Preparation and Evaluation of Capsules containing Standardized Extract of Wild Hawthorn (Crataegus azarolus)

 

Randa Badr¹, Dima Muhammad², Wihad Ibrahim³

¹Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy,

Tishreen University, Lattakia, Syria.

²Department of Pharmacology, Faculty of Pharmacy, Tishreen University, Lattakia, Syria.

³Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy,

Tishreen University, Lattakia, Syria.

 

ABSTRACT:

Wild Hawthorn (Crataegus azarolus) is a popular medicinal plant, rich in polyphenols and flavonoids, and has been associated with many beneficial properties including anticancer, cardioprotective, and antidepressant effects. This study aimed to prepare a standardized aqueous-alcoholic extract of the deried ariel parts of C. azarolus using two different extraction methods ((Magnetic mixer (MA), and ultrasonic-assisted extraction (Us)), determine the total phenolic and flavonoid content of the obtained extracts, and compare it with the international product, and prepare capsules containing this extract to investigate the effect of different types and concentrations of disintegrating agents on the release profile of this capsules. The findings showed that the yield obtained by both extraction methods is almost the same  (MA: 19.9 %, US: 18.4 %) we obtained the flavonoids of the following components (MA extract: powder of the externally marketed drug: prepared dry drug powder) used at a concentration (10 : 350: 352) mg on (36.95: 4.56: 2.299) as a percentage of flavonoids Four different formulations were filled in the hard gelatin capsules using 120 mg of the prepared extract as an active ingredient. The effect of three types of superdisintigrant (croscarmellose Sodiuom ccNa, Microcrystallin cellouse 101, Sodiuom Strach Glycolate SSG) on the dissolution rate of the active ingredient was then investigated. The study showed that the formulation without superdisintigrant released its content faster than others, but all formulations release their content after 45 min. A comparison dissolution test was performed between the Capsules containing the best formulation and the capsules containing a powder of the local Crataegus plant as well as international marketed capsules containing the powder of the Crataegus plant. The study showed that the capsules filled wih the extract released it’s content better than the other after 45 min.

 

 

 

1.    INTRODUCTION:

Crataegus azarolus belongs to the Rosaceae family. It is a deciduous tree or shrub, which constitutes a large part of the forest plants spread in our country in addition to its spread all over the world¹. Various parts of Crataegus have been widely used in traditional medicine for the treatment of different diseases such as Cardiovascular disorders, anti-anxiety, arteriosclerosis, Potential analgesic, diuretic activity, etc., and others^2,3.

 

 

These biological effects are attributed to the presence of phenolic compounds, especially flavonoids, which are concentrated in young leaves and flowers⁴, and they are compounds of polar nature, so they can be extracted with polar grade such as water/alcohol⁵, especially since these solvent are generally classified as safe. This study aimed to prepare aqueous/alcoholic extracts from a plant and determine the total phenolic and flavonoid content in the plant.

 

However as capsules are a convenient way to give powdered herbs or concentrated extracts because they help to mask the bad taste as well as they afford through the fast-dissolving of the gelatin shell the potential for rapid release of the drug. Therfore we aimed to prepare hard gelatin capsules as a dosage form filled either with prepared extracts or with powdered plants, and Study of the effect of superior crumbs on the release of capsules.

 

2.    MATERIALS AND METHODS:

2.1 Chemicals:

Ethanol 95% (Sham lab.), Methanol (Sham lab.), Ammonia (SDFINE-CHEM LTD), Ammonium  Hydroxide (PRO-LAB), Aluminum chloride hexahydrate were purchased from (MERCK), Sodium Nitrite were purchased from (MERCK), Aerosil^® 101 was provided from Degussa AG (Germany), magnesium stearate were purchased from Merck Chemicals Co. (Germany), Croscarmellose Sodium was were purchased  from BASF (Germany), Sodium Starch Glycolate was supplied by MINGTAI Chemical Co. LTD. (Taiwan), All the chemicals used were of analytical grade.  

 

2.2 Plant Material and Preparation:

The leaves and flowers of wild hawthorn (Crataegus azarolus) were collected from shrubs growing in the Al-Adima area of the Syrian coast, and air-dried in the shadow. Plant genus was determined through morphological and microscopic studies. The deried samples were ground into a powder using an electric mill (or grinder) and homogenized by passing it through a 355 µm sieve to get a particle size similar to that of the international product.

 

Aerial powdered parts were extracted using two methods, a magnetic mixer, and an ultrasound bath. Extraction was performed using ethanol 95%/water (10 mg dry weight sample/ 200 ml ethanol 95%/water (160/40 v/v)⁶ for 60 min at room temperature). The obtained extracts were evaporated under reduced pressure using a rotary evaporator, then lyophilized to remove the remaining traces of solvents.

 

2.3 TLC Analysis of Phenolic Compounds of the Obtained Extracts and the International Product:

The phenolic compounds of the produced extracts were determined using TLC plates, and the internationally-marketed product as a standard separation was done using a mobile phase of chloroform/methanol/ammonia 8.5/1.4/0.05 and the vanillin reagent, then the RF⁷ values of the phenolic compounds were calculated.^8,9

 

2.4 Phytochemical Characterization of the Extracts:

2.4.1           Spectrophotometric Determination of Total Phenolic Content:

The total phenolic content of the extracts calculated as gallic acid was determined and compared with that of the international product using the Folin-Ciocalteu method. Samples (50 mg) were dissolved in DMSO (0.1 ml), then sodium carbonate (2%) and Floin-Ciocalteu reagent (in a ratio of 0.1:2 and 1:1 respectively) were added. Absorbance was measured using UV/Vis spectrophotometer at 750nm.

