof.
Lubricants are generally used in order to reduce sliding friction. Suitable lubricants that may be used in the compositions of the present invention include, but are not limited to, sodium stearyl fumarate, magnesium stearate, glyceryl behenate, stearic acid and adipic acid and combinations thereof.
The compositions of the present invention may be in the form of film coated tablets. Film-coatings can be prepared by using agents such as cellulose derivatives, poly(meth)acrylate, polyvinyl pyrrolidone, polyvinyl acetate phthalate, or natural rubbers such as carrageenan, and mixture thereof.
When the pharmaceutical composition of the present invention is a tablet, it may be produced by mixing pharmaceutically acceptable carriers such as an excipient, a disintegrant, and a binder with AP and NAT extracts, together or separately, and directly compressingthe mixture, or the tablet may be produced by granulating a mixed powder by fluidized-bed granulation, stirring granulation, or the like, and then compressing the obtained granules. The tablets so obtained can optionally be film coated with suitable polymers such as HPMC, HPC and PVA, wherein the film coating is carried out by processes known in the art, and typically to a weight gain of about 3-5% by weight of the tablet composition.
When the pharmaceutical composition of the present invention is a capsule, it can be produced by merely filling the granules or a mixture of the AP and NAT extracts and excipients, into appropriate capsule shells.
For liquid dosage forms such as solutions, suspensions and emulsions, the extracts of the AP and NAT can be mixed with suitable vehicle, solubilizer, suspending agent or an emulsifier to prepare the appropriate liquid formulation. The liquid dosage form may contain suitable viscosity enhancing agents such as polymers listed above, gums and gelling agents known in pharmaceutical art to provide a solution that provides a flowable liquid that can be easily dispensed.
The extracts of AP and NAT were combined and tested in mouse model for arthiritis and inflammation. As described in “Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers”, Jul 2005 by the USFDA, the effective dose established in animals can be converted to human equivalent doses (HED) by using a conversion factor. For example, for converting mouse dose in mg/kg to HED in mg/kg, the dose in mouse is to be multiplied by 0.08. Thus, a dose of 1.5mg/kg in mouse converts to HED of 1.2mg/kg. This HED of 1.2mg/kg would mean a dose of 84mg in a human weighing 70kg. Such calculation is routinely employed in the pharmaceutical art.
The components of the pharmaceutical composition are pharmaceutically acceptable, generally regarded as safe (GRAS), conventionally used in the pharmaceutical art, and provide a composition that is stable over its shelf life.
The term “shelf life”, as used herein, refers to the amount of time the pharmaceutical composition may be stored without loss of potency and/or performance profile. In some embodiments of the present invention, shelf life refers to the amount of time the pharmaceutical composition may be stored without loss of about 4%, about 3%, about 2% or about 1% of the potency and/or performance, when stored at room temperature, i.e. about 15oC to about 25oC. The stable compositions provided herein are designed to have shelf life of at least 24 months.
The term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10%.
The specification may use the terms AP and NAT to describe the extracts of Andrographis paniculate and Nyctanthes Arbortristis, respectively.

EXAMPLES
Example 1
Characterization of extracts
a. Arbortristoside (ARB)
The optimized method for the fractionation of Arbortristosides mixture from NAT is as follows:
Step 1. Extraction
Dried, pulverized material (dried powder) of the whole plant (300 gm) of Nyctanthes arbortrisis was transferred in a 2000 ml conical flask. A mixture of methanol and ethanol (1:1) (1500 ml) solution was added to the conical flask and covered with aluminum foil (cold maceration). It was kept aside for one week with occasional shaking every day. It was filtered after a week and the filtrate was collected. The filtrate was concentrated under reduced pressure using a rota evaporator at 70°C. The dried extract was collected, weighed, and the percentage yield was calculated. The extract was preserved and stored in an airtight container till further use.
Step 2. Fractionation Arbortristoside (ARB) mixture
Dried methanol extract (90.66 g) was suspended in 150 ml water and a small amount of ethanol (50ml) was added, if the extract was not dissolved in water. Water suspended extract was fractionated with n-butanol thrice (200 ml x 3) and collected. The n-butanol fraction was then evaporated. Semi-solid n-butanol fraction was stored and preserved in an airtight container, till further use. The presence of Arbortristoside mixture was identified with HPLC.

