INTRODUCTION:

Crystallo-co-agglomeration (CCA) is an innovative technique developed with the intention to come up with the drugs with good micromeritic and mechanical characteristics. The process of CCA involves crystallization followed by simultaneous agglomeration of the drug with the help of a good solvent and/or a bridging liquid and a bad solvent.

This process enables designing of spherical agglomerates containing low dose drugs which are poorly flow able and compressible. The CCA require less processing time and may reduce the manufacturing as well as processing time during compression^1-3.

Yohimbine is generally used as a nutritionalsupplement for athletic performance, erectile dysfunction, weight loss, high blood pressure, diabetic neuropathy, chest pain, and more. Yohimbine HCL has a half life of up to 5 hours and has a bioavailability of 7-87% with a mean value of 33%. Yohimbine HCL usually undergoes hepatic first pass metabolism^4-5.

MATERIALS AND METHODS:

Yohimbine Hydrochloride was supplied by Vital Laboratories Pvt. Ltd, Delhi, India. Methanol, Dichloromethane, PEG 6000, PVA was procured from Loba Chemicals Pvt. Ltd., Mumbai. All other chemicals and reagents used were of analytical grade.

Drug-Excipients Interaction:

The possible physical and chemical interaction between the drug and polymers was tested with the help of FT-IR. The potassium bromide pellet containing Yohimbine Hydrochlorideand optimized agglomerates were prepared separately to record the spectrum in the range of 4000 to 500 cm^-1 using FT-IR spectrophotometer. The peaks were then compared with the standard value to find any interaction that might have occurred^6-7.

Standard Calibration Curve:

The standard calibration curve of Yohimbine Hydrochloride was carried out on UV spectrophotometer by using phosphate buffer as the solvent. An aliquot portion of standard stock solution of Yohimbine Hydrochloridewas diluted with phosphate buffer pH 6.8 to get a series of concentration ranging between 20-100µg/m ^l5. The absorbance was measured at 250 nm against blank^8-9.

Preparation of Yohimbine Hydrochloride agglomerates:

Yohimbine Hydrochloride agglomerates were prepared using a three solvent system as depicted in Table 1 by comprising methanol-dichloromethane-water (good solvent, bridging liquid, bad solvent). In a vessel, PVA was dissolved in sufficient amount of water. Yohimbine Hydrochloride was dissolved in methanol and maintained at room temperature. The latter dispersion containing dissolved polymer under constant stirring condition (300rpm) kept at room temperature. The stirring was continued and bridging liquid dichloromethane was added drop wise to obtain agglomerates, which were then filtered and dried overnight^10-11. Three batches were prepared by changing the concentration of PVA and they are named as A1, A2 and A3_. Three batches were prepared by changing the concentration of PEG 6000 and they are named as B1, B2 and B3

Evaluation of Prepared Batches^12-14:

Quantities of Yohimbine Hydrochloride agglomerates formed:

The prepared agglomerates were air dried for twenty-four hours until the agglomerates were fully dried. The dried agglomerates were then weighed.

Drug content and Percentage yield:

Drug content is that the ratio of experimentally measured drug content to the theoretical value, expressed as percentage (%). An accurately weighed quantity (50 mg) of prepared agglomerates was dissolved in 100 ml of phosphate buffer 6.8. This solution is then appropriately diluted and drug content was estimated by UV spectrophotometer at 250 nm.

The percentage yield (%) of samples was calculated using following equation,

Total weight of agglomerates × 100

% Yield = -------------------------------------------------------

Total weight of drug and polymer

Practical Yield × 100

% Drug Content = ------------------------------------------

Theoretical Yield

In-Vitro Dissolution Study:

Dissolution behavior of pure Yohimbine Hydrochloride and agglomerates was studied using phosphate buffer pH 6.8 as dissolution medium. Drug (50 mg) in muslin cloth was placed in basket containing 300 ml of solution of pH 6.8 for 3 hours. After 30 min of your time interval, 5ml of solution was withdrawn and diluted with buffer 6.8 solution and analyzed at 250.0 nm using UV-spectrophotometer. The share cumulative drug release (% CDR) was then calculated for every batch of agglomerates containing different polymers¹⁵.

