ENHANCEMENT OF SOLUBILITY, BIOAVAILABILITY & EFFICACY … · 2020. 2. 17. · Fig. 2 : Scheme for ion complex formation between diethyl amine and Boswellic acid. Make and Model No.:

Pharmaceutical Resonance 2020 Vol. 2 - Issue 2

© Published by DYPIPSR, Pimpri, Pune - 411 018 ( MH ) INDIA 55

RESEARCH ARTICLE

ENHANCEMENT OF SOLUBILITY, BIOAVAILABILITY & EFFICACY OF BOSWELLIC ACIDS BY ACETYLATION, BETA CYCLODEXTRIN ENCAPSULATION & PREPARING DIETHYL AMINE SALT

Rabindra K. Nanda*, Bhavika Gupta

Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune - 411 018

Dr. Rabindra K. Nanda Professor, Department of Pharmaceutical Chemistry Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune - 411 018 Email : [email protected] Contact : +00 00000 00000

ABSTRACT:

The Boswellic acids contain six important isomers like (1) 3-O-acetyl-11-keto-β-Boswellic acid (AKBA), (2) 11-keto-β-Boswellic acid(KBA) (3) acetylated α and β Boswellic acids(ABA) and (4) α and β-Boswellic acids(BA) which are closely related isomers and are difficult to separate . These isomers exhibit anti-inflammatory activity through 5-LOX pathway and anti-cancer activity by binding to Topoisomerase II. By carrying out in-silico study of their binding to anti-inflammatory and anti-cancer targets, it was found that the AKBA isomer is the most potent and it was confirmed by experimental results published by various groups. Therefore, an attempt was made to carry out acetylation of boswellic acids extract to convert the non-acetylated isomers to acetylated isomers like ABA and AKBA to enhance the potency of the extract. The boswellic acids suffer from poor aqueous solubility. To improve their aqueous solubility, the boswellic acids were encapsulated with beta cyclodextrin. The product was then spray dried to get micronized spray dried powder which exhibit higher aqueous solubility and can be administered in capsules as an oral dosage form. To develop topical formulation, the diethyl amine salt of boswellic acids was prepared and a comparative evaluation of anti-inflammatory activity of all these products was carried out on a rat paw edema model.Anti-cancer activity was evaluated using Human Aspartyl –beta-Hydroxylase(HAAH) as a target enzyme in Real Time PCR.

Key Words : Boswellic acids, Anti inflammatory agents, Anti-cancer activity, TopoisomeraseII, synthesis of acetylated boswellic acids, β- cyclodextrin encapsulation of boswellic acids, solubility enhancement, preparation of diethyl amine salt of boswellic acids.

1. Introduction :

Boswellia serrata (Sallaki or Salai guggal) has a long history of use in the Ayurvedic tradition in managing inflammatory conditions [1-11]. Recent scientific evidence increasingly supports the healthful effects of this plant. Typically the gum oleoresin exudates of Boswellia serrata is reported to contain

sesquiterpenoid essential oils (8‐12% w/w),

polysaccharides (45‐ 60%w/w), and higher terpenoids, (25–35% w/w). The biomarker constituents in the extract of the gum resin are a group of pentacyclic triterpene compounds, known as Boswellic acids [12, 13]. Extracts of Boswellia serrata gum resin are generally resinous in nature,

and Boswellic acids (lipophilic compounds) are insoluble in water.

2. Materials and methods :

A) Acetylation of boswellic acid [14-18]

A methanol extract of gum resin from Boswellia Serrata was subjected to acetylation. This treated fraction is subjected to further ion – pair complex/salt formation with the help of organic base like diethyl amine. The synthesis of the ion – pair complex/salt of Boswellic acid is carried out by two step process ( Fig. 1 ).