 

Standard curve was prepared using different concentrations of gallic acid in (DMSO) ( 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100mg/100ml^10,11. The compilation of the procedure and the measurement of the absorbance were performed as described above. Three replicates were carried out for each sample and the regression equation obtained was: y= 0.0098x + 0.0208, R² = 0.9986.

 

2.4.2 Spectrophotometric Determination of Total Flavonoid Content:

The total flavonoids content of the extracts was determined and compared with that of the international product using the hexahydrate aluminum chloride method. Samples (100mg) were dissolved in ethanol 95% (1ml), then ethanol 30%, sodium nitrate (1M), aluminum chloride (0. 3M)¹², and sodium hydroxide (7.5 ml, 0.75ml, 0.75ml, and 10ml respectivly) were added to 1ml of the obtained solution.  Asorbance measurment were determined using UV/Vis spectrophotometer at 510nm. Ruten was used as an external standard. This flavonoid was dissolved in (Ethanol) and dulated to obtain the following conentrations (0.006, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06mg/ml)¹³. The following procedure was carried out as mentioned above, and three replica assays were performed for each sample. The regression equation obtained was: y = 3.1798x+0.0335, R² = 0.9803.

 

2.5 Formulation and Evaluation of Hard Gelatin Capsules:

Four different formulations listed in table 1 were filled in the hard gelatin capsules (size 00) using 120mg of the prepared extract as an active ingredient. To investigate the effect of the superdisintegrant on the release of the active ingredient we used three different type (corsscarmelose, Micorcystalline celloulose 101,  Sodiuom Strach Glycolate) in concentration 15% of the whol formulation.

 

In addition another formutaion using just the powdered local plant was used to elucidate the difference  in releas profile of  the international product.^14,15

 

Table 1. Composition of different formulations of Crataegus azarolus capsules

 

2.5.1  In vitro release studies:

In vitro release studies of the capsules (n = 3*2) were performed in a USP Basket-type apparatus at 50rpm using 350mL of distilled water at 37°C. Five milliliter samples were withdrawn at regular intervals (5, 15, 30, 45) and replaced with distilled water. The samples were evaluated after proper dilution and mixing with a suitable marker pectrophotometrically at 510nm (λmax)¹¹.

 

3.    RESULTS AND DISCUSSION:

3.1 Morphological and Microscopic Examination:

The plant genus, Crataegus azarolus (Fig 1A), was determined through morphological examination based on the Flora of Syria, Palestine, and Sinai, as well as a microscopic study which revealed wide-lumen thick-walled Trichoma capillary cells (Fig 1B).

 

Fig 1. (A) Crataegus azarolus, (B) Trichoma capillaries of Crataegus azarolus

 

3.2 TLC Analysis of Phenolic Compounds:

TLC results analysis of the obtained extracts using the vanillin reagent showed a similar profile to that of the international product. The RF values for each observed spot were close and in the same order, confirming that the plant used in this study shares high similarity to that found in the international product.

 

3.3 Influence of Extraction Conditions on the Extraction Yield:

The extraction yield varied with the applied extraction technique. The magnetic mixer-assisted extraction provided a higher yield of 19.9 %in comparison to the ultrasonic-assisted extraction which gave a yield of: 18.4%.

3.4 Total Flavonoid Content:

The total flavonoid content of the obtained extracts and the international product are shown in Table 3.

 

Table 3. The percentage of flavonoids in all samples

 

The result corresponds to a monograph in which the dry leaves and flowers contain about 1% flavonoids¹⁶

 

Table 4. the flavonoid content of the obtained extracts and the international product)

 

The result corresponds to a monograph for a dry hydroalcoholic extract of at least 6% flavonoids^16,17

 

3.5 Dissoltuion Studies:

3.5.1 The effect of the disintegrant:

Results obtained from the in vitro release profile of the prepared formulas using two capsules each time showed that all formulation released its content during 45 Min. It is notable to notice that formulation 1 which didn’t contain a disintegrant release its active ingredient faster at 5-15-30 min with p-value > 0.05. This could be attributetd to the effect of the highly soluble ingredients which bind water molecules and increases the viscosity of the medium and thus delays disintegration.¹⁸

 

Fig 5. Dissolution rate of the Formulations

 

4.     Comparison of the dissolution results of capsules of the internationally marketed product with the capsules of local plant powder and with formula F1:

Figure 9 depicts that the release of flavonoids from the plant’s powder after 15 minutes was the best, but after 45 minutes Formulation 1 released its content more than the other. Although the formulation didn’t present a meaningful imp significant improvement with regard to the rate of dissolution, but we cannot forget that the herbal extract help the patient to avoid taking more capsules to reach the required pharmaceutical effect.

 

 

Fig 7 Dissolution rate of formula F1 , internationally marketed product and capsules with local plant powder.

 

5.    CONCLUSIONS:

the results of the current phytopharmaceutical evaluations confirmed that this (aqueous: alcoholic) extract (80:20) is in agreement with the 2016 European Union Herbal monograph 2016. The release study showed the negative effect of the disintegrant on the release of the active ingredient therefore we considered Formula F1 as the best formulation. In addition, capsules with local herbal powder showed better dissolution rate than the international product.

 

6.    ACKNOWLEDGMENTS:

The authors extend their thanks to Al-Andalus University, Faculty of Pharmacy, for providing laboratory facilities for the implementation of this research work, and Damascus University, Faculty of Science, and Leishmania Laboratory for providing the freeze-dryer for the implementation of this research work.

 

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Accepted on 28.05.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(1):175-178.