Quantification of ARB by HPLC
The n -butanol fraction of NAT was taken further for HPLC analysis.
Preparation of sample & standard solution: 10 mg standard and sample were dissolved in 10 ml Acetonitrile: Water (95:5) v/v, additionally a small amount methanol was added for proper dissolving and kept for sonication for 10 mins. From the above stock solution, 0.1ml was taken and volume was made up to 10ml (10 µg/ml).

Table 1: Optimized Chromatographic conditions
Stationary phase RP-C18e, Hibar 250-4,6.(5 um)
Mobile Phase Water:Acetonitrile (95:5v/v)
Elution mode gradient (50:50% v/v)
Flow rate 1 ml/min
Detection 210nm PDA (UPLC)
Injection volume 20 µl
Column temperature Room temperature
Retention time (i) 3. 342 min (ii) 3.649 min

HPLC software and system details:
LC Model: SPD-M20A 230V
CAT NO: 228-45005-38
SeriaL No: L20154503598 AE (MADE IN JAPAN)
Mfg by: SHIMADZU Corporation, Japan.
Software: LC Solution (UPLC)

Table 2: Quantification of ARB
Standard /Sample Concentration (µg/ml) Retention time (mts) Peak area
Average peak area Percentage of ARB
*Standard (p-methoxy cinnamic acid) 10 3.641
3.638 370422
285783 285783 97%
n-butanol fraction (Peak 2) 3.654
3.648 247558
300289 273924 83.4%
Standard (beta sitosterol) 3.729
3.663 190529
246345 218437 More
Than
40 %
n-butanol fraction (Peak 1) 3.348
3.345 53922
59646 56784 25.99%

As per the quantification analysis, the n-butanol fraction of NAT contains 83.4% of iridoid glycosides and 25.99% Beta-sitosterol.

b. Beta-sitosterol
The optimized method for the fractionation of Beta-sitosterol (alone) from the mixture in NAT is as follows –
Step 1: Extraction
A weighed quantity of 1000 g of Nyctanthes arbortrisis whole plant pulverized material (powder dried) was transferred in a 2000 ml conical flask. 1500 ml of methanol 85% was added in a conical flask and covered with aluminum foil (cold maceration). This was kept aside for one week, and shaken every day. After one week, the extract was filtered and the filtrate was collected. The filtrate was concentrated under reduced pressure by using a rota evaporator at 60°C. The dried extract was collected and weighed. The extract was preserved and stored in an airtight container till further use.

Step 2: Fractionation of beta-sitosterol
The required quantity of dried extract was suspended in water and a small amount of ethanol was added. The water suspended extract was fractionated with petroleum ether thrice. The petroleum ether fraction was collected and evaporated. The solid petroleum ether fraction was stored and preserved in an airtight container.
Quantification of beta-sitosterol by HPLC
The petroleum ether fraction of NAT was subjected to HPLC analysis.
Preparation of sample & standard solution:
10 mg standard and sample were dissolved in 10 ml Acetonitrile: Water (95:5) v/v. Additionally, a small amount methanol was added for proper dissolution and sonicated for 10 mins. From the above stock solution, 0.1ml was taken and volume was made up to 10ml, to give a solution of strength 10 µg/ml.
Table 3: Optimized Chromatographic conditions
Stationary phase RP-C18e, Hibar 250-4,6.(5 um)
Mobile Phase Water:Acetonitrile (95:5v/v)
Elution mode gradient (50:50% v/v)
Flow rate 1 ml/min
Detection 210nm PDA (UPLC)
Injection volume 20 µl
Column temperature Room temperature
Retention time (i) 3. 342 min (ii) 3.649 min

HPLC software and system details:
LC Model: SPD-M20A 230V
CAT NO: 228-45005-38
SeriaL NO: L20154503598 AE (MADE IN JAPAN)
Mfg by: SHIMADZU Corporation, Japan.
Software: LC Solution (UPLC)

Table 4: Quantification of beta-sitosterol
Standard /Sample Concentration (µg/ml) Retention time
(mts) Peak area Average peak area Percentage of beta-sitosterol
Standard (beta sitosterol) 10 3.729
3.663 190529
246345 218437 More
than 40%
Beta-sitosterol (NAT-1) 3.638
3.655 75684
80333 78009 35.7%
Beta-sitosterol (NAT-2) 3.631
3.643 77115
67921 72518 33.2%