Procedure for acetylation :

5.0 gm crude powder of Boswellia serrata extract was taken in 100.0 ml beaker. To it, 20.0 ml of methanol was added. The mixture was stirred for 5.0 minutes till all the mixture dissolved completely. 5.0 ml of Acetyl chloride was added slowly to the beaker. This reaction is a spontaneous reaction carried out at room temperature till the colour changes slightly yellow to blackish. Water was added to the methanolic acetylated solution, a white precipitate is formed. Precipitated acetylic Boswellic acid derivatives



containing enriched fraction of ABA/AKBA were dried. The obtained solid was filtered and dried in oven at 40 – 50 0C.The dried product was then subjected for further characterization studies with the help of HPTLC ,IR and Mass spectral methods.

Step 1 : Acceleration Process

Fig. 1 : Scheme for the Acetylation process of Boswellic acid derivatives.

Where, ABA = R1 & R2 - H AND R3 - Acetyl (CH3 - COO-). BBA = R1 & R2 - H AND R3 - OH. KBA = R1/R2 = O AND R3 - OH. AKBA = R1/R2 = O AND R3 - Acetyl (CH3 - COO-).

Step II - Procedure for the preparation of Diethyl amine salt [19] :

New and novel salts or ion pair complexes were obtained by reaction between diethyl amine with selectively enriched 3-O-acetyl-11keto-β- Boswellic acid (AKBA,) and 3-O-acetyl -β Boswellic acid (ABA) compounds obtained .These salts or ion pair complexes are useful for anti-inflammatory and analgesic treatment of joints similar to the Diclofenac:Diethyl ammonium salt used in the topical cream or gel preparation.

Where, R1 and R2 = H (ABA), & R1 and R2 = O (AKBA).

Fig. 2 : Scheme for ion complex formation between diethyl amine and Boswellic acid.

Step III – Different solvents used to increase yield:

This is a simple method by which salts or ion-pair complexes of Boswellic acids with heteroatom bases (also referred to as ‘organic base’) can be made. The acetylated product was then dissolved in different solvents like dichloromethane, toluene, benzene, methanol etc to check the increase in the percentage yield of the product. To the acetylated solution of Boswellic acid (2.0 gm) in methanol was added diethyl amine free base (8.6 ml, 0.4 gm) solution and

stirred at room temperature for 1.0 hr. Then the solvent was evaporated under reduced pressure and dried to give diethyl amine powder of Boswellic acid in white colour.

B) Characterization of boswellic acid derivatives :

1) Melting point:

Instrument Used: Veego Melting Point Apparatus. Make and Model No.: VMP PM, 32/1104.

All the melting points reported (0C) here are uncorrected.

2) High Performance Thin Layer Chromatography (HPTLC) :

In order to ascertain the purity and homogeneity of the synthesized compound, thin layer chromatography was carried out using silica gel (G – 60 mesh). The solvent system used includes Ethyl acetate: Toluene (06: 04 v/v). The spots were visualized by exposure to iodine vapour. The Rf value was calculated for each compound by the formulae given below.

3) Rotational and vibrational studies :

Instrument Used: FTIR Spectrophotometer with Diffuse Reflectance attachment.

Make and Model No. : Shimadzu 8400S.

Infrared absorption spectra of the synthesized compounds were observed in the frequency range of 4000 – 400 cm-1.

4) 1H NMR Studies :

Instrument Used: NMR Spectrophotometer (IICB KOLKATA)

Make and Model No.: Bruker Avance II 400 NMR.

The synthesized compounds were subjected to 1H NMR studies.

5) Mass Spectra :

Instrument Used: MASS Spectrophotometer (IICB KOLKATA)

Make and Model No.: NBM 146 MASS.(TOF MS ES+)

The synthesized compounds were subjected to MASS spectral studies.

1) Formulation and development



2) Preparation of solid forms of β-CD complex with boswellic acid derivative :

Spray drying operations were performed by Lab Spray Dryer (Lab-Ultima LU 222 advanced). This method involve generation of solid form of β-CD complex with Boswellic acid by spray drying of solution of drug in methanol, dichloromethane , and water system in ratios of 80:15:05 of methanol , DCM, water respectively. While spray drying parameters like feed pump flow rate(1ml/min), inlet-outlet temperature(550Cand500C), cool temperature(400C) and aspiration flow rate(45Nm3 /hr)were optimized and kept constant. By using above parameters, spray drying process was carried out and following batches were prepared.