X-ray diffraction study (XRD):

X-ray diffraction spectra of Yohimbine Hydrochloride and ready agglomerates were recorded with x-ray diffractometer employing a voltage of 45 Kv and a current of 40 mA. The instrument was operated in continuous scan mode over 2θ range at 20°- 80°. The relative intensity 1/10 and interplanar distance (d) like the 2θ values were reported and compared¹⁶.

Table 1: Formulation Chart of Yohimbine Hydrochloride agglomerates

Formulation	A1	A2	A3	B1	B2	B3
Drug (gm)	0.2	0.2	0.2	0.2	0.2	0.2
PVA (gm)	0.25	0.50	0.75	-	-	-
PEG 6000 (gm)	-	-	-	0.25	0.50	0.75
% Water- %Methanol-%Dichloromethane	35.48- 32.26- 32.26	35.48- 32.26- 32.26	35.48- 32.26- 32.26	35.48- 32.26- 32.26	35.48- 32.26- 32.26	35.48- 32.26- 32.26

Scanning electron microscopy (SEM):

Scanning electron microscopy of Yohimbine Hydrochloride and prepared agglomerates were taken using scanning electron microscope. The form and surface morphology were observed using SEM. The agglomerates were observed at various magnifications so as to research the effect of additives on surface morphology and agglomeration efficiency.

Evaluation of Micromeritics Properties of Optimized Agglomerates^17-18:

Tapped density:

Agglomerates (10g) were accurately weighed and transferred to a suitable graduated cylinder. The cylinder was then gently tapped to the base on a slightly resilient surface, such as a rubber pad or book, until the height of the sample in the cylinder reached at a minimum that is the sample height does not reduce with further tapping. Volume of sample in cc (ml) was read off.

D_t= M/V_t

Where, D_t = Tapped density (g/ml), M = Mass of powder (g), V_t = Tapped volume of powder (ml)

Bulk density:

Sample (10g) was passed through a sieve with aperture of 1.0 mm. In a measuring cylinder, sample up to 250 ml was introduced gently without compacting. The unsettled apparent volume was read and bulk density was calculated using following formula.

D_b= M/V_o

Where, M= Mass of powder (gm/cc), V_o = Bulk volume of powder (ml)

Compressibility index (CI):

Itis amanifestation of the compressibility of a powder. And it was calculated by using following equation,

I = (1-V/V_o) × 100

Where, V= the volume occupied by the sample of powder after being subjected to standardized tapping procedure andV_o= the volume before tapping

Hausner’s ratio:

Hausner’s ratio is defined as a ratio of a tapped density to bulk density. It’s a measure of relative importance of interparticulate interactions. Tapped density and bulk density were measured and therefore the hausner’s ratio was calculated using the subsequent equation.

Hausner ratio = Bulk density/Tapped density

Angle of repose:

Angle of repose is defined as the maximum angle possible between the surfaces of pile of powder and horizontal plan. The angle of repose for the powder of every formulation was determined by the funnel method. The powder was made to permit effuse of the funnel opening fixed at a height of 2 cm from the surface on a plane paper kept on the horizontal platform. After this, gradual addition of the powder from the funnel mouth was done which forms a pile of powder at the surface, this was continued until the pile touch the tip of the funnel. A circle was drawn round the pile base to calculate the radius of the powder cone was measured. Angle of repose was calculated with the utilization of the subsequent equation.

Ө = tan^-1[h/r]

Where, h = height of pile formed, r = radius of pile formed

RESULT AND DISCUSSIONS:

Drug-Excipients Interaction:

From the results of the FT-IR study as shown in Figure 1, it was found that there were no physical or chemical interactions between the drug and the polymer as there was no disappearance, mismatch or formation of any new peak when matched with the standard reference.