Both samples (NAT-1 and NAT-2 obtained from two sources) contain around 35% of beta-sitosterol.

c. Andrographolide
Extraction and Fractionation of Andrographolide (ANG)
1000 gm of Andrographis paniculata (whole plant) powder was weighed and transferred to 5000 ml conical flask. Equal amount of (1500:1500 ml) Dichloromethane: Methanol (1:1) was added to it. The conical flask was covered with Aluminium foil and kept aside for 10 days (cold maceration). The mixture was shaken twice a day. The solvent was filtered and evaporated using Rota evaporator. The whole dried extract was collected and weighed. The dried extract was washed with toluene several times (greenish crystal were present). The whole extract was then dissolved in hot water and ethanol (if required). A small amount of charcoal was added and the mixture was boiled for 5 min and filtered. The filtrate was collected and partitioned with petroleum ether, ethyl acetate and dichloromethane until it became colourless. The dichloromethane fraction was evaporated up to 70% of its volume, and was kept aside overnight. The separated Andrographolide crystals were filtered and dried.
Quantification of Andrographolide (ANG) by HPLC
Preparation of standard solution:
From 1mg/ml reference standard stock solution a final concentration of 100 µg/ml was prepared using mobile phase [Methanol: Water (1:1)].
Preparation of sample solution:
10mg of test substance was dissolved in the mobile phase [(Methanol and Water) 1:1]. Further, 1 ml of this solution was diluted to 10 ml with mobile phase to obtain stock solution. From the above stock solution, 1 ml was diluted to 10 ml and sonicated for 15 minutes, followed by HPLC analysis.
Table 5: Optimized Chromatographic conditions
Stationary phase Shimpack C18 (250 x 4.6 mm i.d., 5µ)
Mobile Phase Methanol: Water
Elution mode gradient (50:50% v/v)
Flow rate 1 ml/min
Detection 223 nm
Injection volume 10 µl
Column temperature Room temperature
Retention time 3.7 min (ANG)
System Shimadzu HPLC Systems (Model: LC-20AD)
Data station LC Solution

Table 6: Quantification of Andrographolide (ANG)
Standard /Sample Concentration (µg/ml) Retention time
(mts) Peak area Percentage of ANG
Standard 100 µg/ml 3.717 8405 99.87
Sample AP-MP-1
Method 1 - 3.719 25197 29.9

Sample AP-MP-1 contains around 29.9% of andrographolide (ANG).

Example 2
Determination of Andrographis paniculata (AP) and Nyctanthes arbrotristis (NAT) having antiarthritic effects against Freund’s adjuvant-induced arthritis in rats.
A. Materials and method
Animals: The following laboratory settings were employed to house the 78 male Wistar rats: temperature range of 22±3°C, relative humidity range of 54–78%, and a cycle of 12 hours of light and 12 hours of darkness. The following laboratory settings were employed to house the 78 male Wistar rats: temperature range of 23.4–28.8°C, relative humidity range of 54–78%, and a cycle of 12 hours of light and 12 hours of darkness. The rats were given extracts perorally (p.o). The animals were divided into 13 groups of six, and the specifics of each group’s treatment are provided below:
Group-1: Normal Control (0.5% CMC, 10ml/kg, p.o., daily)
Group-2: Disease Control (0.5% CMC, 10ml/kg, p.o., daily)
Group-3: AP (15 mg/kg, p.o., daily)
Group-4: AP (30 mg/kg, p.o., daily)
Group-5: AP (60 mg/kg, p.o., daily)
Group-6: NAT (75 mg/kg, p.o., daily)
Group-7: NAT (150 mg/kg, p.o., daily)
Group-8: NAT (300 mg/kg, p.o., daily)
Group-9: AP (15 mg/kg, p.o., daily) and NAT (75 mg/kg, p.o., daily)
Group-10: AP (30 mg/kg, p.o., daily) and NAT (150 mg/kg, p.o., daily)
Group-11: AP (60 mg/kg, p.o., daily) and NAT (300 mg/kg, p.o., daily)
Group-12: Methotrexate (2 mg/kg, i.p., twice a week)
Group-13: Sulfasalazine (150 mg/kg, p.o., daily)
On day 1, all groups except group 1 animals were injected into the subplantar region of the left hind paw with 0.1 ml of complete Freund’s adjuvant. Dosing with the test compounds or the standard is started on the same day and continued for 12 days. Paw volumes of both sides and body weight were recorded on the day of injection, whereby paw volume is measured plethysmographically. On day 5, the volume of the injected paw is measured again, indicating the primary lesion and the influence of therapeutic agents on this phase. The severity of the adjuvant induced disease is followed by measurement of the noninjected paw (secondary lesions on day 21) with a plethysmometer. Purposely, from day 13 to 21, the animals will be not dosed with the test compound or the standard. On day 21, the body weight is determined again and the severity of the secondary lesions is evaluated visually and graded according to the following scheme score:

B. Lesion Score
Table 7: Lesion score
Name of the body part Evaluation criteria Score
Ears absence of nodules and redness 0
presence of nodules and redness 1
Nose no swelling of connective tissue 0
intensive swelling of connective tissue 1
Tail absence of nodules 0
presence of nodules 1
Forepaws absence of inflammation 0
inflammation of at least 1 joint 1
Hind paws absence of inflammation 0
slight inflammation 1
moderate inflammation 2
marked inflammation 3

The following parameters were measured and analysed
A. For primary lesions: The percent inhibition of paw volume of the injected left paw over disease control was measured at day 5.
B. For secondary lesions: The percentage inhibition of paw volume of the non-injected right paw over disease control was measured at day 21.
C. An arthritic index is calculated as the sum of the scores as indicated above for each animal. The average of the treated animals was compared with the control group.
D. The total percentage change is calculated as follows by addition of percent inhibition of the injected paw on day 5 + percent inhibition of the non-injected paw on day 21 + percent inhibition of the arthritic index.
C. Results
2.1 Effect of AP and NAT on clinical signs and body weight
All the animals except G1, normal control, showed pain symptoms such as decreased activity, and abnormal gait after Freund’s adjuvant administration. There was no significant difference in body weight was observed in all the treatment groups when compared to G2, disease control (P>0.05).
Table 8: Effect of Andrographis paniculata (AP) and Nyctanthes Arbrotristis (NAT) on body weight (g) of arthritic rats
Day-0 Day-7 Day-14 Day-21
G1: Normal Control 182.1±15.4 188.5±15.6 194.6±16.9 201±18.4
G2: Disease Control 177±13.9 180.1±14.1 183.1±14.2 185.3±14
G3: AP 15mg/kg 169.3±11.6 176.1±12.9 182±12.7 188.8±13.3
G4: AP 30 mg/kg 166.5±13.6 172.6±14.5 178.5±13.9 185±14.2
G5: AP 60 mg/kg 177.8±14 184.6±14.1 190.6±13.6 197.8±13.4
G6: NAT 75 mg/kg 174.5±13.9 181.6±14.8 188.6±15.8 195.6±15.2
G7: NAT 150 mg/kg 166.6±9.9 172.8±10.4 179.3±11 183.6±10.6
G8: NAT 300 mg/kg 173.5±13.9 178.8±13.8 185±14.4 191±13.2
G9: AP (15mg/kg) + NAT (75mg/kg) 175±16.3 180.8±15 186.6±15.2 192.8±14
G10: AP (30mg/kg) + NAT (150mg/kg) 170±14.1 176.1±16 181.6±15.4 188.5±15.4
G11: AP (60mg/kg) + NAT (300mg/kg) 172.5±12.8 179±12.5 185±12 191.3±11.4
G12: Methotrexate 2 mg/kg 182±11 187.6±10.5 193±10.5 199±10
G13: Sulfasalazine 150 mg/kg 179.5±4.7 185.8±6 192±5.5 198.1±6.1
Values are Mean ± SD, n=6, *: p<0.05 when compared to G2 (Disease Control). #: p<0.05 when compared to G1 (Normal Control).