Sr. Methanol Dichloro -methane

Water Batch

1 80% 20% 0% R-1

2 80% 15% 5% R-2

Table 1 : Batches of spray drying prepared by Methanol, DCM, Water solvent system.

3) Characterization of β-CD complex with Boswellic Acid Derivative :

The characterization of drug was carried out by conducting various tests as follows:

i) Assessment of Organoleptic properties of viz. colour, appearance.

ii) Melting point determination.

iii) Infrared spectroscopy (IR).

iv) Powder SEM (scanning electron microscopy).

C) Biological studies :

1. Anti-inflammatory activity :

The Biochemical Estimation Kits were procured from Biolab Diagnostics (I) Pvt, Ltd, Boisar, India. The λ Carrageenan Na was obtained from S.D. Fine Laboratories.The Diclofenac sodium was obtained from Hindustan Antibiotics Ltd., India. The boswellic acids were obtained from Manakarnika, Pimpri-chinchwad, Pune.

The Instruments used were (1) Analytical weighing balance of Shimadzu (2)Cooling centrifuge of Remi (3) Plethysmometer of UGO Basile 7140, Italy make and Shimadzu 1700 UV-Visible spectrophotometer for the experiments.

Albino Wistar rats of either sex weighing 150-250 g were used for present investigation. They were kept

in polypropylene cages in an air-conditioned area at 22±3º C in 10-14 h light dark cycle. They were provided with balanced feed and water ad libitum.

The experimental protocol was approved by IAEC (Institutional animal ethics committee). Laboratory animal handling and experimental procedures were performed in accordance with CPCSEA guidelines (Approval number: 198/99).

Experimental procedure:

To evaluate T1, T2, and T3 in carrageenan induced rat paw edema model was used as follows:The samples were labelled as STD- Standard (Diclofenac Sodium), T1- Boswellia serrata extract, ,T2- Beta-CD complex of Boswellic acid derivative, T3- Boswellic acid diethyl amine salt.

The Wistar albino rats weighing 160-200 g were used. The overnight fasted animals were divided into Five groups (n=6) as follows:

Carrageenan was injected into right hind paw. Paw volume was measured by using Plethysmometer at the time interval of 0 min, 1hr, 2hr, 3hr, 4hr, 5hr and 24hr after administration of Carrageenan. Results were expressed as,

Edema volume = Vt - Vc.

Vt = Paw volume in ml, at time t, after Carrageenan administration. Vc = Paw volume in ml, before Carrageenan administration. Inhibition rate (%) = Ec- Et/ Ec × 100.

Ec = Edema volume of control group. Et = Edema volume of treated group.

Groups (n=6)

Treatment Evaluation

I (Control) 1%Carboxy methyl

Cellulose

Change in paw edema

Volume Percent

inhibition

II (STD) Diclofenac sodium

(10mg/kg, p.o.)

III (T1) 100 mg/kg

IV (T2) 100 mg/kg

V (T3) 100 mg/kg

Table 2 : Exper imental protocol for Carrageenan induced paw edema in rats.

STD- Standard (Diclofenac Sodium), T1- Boswellia serrata extract, ,T2- Beta-CD complex of Boswellic acid derivative, T3- Boswellic acid diethyl amine salt.



2. Anticancer activity detection assay:

Human aspartyl / asparaginyl beta hydroxylase (HAAH) is an enzyme that in humans is encoded by the ASPH gene.

Fig. 3 : 3D structure of aspar tate β - hydroxylase(HAAH).

Clinical significance of aspartate β – hydroxylase(HAAH):

1. The over-expression of HAAH was recognized as an indicator of carcinoma in humans.

2. Further research has correlated elevated HAA levels (variously in affected tissue or blood serum) with hepato-cellular (liver) carcinoma adenocarcinoma (pancreatic cancer), colorectal cancer, prostate cancer and lung cancer.