Figure 1: a) FT-IR of Pure drug Yohimbine Hydrochlorideb) FT-IR of Optimized Formulation

Standard Calibration Curve:

From the scanning of Yohimbine Hydrochloride in phosphate buffer (pH 6.8), it was concluded that the drug had λ max of 250 nm. From the standard calibration curve of Yohimbine Hydrochloride in phosphate buffer (pH 6.8), it was found that the Yohimbine Hydrochloride obeys beer’s- lambert’s law in the range of 10 -100 µg/ml. The equation of linear line was found to be, y= 0.014X + 0.139, R²= 0.997. Correlation coefficient (R²) value indicates the linear correlation between concentration and absorbance as shown in Figure 2. Thus, the method could be used successfully for analysis of Yohimbine Hydrochloride content.

Figure2: Standard Calibration Curve of Yohimbine Hydrochloride

Quantities of Yohimbine Hydrochloride– PVA agglomerates formed:

The estimation of quantity of agglomerates prepared by using PVA as a polymer was found to be 0.107gm for 0.25gm of PVA, 0.134gm for 0.50gm of PVA and 0.126gm for 0.75gm of PVA. The quantities of agglomerates formed with the use of PEG 6000 as a polymer were found to be 0.119gm for 0.25gm of PEG 6000, 0.168gm for 0.50gm of PEG 6000 and 0.148gm for 0.75gm of PEG 6000.

Drug content and percentage yield:

All the batches of PVA containing agglomerates showed drug content (loading) in the range of 28.75% - 32.40% and percentage yield in the range of 47.50% - 63.36%. While the batches of PEG 6000 containing agglomerates showed drug content in the range of 40.75% – 46.30% and percentage yield in the range of 60.50% – 74.33%. These results in Table 2 indicated good loading efficiency and insignificant drug loss for batches of PEG 6000 which was mainly because of slight solubility of drug in poor solvent. The variation in percentage yield might be attributed to drug loss during agglomeration. During stirring the drug stuck to the wall of the vessel and remained un-agglomerated.

Table 2: Drug content and percentage yield of Yohimbine Hydrochloride agglomerates

Formulation	%Drug content* (+) SD	*% Yield(+) SD**
A₁	28.75 + 0.08	47.50 + 0.04
A₂	43.27 + 0.10	67.07 + 0.09
A₃	32.40 + 0.11	63.36 + 0.01
B₁	40.75 + 0.08	60.55 + 0.08
B₂	59.17 + 0.02	84.46 + 0.04
B₃	46.30 + 0.03	74.33 + 0.01

In-Vitro Dissolution Study:

Dissolution of optimized agglomerates and pure Yohimbine Hydrochloride in previously optimized media was performed using USP type II apparatus. An aliquots of dissolution were subjected for analysis by UV-spectrophotometer. Dissolution profile of PVA containing agglomerates showed drug release in the range of 47.77% – 54.24%. Dissolution profile of PEG 6000 containing agglomerates showed drug release in the range of 54.31%- 59.10%. From the above discussion, it was concluded that agglomerates containing PEG 6000 had higher dissolution rate as compared to the agglomerates prepared using PVA. Hence, these optimized agglomerates were selected for further evaluations as shown in Table 3 and Figure 3. From this it can be concluded that that as the polymers concentration increases, drug release also increases. This is true for all the formulations containing PVA and PEG 6000.

Figure 3: % Cumulative drug release of pure drug and prepared agglomerates.