2.2 Effect of AP and NAT on paw volume
In Freund’s adjuvant non-injected paw, the test item AP and NAT treated groups (G3-G8) and their combination treated groups (G9-G11) show a significant dose dependent prevention of Freund’s adjuvant induced changes in paw volume on Day 15 and 21 when compared to vehicle treated disease control group (G2) (p<0.05). The highest percentage of inhibition of secondary lesions (16.8%) was observed with animals treated with mid dose (30 mg/kg) of AP, followed by animals treated with mid dose (150mg/kg) of NAT (16.7%) and low dose of AP and NAT (15 & 75 mg/kg) combination (15.4%). The above results were comparable to standards, methotrexate and sulfasalazine treated groups.

In Freund’s adjuvant injected paw, the test item AP and NAT treated groups (G3-G8) and their combination treated groups (G9-G11) show a significant dose dependent prevention of Freund’s adjuvant induced changes in paw volume on Day 5, 10, 15 and 21 when compared to vehicle treated disease control group (G2) (p<0.05), except AP low dose (15mg/kg) treated animals on day 5 and NAT low dose (75mg/kg) treated animals on day 10, which show only a non-significant protection (p>0.05). The highest percentage of inhibition of primary lesions (20.5%) was observed with animals treated with high dose (60 and 300 mg/kg) of AP and NAT combination, followed by animals treated with high dose (300mg/kg) of NAT (16.0%) and high dose (60 mg/kg) of AP (15.0%).
Table 9: Effect of Andrographis paniculata (AP) and Nyctanthes Arbrotristis (NAT) on paw volume of arthritic rats
Non-Injected paw Injected paw
Day-0 Day-5 Day-10 Day-15 Day-21 Day-0 Day-5 Day-10 Day-15 Day-21
G1: Normal Control 1.4±0.1 1.5±0 1.5±0.1 1.5±0.1 1.5±0.1 1.4±0.1 1.5±0.1 1.5±0.1 1.5±0.1 1.5±0.1
G2: Disease Control 1.4±0.1 1.5±0.1 1.5±0.1 1.6±0.1# 1.7±0.1# 1.4±0.1 1.8±0.1# 2.3±0.1# 2.7±0.1# 3.2±0.1#
G3: AP 15mg/kg 1.3±0 1.3±0 1.4±0* 1.4±0* 1.4±0* 1.3±0 1.7±0.1 2±0.1* 2.4±0.1* 2.7±0*
G4: AP 30 mg/kg 1.3±0 1.3±0 1.3±0* 1.4±0* 1.4±0* 1.3±0 1.6±0.1* 1.9±0.1* 2.3±0.1* 2.6±0.1*
G5: AP 60 mg/kg 1.4±0.1 1.4±0.1 1.4±0.1 1.5±0.1* 1.5±0.1* 1.4±0.1 1.6±0.1* 1.7±0.1* 1.9±0.1* 2.1±0.1*
G6: NAT 75 mg/kg 1.4±0.1 1.4±0.1 1.4±0.1 1.4±0.1* 1.4±0.1* 1.4±0.1 1.7±0.1* 2.1±0.1 2.5±0.1* 2.9±0.1*
G7: NAT 150 mg/kg 1.3±0 1.3±0 1.3±0* 1.4±0* 1.4±0* 1.3±0 1.6±0* 1.9±0* 2.2±0* 2.5±0*
G8: NAT 300 mg/kg 1.4±0.1 1.4±0 1.4±0 1.4±0* 1.4±0* 1.4±0.1 1.5±0.1* 1.7±0.1* 1.9±0.1* 2.1±0.1*
G9: AP (15mg/kg) + NAT (75mg/kg) 1.4±0.1 1.4±0.1 1.4±0.1 1.4±0.1* 1.4±0.1* 1.4±0.1 1.5±0.1* 1.7±0.1* 1.9±0.1* 2.1±0.1*
G10: AP (30mg/kg) + NAT (150mg/kg) 1.3±0 1.3±0 1.4±0 1.4±0* 1.4±0* 1.3±0 1.5±0.1* 1.7±0.1* 1.8±0.1* 2±0.1*
G11: AP (60mg/kg) + NAT (300mg/kg) 1.4±0 1.4±0 1.4±0 1.4±0* 1.4±0* 1.3±0 1.5±0* 1.6±0* 1.7±0* 1.8±0*
G12: Methotrexate 2 mg/kg 1.4±0 1.4±0 1.5±0 1.5±0 1.5±0* 1.4±0 1.6±0* 1.7±0.1* 1.9±0.1* 2±0.1*
G13: Sulfasalazine 150 mg/kg 1.4±0 1.4±0 1.5±0 1.5±0 1.5±0* 1.4±0 1.6±0* 1.7±0* 1.9±0* 2.1±0*
Values are Mean ± SD, n=6, *: p<0.05 when compared to G2 (Disease Control). #: p<0.05 when compared to G1 (Normal Control).