3. The pancreatic study showed elevated HAA only in diseased tissue, but not in adjacent normal and inflamed tissue.

Polymerase chain reaction: (PCR)

Real-time PCR, also called quantitative PCR or qPCR, can provide a simple and elegant method for determining the amount of a target sequence or gene that is present in a sample.

Rn is the fluorescence of the reporter dye divided by the fluorescence of a passive reference dye; i.e., Rn is the reporter signal normalized to the fluorescence signal of ROX™.

Ct (threshold cycle) is the intersection between an amplification curve and a threshold line (Figure 1B). It is a relative measure of the concentration of target in the PCR reaction.

Materials and Methods :

Chronic myeloid leukemia Blood was procured from S.N. Gene Lab (Surat), Reverse Transcriptase Enzyme from In-vitrogen,Curcumin and boswellic acids and its semi-syntetic derivatives from Lab.

To validate the synthesized derivative for anti - cancer expression, PCR is performed as follows:

Procedure:

From Colorectal cancer patient ,1.0 ml blood was taken in a test tube. To it added 4 ml Karyotyping media and diethylamine salt of boswellic acid conc. (16m mole).

The mixture was then subjected to incubation for the period of 48.0 hrs in the thermostat.

After incubation, the RNA moieties present in the blood sample were extracted with the help of RNA Extraction Kit.

To the extracted RNA, Reverse Transcriptase Enzyme was added in order to convert RNA into Complementary DNA i.e. c DNA.

To the above made c DNA, the ASPH genes were added and the resultant mixture was then subjected to PCR to check the expression of anti-cancer agents.

Fig. 4 : Graphical representation of real time PCR data.

RESULT AND DISCUSSION :

A) Acetylation of boswellic acid

Step I – Acetylation :

Acetylation converts less potent Boswellic acids present in the fraction to AKBA and ABA which are more potent isomers for ani-inflammatory and ani- cancer activity.

Step II - The process of salt formation:

New salts or ion pair complexes obtained by reaction between Boswellic acid or selectively enriched 3-

http://en.wikipedia.org/wiki/Blood_serum

http://en.wikipedia.org/wiki/Blood_serum

http://en.wikipedia.org/wiki/Adenocarcinoma

http://en.wikipedia.org/wiki/Pancreatic_cancer

http://en.wikipedia.org/wiki/Colorectal_cancer

http://en.wikipedia.org/wiki/Colorectal_cancer

http://en.wikipedia.org/wiki/Prostate_cancer

http://en.wikipedia.org/wiki/Lung_cancer



Fig. 5 : Flowchar t for PCR study.

0acetyl-11keto-beta Boswellic acid (AKBA,) and 3-oacetyl beta Boswellic acid (ABA) compounds obtained through a new improved process, and an organic amine, more particularly diethyl amine. These salts or ion pair complexes are useful in for anti-inflammatory and analgesic treatment of joints and cancer prevention or cancer therapeutic agents. The purity and identity of synthesized compounds were determined by using Chromatographic technique and Spectral data:

Step III - Different solvents used to increase yield : ( Table 3 )

B) Characterization of boswellic acid derivatives [20-25]

1) Melting Point:

Melting point of synthesized derivatives are given in table No.3

2) HPTLC study of acetylation process :

Sr. Solvents Used Mode of Reaction Time (Min.) Yield (%) Melting Point

(0C)

1 Methanol MW Irradiation-(3) 3.0 80.23 190-193

2 DCM MW Irradiation -(3) 3.0 67.64 192-194

3 Toluene MW Irradiation -(3) 3.0 84.11 189-190

4 Benzene Conventional Heating 30.0 79.34 191-192

5 Methanol Conventional Heating 30.0 91.14 188-192

Table 3 : Physico – chemical data of the synthesized salt.

Fig. 6 : HPTLC chromatogram showing separated and increase peak height of ABA, AKBA after Acetylation.

Peak no. RF value % of

isomers

β-

Boswellic

Acid

derivative

T1 T4 T1 T4 T1 T4

5 4 0.45 0.47 19.65 - BBA

6 - 0.50 - 6.47 6.22 KBA

7 5 0.58 0.60 26.64 32.37 ABA

8 6 0.62 0.64 17.77 26.69 AKBA

Table 4 : The RF value and % of β- Boswellic Acid derivative T1 (Boswellia Serrata extract), T4 (Acetylated Boswellia Serrata extract).