Table 3: Percentage CDR of agglomerates

Time (Hrs)	Percentage cumulative drug release of Yohimbine Hydrochloride (% CDR)	%CDR of formulations
Time (Hrs)		A1	A2	A3	B1	B2	B3
0	0	0	0	0	0	0	0
0.5	22.95	20.92	21.64	22.92	19.17	20.42	20.14
1	30.01	25.74	27.52	29.45	31.36	29.17	26.26
1.5	36.00	33.07	35.31	37.56	37.95	38.11	37.54
2	42.90	37.31	40.83	42.82	43.14	50.25	48.20
2.5	47.71	41.40	48.35	51.36	45.44	56.68	53.47
3	53.33	47.77	51.06	54.24	54.31	59.10	57.02

Evaluation of Optimized Batch of Yohimbine Hydrochloride Crystallo-Co-Agglomerates:

X-ray diffraction study (XRD):

The XRD pattern of Yohimbine Hydrochloride in Figure 4 exhibited intense, sharp well resolved peaks whereas XRD pattern of agglomerated crystals exhibited less intense and denser peaks compared to Yohimbine Hydrochloride. The XRD pattern of Yohimbine Hydrochloride showed its characteristics peaks at 2θ of 5.77, 10.88, 12.09, 13.03, 16.79, 18.02, 18.60, 19.47, 20.26, 21.00, 22.90 and 23.36. Intensities of characteristics peaks of drugs were decreased in agglomerates which might be due to differences in crystallinity of Yohimbine Hydrochloride in agglomerate. These observations indicated reduction in crystallinity or partial amorphization of drugs in its agglomerated terms.

Figure4: a) X-Ray diffractogram of Yohimbine Hydrochloride. b) X-ray diffraction pattern of optimized batch

Scanning electron microscopy (SEM):

An examination of the SEM of Yohimbine Hydrochloride confirmed that the pristine Yohimbine Hydrochloride was significantly smaller in particle size and blade or plate shaped elongated crystals with fines which hindered the flowability and compressibility. Improved flowability of agglomerates was mainly because of good sphericity of modified crystals obtained by CCA as evident by cylindrical shape of crystals/ agglomerates. SEM of Yohimbine Hydrochloride crystals in Figure 5 revealed no evidence of porosity with smooth surface where as agglomerated Yohimbine Hydrochloride have shown cylindrical crystals with clear evidence of rough surface and porosity.

Figure5: a) SEM of pure Yohimbine Hydrochloride. b) SEM of agglomerates

Particle size distribution:

The geometric mean diameters of agglomerates (669.3nm) were definitely larger than those of pure Yohimbine Hydrochloride. The result of the study in Figure 6 indicated that the original crystals were uniformly agglomerated by the CCA process employed.

Figure 6: Histogram for particle size analysis of optimized agglomerates

Evaluation of Micromeritics Properties of Optimized Agglomerates:

From the results of micromeritics studies as shown in Table 4, it can be concluded that the agglomerates showed improvement in flow property when compared to pure Yohimbine Hydrochloride. Among different agglomerates prepared, formulation containing PEG 6000 showed maximum flowability as evident by low values of angle of repose (θ), hausner’s ratio and compressibility index.

CONCLUSION:

Yohimbine Hydrochloride–polymer agglomerates were successfully prepared by crystallo-co-agglomeration technique. CCA technique can be successfully employed as an alternative to conventional wet agglomeration. This study showed that, it is possible to quantify differences between pristine Yohimbine Hydrochloride and Yohimbine Hydrochloride agglomerates by a quick and simple screening method. The micromeritics of the agglomerates such as flowability, packability and compactability were dramatically improved. The dissolution and disintegration rate of agglomerate was increased in presence of PEG 6000. From the above investigation, it can be concluded that this method is proficient for creating spherical agglomerates with enhanced micromeritics, mechanical and conventional properties.

CONFLICT OF INTEREST:

None.

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Received on 01.09.2021 Modified on 03.12.2021

Res. J. Pharma. Dosage Forms and Tech.2022; 14(2):105-110.

DOI: 10.52711/0975-4377.2022.00016

Sample	Bulk density (gm/cc)	Tapped density(gm/cc)	Compressibility index (%)	Hausner’s Ratio	Angle of Repose (θ)
Pure Drug	0.18 +0.003	0.50 +0.004	22.66 +0.11	1.28 +0.03	24.56 +0.22
Agglomerates	0.33 +0.006	0.73 +0.023	16 +0.66	0.42 +0.06	13.37 +0.32