2.3 Effect of AP and NAT on arthritic index and total percentage inhibition
The test item AP and NAT treated groups (G3-G8) and their combination treated groups (G9-G11) showed a significant dose dependent prevention of Freund’s adjuvant induced changes in arthritic index when compared to vehicle treated disease control group (G2) (p<0.05). The lowest total score (1.3) and highest percentage of inhibition (74.9%) was observed with animals treated with high dose (60 and 300 mg/kg) of AP and NAT combination. Further, the total percentage inhibition (which is a sum of percentage inhibition of arthritic index and primary & secondary lesions) was also highest (109.5 %) in animals treated with high dose (60 and 300 mg/kg) of AP and NAT combination.
Table 10: Effect of Andrographis paniculata (AP) and Nyctanthes Arbrotristis (NAT) on arthritic index
Arthritic Index
Ears Nose Tail Fore paws Hind paws Total Score
G1: Normal Control 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0
G2: Disease Control 1.0±0.0 1.0±0.0 0.3±0.5 0.3±0.5 2.6±0.5 5.3±0.5#
G3: AP 15mg/kg 0.3±0.5 0.5±0.5 0.1±0.4 0.1±0.4 2.0±0.0 3.1±0.9*
G4: AP 30 mg/kg 0.1±0.4 0.1±0.4 0.00±0.0 0.00±0.0 1.8±0.4 2.1±0.9*
G5: AP 60 mg/kg 0.1±0.4 0.1±0.4 0.00±0.0 0.00±0.0 1.3±0.5 1.6±1.2*
G6: NAT 75 mg/kg 0.1±0.4 0.5±0.5 0.3±0.5 0.1±0.4 2±0.6 3.1±1.1*
G7: NAT 150 mg/kg 0.3±0.5 0.6±0.5 0.3±0.5 0.1±0.4 1.8±0.7 3.3±1.3*
G8: NAT 300 mg/kg 0.1±0.4 0.6±0.5 0.1±0.4 0.1±0.4 1.5±0.5 2.6±1.0*
G9: AP (15mg/kg) + NAT (75mg/kg) 0.1±0.4 0.5±0.5 0.3±0.5 0.1±0.4 1.5±0.5 2.6±1.2*
G10: AP (30mg/kg) + NAT (150mg/kg) 0.3±0.5 0.6±0.5 0.3±0.5 0.1±0.4 1.3±0.5 2.8±1.3*
G11: AP (60mg/kg) + NAT (300mg/kg) 0.00±0.0 0.3±0.5 0.00±0.0 0.00±0.0 1.0±0.0 1.3±0.5*
G12: Methotrexate 2 mg/kg 0.2±0.4 0.3±0.5 0.1±0.4 0.00±0.0 1.6±0.8 2.3±0.5*
G13: Sulfasalazine 150 mg/kg 0.1±0.4 0.3±0.5 0.3±0.5 0.00±0.0 1.5±0.5 2.3±0.8*
Values are Mean ± SD, n=6, *: p<0.05 when compared to G2 (Disease Control). #: p<0.05 when compared to G1 (Normal Control).