Gum resin of Boswellia species known as frank-incense has been used as anti-inflammatory agent in Traditional Ayurvedic Medicine in India. A detailed study on structural requirement for Boswellic acid indicated that from all of the acid derivatives, 3-0-acetyl-11- keto-β-Boswellic acid (AKBA) shows most pronounced docking score with anti-inflammatory and anti-cancer activity. The acetylation process done on the extract of boswellic acid derivatives showed the significant results as follows : The HPTLC analysis shows that the % age of Boswellic acid derivative (ABA/AKBA) is increased.



Peak no.

RF value % of isomers

β- Boswellic

Acid derivative

T1 T4 T1 T4 T1 T4

5 4 0.45 0.47 19.65 - BBA

6 - 0.50 - 6.47 6.22 KBA

7 5 0.58 0.60 26.64 32.37 ABA

8 6 0.62 0.64 17.77 26.69 AKBA

Table 4 : The RF value and % of β- Boswellic Acid derivative T1 (Boswellia Serrata extract), T4 (Acetylated Boswellia serrata extract).

3) Rotational and vibrational studies : i. IR Spectrum of Boswellic acid derivatives.

Fig. 7: Boswellic acid der ivatives [where, ABA = R1 & R2 – H AND R3 – Acetyl (CH3 – COO-); BBA = R1 & R2 – H AND R3 – OH; AKBA = R1/R2 - =O AND R3 – Acetyl (CH3 – COO-); KBA = R1/R2 - =O AND R3 – OH].

Sr. Bond IR Range(cm-1)

1. -C-H (stretching) 2923, 2947

2. -C=O - (CH3=C=O) 1741

3. -C=O - (COOH) 1699

Table 5 : IR of Boswellic acid derevatives.

ii) Acetylated Diethylamine Salt of the Boswellic Acid Derivatives:

Fig. 8 : Acetylated Diethylamine Salt of the Boswellic Acid Derivatives (where, R1 and R2 = H (ABA), & R1 and R2 = O (AKBA)

4) Mass Spectrum :

Sr. Molecular Weight

(m/z ratio)

Characteristic Fragment

1. 585 Diethyl amine salt of

AKBA

2. 567 Loss of water (18)

3. 297

(Base peak) due to retro-Diels-Alder

fragmentation

Table 7 : Data for the mass spectrum of the Boswellic acid derivatives.

5) 1H NMR Study :

Table 8 : Data for the 1H NMR spectrum of the boswellic acid derivatives.

Sr. Chemical

Shift in ppm

Proton signal Characteristics

1. 5.535 Ethylene (12-13)

2. 1.66- 1.95 CH2-CH2

3. 0.93-1.12 Methyl attached to CH2 and

methyl in the ring.

4. 2.09 Acetyl group at 3-position

All the compounds were characterized on the basis of their physico-chemical properties like melting point, Rf values and spectral and chromatographic data.

The IR and MASS spectra of the synthesized derivatives showed characteristic band absorption for –C=O stretching group (of –CH3 – COO-) at 1700 - 1742 cm-1, which indicates the acetylated functional moiety, -CH stretching of the aromatic ring (2900 - 2972 cm-1), -C=O (of - COOH) at 1633 – 1699 cm-

1.Mass and NMR showing characteristic mass value (585) and ethylene signal in NMR of AKBA diethyl amine salt respectively.

C) Formulation and development [26] :

Characterisation of Boswellic Acid complexed with β-CD:

1) Organoleptic Properties: The colour of the powder was white and it was odourless.

2) Melting Range:

Melting point of Boswellic Acid Derivative: was found to be 136-1370C. The reported range for Boswellic Acid Derivative: was between136-1380C. Hence, observed melting point values are in good agreement with the reported value.