Table 11: Effect of Andrographis paniculata (AP) and Nyctanthes Arbrotristis (NAT) on percentage inhibition of primary & secondary lesions in arthritic rats
Percentage Inhibition
Primary Lesion-Injected Paw Secondary Lesion-Non-Injected Paw Arthritic Index Total % inhibition
G1: Normal Control - - - -
G2: Disease Control - - - -
G3: AP 15mg/kg 8.4±5.8 15.6±4.9 40.5±18.4 64.7±12.1
G4: AP 30 mg/kg 10.4±6.9 16.8±5.5 59.3±18.4 86.7±12.5
G5: AP 60 mg/kg 15±8.5 11.3±7 68.7±22.7 95.1±34
G6: NAT 75 mg/kg 5.1±6.5 14.1±6.4 40.5±21.9 59.8±24.6
G7: NAT 150 mg/kg 10.9±3.8 16.7±3.7 37.4±25.6 65.1±28.8
G8: NAT 300 mg/kg 16±5.8 13.4±5.4 49.9±19.3 79.4±14.3
G9: AP (15mg/kg) + NAT (75mg/kg) 15.6±7.7 13±8.1 49.9±22.7 78.6±14
G10: AP (30mg/kg) + NAT (150mg/kg) 18.4±6.4 15.4±5.6 46.8±24.9 80.6±25.3
G11: AP (60mg/kg) + NAT (300mg/kg) 20.5±4.5 14±5.5 74.9±9.6 109.5±16.4
G12: Methotrexate 2 mg/kg 13.5±4.4 10±4.7 56.2±9.6 79.8±5.7
G13: Sulfasalazine 150 mg/kg 14.4±3.2 10.4±1.7 56.2±15.3 81.1±14.7

The results showed a significant antiarthritic activity for the combination of AP and NAT fractions. Based on the study results, it may be concluded that AP and NAT fractions show a significant dose dependent antiarthritic activity against Freund’s adjuvant induced arthritis in rats. Further, the combination of AP and NAT extracts at highest doses shows a maximum antiarthritic activity.

These effective doses in mice convert to human doses of AP of 1.2 to 6mg/kg, i.e. about 84mg to about 420 mg in an average human weighing 70kg; and human doses of NAT of 6 to 24mg/kg, i.e. about 420mg to about 1680mg in an average human weighing 70kg.

References:
1. Burgos RA, Hancke JL, Bertoglio JC, Aguirre V, Arriagada S, Calvo M, Cáceres DD. Efficacy of an Andrographis paniculata composition for the relief of rheumatoid arthritis symptoms: a prospective randomized placebo-controlled trial. Clin Rheumatol. 2009 Aug;28(8):931-46. doi: 10.1007/s10067-009-1180-5. Epub 2009 Apr 29. PMID: 19408036.
2. Fenfen Li, He Li, Shasha Luo, Yantao Ran, Xiaoqian Xie, Yale Wang, Ming Zheng, Mengzhen Wang, Zeyue Zhao, Xiaotian Li, Evaluation of the effect of andrographolide and methotrexate combined therapy in complete Freund's adjuvant induced arthritis with reduced hepatotoxicity, Biomedicine & Pharmacotherapy, Volume 106, 2018, Pages 637-645, ISSN 0753-3322, https://doi.org/10.1016/j.biopha.2018.07.001.

,CLAIMS:We claim:
1. A composition for treating inflammatory conditions, the said composition comprising:
(i) about 80mg to about 450mg of an extract of Andrographis paniculata,
(ii) about 400mg to about 2000mg of an extract of Nyctanthes arbortristis, and
(iii) pharmaceutically acceptable carriers.

2. The composition as claimed in claim 1, wherein the extract is an aqueous, alcoholic or hydroalcoholic extract of Nyctanthes arbortristis and Andrographis paniculata.

3. The composition as claimed in claim 1 , wherein the pharmaceutically acceptable carriers are selected from the group comprising diluents, disintegrants, binders, lubricants, solvents, cosolvents, isotonicity agents, chelating agents, buffers, pH-adjusting agents, solubilizers, stabilizers, dispersion agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, flavouring agents.

4. The composition as claimed in claim 1, wherein the composition is in the form of a dosage form selected from the group comprising capsules, tablets, granules, powders filled in sachet, emulsions, oral liquid solutions, oral liquid suspensions, syrups, lozenges, solid dispersion, oral films and dry suspensions for reconstitution.

5. The composition as claimed in claim 1, wherein the composition is in the form of a tablet.

6. The composition as claimed in claim 1, wherein the inflammatory disease is Rheumatoid arthritis.

7. The composition as claimed in claim 6, wherein the composition is administered to a subject such that the composition comprises about 100mg of the Andrographis paniculata and about 300mg of Nyctanthes arbortristis.