3) Infrared Spectrum of β-CD complex with Boswellic Acid derivative:

FTIR spectrum has been successfully used for identification and structural analysis of organic compounds. IR spectrum of Boswellic Acid derivative was taken and is given in Table no 11.

The IR spectrum of Boswellic Acid derivative reveals the presence of major functional groups in the structure of Boswellic Acid derivative supporting its identity.

Fig. 9 : Structure of Boswellic Acid with beta-CD.

Table 9 : Interpretation of IR Spectrum of β -CD Complex with Boswellic Acid.

IR Frequency

(cm−1)

Corresponding functional group and

type of molecular vibration

Inference

3330, 3353, 3384, 3454

-OH group β -

Cyclodextrin

2923 (-CH) Stretching

Boswellic Acid

1741 -- CH3-C=O

1688 -C=O (of -COOH)

iv) SEM:

Extracts of Boswellia serrata gum resin are generally resinous in nature, and Boswellic acids (lipophilic compounds) are insoluble in water. β-cyclodextrins form the inclusion complexes with Boswellic Acid by taking up lipophilic moiety of the molecule, into the central cavity. Spray drying technique is used to improve the quality and purity of β-CD- Boswellic Acid powder. The aqueous solubility of β-CD complex of Boswellic acid has improved considerably as compared to precipitated powder without β-CD. than the normal Boswellic acids. The particle size is also smaller (5.0 to 10.0 um) and is a free flowing powder.

SEM of Boswellic Acid derivative of β -CD Complex with Boswellic Acid derivative:

Fig. 10 : SEM showing the par ticle size of drug powder is found between (5.0 to 10.0µm).

D) Biological studies [27-38]

1) Anti-Inflammatory Activity:

Carrageenan induced paw edema in rats:

Diclofenac sodium treated group which is used as standard showed significant inhibition (p<0.01) of paw edema from 0.5 hr. to 24th hr as compared to control group.

Groups treated with T1 (Boswellia serrata extract crude powder), T2 (Beta-CD complex with Boswellic Acid), T3 (diethyl amine salt of Boswellic Acid) showed significant decrease (p<0.01) in paw edema volume from 0.5 hr to 24th hr when compared to control group.

Group treated with T3 showed significant decrease (p<0.05) and (p<0.01) in paw edema volume at 0.5 hr to 1st and from 2nd to 24th hr, respectively when compared to control group.

Fig. 11 : Results are presented as mean ± SEM (n=6). The data was analysed using one way Analysis of Variance ANOVA followed by Dunnett test. *p<0.05, **p<0.01 when compared with control group.

Std. - Diclofenac sodium 10.0 mg/kg p.o., T1- Boswellia Serrata Extract (100.0 mg/kg p.o.), T2 – β - CD complex with Boswellic Acid (100.0 mg/kg p.o.), T3 - Diethyl amine salt of Boswellic (100.0 mg/kg p.o.).

Fig. 12 : Results are presented as mean ± SEM (n=6). The data was analysed using one way Analysis of Variance ANOVA followed by Dunnett test. *p<0.05, **p<0.01 when compared with control group.

Std. - Diclofenac sodium 10.0 mg/kg p.o., T1- Boswellia Serrata Extract (100.0 mg/kg p.o.), T2 - β -CD complex of Boswellic Acids (100.0 mg/kg p.o.), T3 - Diethyl amine salt of Boswellic Acids(100.0 mg/kg p.o.).

2) Anticancer activity detection assay by PCR:

Cycle time is inversely proportional to expression. So if the number of cycle time is more then drug activity will be more.



Groups

Paw edema volume in ml at

0.5 h 1 h 2 h 3 h 4 h 5 h 24 h

Series I - Control

0.44±0.007 0.50±0.01 0.54±0.009 0.54±0.00

6 0.56±0.008 0.57±0.009 0.27±0.009

Series II - Std.

0.30±0.008**

0.35±0.07**

0.34±0.009**

0.32±0.008**

0.27±0.008**

0.24±0.004**

0.08±.007**

Series III - T1 100

0.41±0.006 0.48±0.00

9 0.49±0.008

0.51±0.007

0.53±0.007 0.53±0.008 0.25±0.006

Series IV - T2 100

0.35±0.007**

0.40±0.007**

0.41±0.009**

0.41±0.001**

0.38±0.010**

0.35±0.007**

0.16±0.004**

Series V -T3 100

0.35±0.011**

0.38±0.008**

0.39±0.006**

0.37±0.012**

0.35±0.025**

0.33±0.005**

0.13±0.009**

Table 10 : Effect of T1, T2 and T3 on Paw edema volume in Car rageenan induced paw edema in rats.

Fig. 11 : Effect of T1, T2 and T3 on paw edema volume in Carrageenan induced paw edema in rats.

Groups Percent inhibition of paw edema at

0.5 h 1 h 2 h 3 h 4 h 5 h 24 h

I - Control - - - - - - -

II - Std. 31.81 30 34.61 40.74 51.78 57.89 70.37

III - T1 25 6.81 4.00 5.7 5.50 5.35 7.01 7.40

IV - T2 100 20.45 20.00 21.15 24.07 32.14 38.59 40.74

V - T3 100 20.45 24.00 25.00 31.48 37.5 42.10 51.85

Table 11 : Effect of T1,T2,T3 on % inhibition of paw edema in Carrageenan induced paw edema in rats.

Fig. 12 : Effect of T1,T2 and T3 on % inhibition of paw edema in Carrageenan induced paw edema in rats.

Fig. 13 : PCR Study showing cycle time for the CML blood without drug.

Fig. 14 : PCR Study showing cycle time for the CML blood with synthesized diethyl amine salt of Boswellic acid.



Fig. 15 : PCR Study showing cycle time for the CML blood with curcumin.

DRUG Cycle Time (CT)

Control 23

Standard (CURCUMIN) 28

Diethyl amine salt of Boswellic acid.

26

Table 12 : Results of PCR

Conclusion:

The docking results of AKBA was compared with that of the other derivatives of boswellic acid, based on various parameters such as dock score, bond interactions and binding free energy (39). The docking result showed that the AKBA have favorable interactions with active site residues and also have favorable dock score and binding free energy, indicating that AKBA is having better anti-inflammatory and anti-cancer activity. The extract was per acetylated to increase the concentration of acetylated isomers. The Diethyl amine salt of boswellic acids was prepared similar in line with diclofenac diethyl amine salt. The docking studies carried out on 4 isomers of Boswellic acid on LOX enzyme (PDB Code: 1lox) indicated the best binding for AKBA isomer.

The order of binding is as follows:

AKBA >> KBA >> BBA >> ABA.

An attempt to separate all the four isomers by column chromatography was unsuccessful to get pure isomers. Hence, Acetylation reaction was carried out to increase the percentage of acetylated derivative of Boswellic acid which shows higher activity as per animal studies. Acetylated derivative was further converted to diethyl amine salt similar to Diclofenac diethyl amine salt. Acetylated derivative was further characterised by HPTLC, MASS, NMR, IR etc.

Spray dried powder of Boswellic acid with β – CD gives free flowing powder with enhanced solubility in water.

Anti – inflammatory studies carried out on rat paw edema model shows following order of activity AKBA – Diethyl amine salt (T3) >> Spray dried B – CD Complex of B.A. (T2) >> Boswellic acid extract (T1).

T2 (β -CD complex of Boswellic acid) and T3 (Diethylamine salt of boswellic acid) exhibited significant anti-inflammatory and anti-arthritic activities in the acute and chronic models of inflammation. T2 and T3 showed dose-dependent inhibition of second phase of carrageenan-induced rat paw edema, suggesting the inhibition of prostaglandins release.

The assay was performed to check the anti-cancer activity of diethylamine salt of Boswellic acid by real time PCR. It showed that salt of boswellic acid reduces the expression of cancer.

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ENHANCEMENT OF SOLUBILITY, BIOAVAILABILITY & EFFICACY … · 2020. 2. 17. · Fig. 2 : Scheme for ion complex formation between diethyl amine and Boswellic acid. Make and Model No.: