1. INTRODUCTION:

Health foods and dietary supplements have Aloe-vera (AV) components as anthraquinones for a very long time^1,2. Requirements and acceptance of herbal treatments are increasing day by day due to their minimal side effects^3,4. The common constituents present in the plant are phenolic compounds, vitamins, enzymes, polysaccharides, and organic acids, which are water- and fat-soluble^5,6. Aloe-emodin (AEM), the plant's principal ingredient is used to cure a variety of ailments.

For determining the AEM amount in AV extracts and associated products have done by analytical technique HPLC with both UV absorption and fluorimetric detector. The approach works well and demonstrates good sensitivity and precision⁷.

AEM's limited bioavailability is being attempted to be addressed by structural alterations and nanocarrier systems in line with the goal of creating prospective therapies from natural compounds⁸. Several researchers have also reported that AEM has anti-bacterial, anti-cancer, antioxidant, neuroprotective, hepatoprotective, anti-inflammatory and others also^9-14.

HPLC-ESI-Q-TOF-MS was used to analyse the extract of Rheum cordatum plant in different solvents¹⁵. The UHPLC technique was used for the simultaneous estimation of five anthraquinones, including AEM, in rhubarb-based medicine¹⁶ . An HPLC-DAD analytical technique was employed for the determination of AEM in Yiqing samples¹⁷. HPLC has been used to quantify aloins and AEM in AV raw materials and finished products¹⁸. From AV leaf extract, bioactive compounds were determined by improved chromatographic methods ¹⁹. Drug release studies of AV formulations using a quality-by-design approach have been reported^20,21.

The antimicrobial activity of AEM has been reported against Staphylococcus epidermis and gram-positive pathogenic species. The minimum inhibitory concentration (MICs) and minimum bactericidal concentration (MBCs) were reported 4–32 and 32–128 μg/mL, in order. Against gram negative-bacteria MICs and MBCs were reported 128–256 and above 1024 μg/mL, respectively²². The AV extract has been shown to be effective towards all uropathogenic bacteria, with the exception of an E. coli strain that is multiresistant (E. coli 1449)²³. Diverse environmental circumstances shown considerable changes regarding the antioxidant capability of plants of various species, with all extracts demonstrating significant antioxidant capacity ranging from 56 to 80%²⁴. In-vitro anthelmintic activity, alcoholic extract as compared to the aqueous extract may have contributed to its superior performance. It may be inferred that the Boswellia serrata + Aloe barbadensis combination has a combinatorial anthelmintic impact in both alcoholic and aqueous extract²⁵.

QbD is a very important tool for optimising chromatographic conditions during the method development and validation processes to estimate active components in varied samples. In chromatographic analytical techniques, the HPTLC, HPLC, or UHPLC methods use the trial-and-error or "one factor -at- a-time (OFAT) approach, in which the independent variables are screened and selected to run a series of trials until no sharp and symmetric peak is found^26-28.

The present study object is to develop a chromatographic method by RP-HPLC with QbD tools for the quantification of AEM in herbal extract and marketed herbal formulation. The exiting analytical techniques are helpful to design the present research work.

This is an important study in terms of quantifying AEM in AV extract and marketed herbal formulations using the RP-HPLC analytical technique. After quantifying the AME the in-vitro study, antimicrobial, antioxidant, and anti-helminthic activity have been performed.

2. MATERIALS AND METHODS:

2.1 Drugs and Chemicals:

From Sigma Aldrich (St. Louis, MO, USA), an AEM reference sample was obtained. For the aqueous solution, analytical-grade reagents and water were used. Milli-QR gradient A10R (Millipore, Germany) was used for the preparation of a buffer having a pore size of 0.22 μm.

2.2 Instrument and Chromatographic conditions:

Waters 1525 binary pump separation module (Waters, USA) made up the HPLC system. A 10µl sample loop was employed for the auto sampler injection system (Waters 2707). Filter with a 0.45µm pore size from Millipore in Bangalore, India. The gradient flow by symmetric C₁₈column (4.6 × 75mm, 3.5μm spherical particles) was controlled using the Waters 515 solvent delivery system.

2.3 QbD approach for HPLC method development:

Risk Assessment studies: Tthe separation and quantification of analytes are influenced by different factors like mobile phase composition, column configuration, flow rate, column temperature, detecting wavelength, and volume of sample, all of which affect the instrument's performance. The given factors, with the help of the fishbone identify the critical attributes (CAA), and then after a systemic risk analysis was carried out^30,31.

2.4 Optimization:

13.0.3.0 Software (Stat-Ease Inc., Minneapolis, USA) was used for the optimization of chromatographic conditions. Effects of factors' interaction among themselves had been observed in the rest of the twelve trials. In the chromatographic optimization, three factors and two responses were considered^32,33.

2.5 Method Development:

The composition of the mobile phase was orthophosphoric acid methanol (0.1% OPA) and water (70:30, v/v) have a pH of 3.5. The flow rate of the mobile phase was 1mL/min at 28ºC, and a volume of 20 µL of sample was used for the analysis. The emitted light has been detected at 256nm. For the analysis, the runtime was fixed 7 minutes. Retention time for AME was 4.628 minutes, and peak’s tailing factor value was 0.99.

2.6 Linearity studies:

Five dilutions for calibration curve were considered over concentrations ranging from 10 to 50µg/mL³⁴

2.7 Method Validation:

ICH (Q2A) Food and Drug Administration FDA guidelines were followed Validation parameters like linearity, accuracy, precision, ruggedness, limit of detection, and limit of quantification were conducted to complete the validation^35-42.

2.7.1 Accuracy:

A standard drug of AEM was added to the pre-analyzed sample (10.0µg/mL of AEM) in three concentration levels (80%, 100%, and 120%) in triplicate. In term of % recovery result are presented.

2.7.2 Precision:

An intra-day precision experiment was studied by analysing three concentrations of AEM (10, 20, and 30 µg/mL) on the same day in triplicate, and for the inter-day precision analysis, the same three concentrations were studied for three days over a week.

2.7.3 Selectivity and specificity:

Examine the chromatograms and confirm the absence of interfering peaks at the retention time of AEM.

2.7.4 Ruggedness: To check the ruggedness parameter, experiments were conducted in the chromatographic conditions of the developed analytical method by two analysts.

2.7.5 Robustness: A small amount was deliberately changed from the original value for these three chromatographic conditions. Experiment was conducted by 10µg/mL AEM at one point, one factor was changed.

2.8 In-vitro study:

2.8.1 Antimicrobial activity:

To purify the AV extract and obtain AEM for antimicrobial activity research, an alcohol/salt aqueous two-phase system was used. Agar diffusion was used to test the antimicrobial activity of AEM that got from AV extract against S. aureus (Gram positive bacteria), E. coli (Gram negative bacteria), E. cloacae (Gram negative bacteria), and C. albicans (Fungi).^43,44

Serrano R. et al. method was used for the culturing of medium. Minimum bactericidal concentrations (MBCs) and Minimum inhibitory concentrations (MICs)are estimated by dilution method⁴⁶.

2.8.2 Antioxidant activity:

DPPH (2, 2-Diphenyl-1-picrylhydrazyl) ASSAY: The DPPH radical scavenging activity was assessed using the approach described by Brand-Williams et al⁴⁷. The %AA of AEM, AV extracts and herbal formulation samples have been examined over the concentration range (10-50µg/ml). For each sample five dilutions were prepared and observe the reading.

Whole experiments results are presented in table and figures.

Ao - As

AA% = --------------------- X 100

Ao = Absorbance of control

As = Absorbance of sample

2.8.3 Anti-helminthic activity:

The in-vitro anthelmintic study was conducted in accordance with the standards has been prescribed by authors in research article^48-51. Normal Control Group using normal saline 0.5% w/v. Standard: Standard Group Albendazole 20mg/ml kg bw. Samples: AEM, AV extract and Herbal formulation. Each Petri dish was filled with Pheretima Posthuma adult Indian earthworms (n=3, or 4). The worms have always been watched until they became paralysed and then died. Average times for mobility loss (paralysis) and total loss of movement.

3. RESULTS:

3.1 Risk Assessment and Optimization:

Risk assessment studies’ goal was to recognise and line up the causes that affect the critical quality attributes (CQAs). During risk assessment studies, different factors were analyzed; among these three factors, we have considered them for the trial. As responses, retention time and tailing factor were used.

3.2 BBD optimization:

3.2.1 Retention time:

The study's findings are presented in Table 1: Independent variables response by application of Box-Behnken Design in 3-2 factors-response. Retention time was optimized, and it was observed in five trials out of seventeen in the table.

Table 1: Independent variables response by application of Box-Behnken Design in 3-2 factors-response

	Dependent variables			Independent variables
Run	Factor A	Factor B	Factor C	Response 1	Response 2
1.	67	0.8	256	4.551	0.74
2.	73	0.8	256	4.882	0.82
3.	67	1.2	256	4.785	0.78
4.	73	1.2	256	4.484	0.74
5.	67	1	254	4.872	0.81
6.	73	1	254	4.743	0.78
7.	67	1	258	4.376	0.72
8.	73	1	258	4.688	0.78
9.	70	0.8	254	4.726	0.79
10.	70	1.2	254	4.743	0.79
11.	70	0.8	258	4.494	0.75
12.	70	1.2	258	4.353	0.72
13*.	70	1	256	4.628	0.99

Factor A: Percentage of 1% OPA methanol (%), Factor B: Flow rate (ml/min), Factor C: ʎmax (nm), Response 1: Retention time (minutes), Response 2: Tailing Factor.

13*-17* Optimised conditions (These trials are reproducible, which indicate that in optimised chromatographic conditions the responses are same).

The inbuilt ANOVA analysis reveals that a quadratic model is significant. The high ‘f’ value (75.96) and model terms with values less than 0.0500 confirm that the model is significant. Table 2: Model Summary Statistics,

"Model Summary Statistics with Values and Fit Summery has given," also from ANOVA analysis, the value of adequate precision was 30.57; it measures the signal-to-noise ratio. This model can be used to navigate the design space. The inbuilt final equation of the model is A (+0.0266), B (-0.0360), C (-0.1466), AB (-0.1580), AC (+0.1102), BC (-0.0395), A2 (+0.0691), B2 (-0.0216), and C2 (-0.0274). Based on this equation.

3.2.2 Tailing factor:

The optimised responses' tailing factor values explain the symmetry and shape of the peaks. Table 1 shows an inbuilt ANOVA quadratic model created using the BBD application. Five of the seventeen trials have been optimized. The f-value and model term p-value were 1925.92 and 0.0500, respectively. Table 2 contains model summary statistics and a fit summary. The final equation of the model is A (+0.0088), B (-0.0087), C (-0.0250), AB (-0.0300), AC (+0.0225), BC (-0.0075), A2 (-0.1050), B2 (-0.1150), and C2 (-0.1125).

3.3 Method development:

By optimised chromatographic conditions a standard solution (30µg/ml) of AEM has run in given instrument conditions. A volume of 20µL samples (n=3) were injected. A sharp and symmetric peak of standard is shown in figure 1, which indicates no interference in the optimised retention time.

Figure 1: Chromatogram of 30µg/mL standard solution of aloe emodin

3.4 Validation Parameters:

3.4.1 Linearity:

Five standard solutions were tested over the concentrations ranging from 10 to 50µg/mL. r2 value was 0.9981, with intercepts and slope values of 0.0662x - 0.01078. %RSD value was in range 6.06 to 1.33.

3.4.2 Accuracy:

Concentrations of 80%, 100%, and 120% were analysed in addition to the previously analysed sample of 10 µg/mL. The percentage recovery was established at 98.06, 98.95, and 99.86, with %RSD values of 0.67, 0.81, and 0.75 at each level.

3.4.3 Precision:

The inter-day (n=3) % RSD values were 1.98, 0.52, and 2.08 for concentrations of 10, 20, and 30µg/mL respectively, and the intra–day (n=3) %RSD values were 1.14, 0.61, and 2.18 for concentrations of 10, 20, and 30 µg/mL, respectively.

3.4.4 Ruggedness:

The ruggedness experiment was performed by two analysts with a AME concentration of 20µg/mL. Here, percentages of recovery of the samples were calculated. By both analysts which were given good result. First analyst was producing % recovery 98.89 and %RSD 1.56 and by analysts II its value was 97.62 and 1.81. Less than 2% of %RSD value suggests that the developed is good.

3.4.5 Robustness:

For the experiment (n=3), the concentrations of AME 20 μg/mL was used, results of the intended changes in parameter buffer pH, flow rate, and mobile phase composition in terms of average SD, and %RSD. Less than 2% of %RSD value suggests that the developed approach is resilient.

3.5 Application:

3.5.1 Analysis of Aloe vera extract:

A dilution of 10.0µg/mL of the extract was prepared from the stock solution. 10mL of this solution was injected into the column, and a response was observed between the peak area and retention time. The strength was determined by the linearity curve. The procedure was repeated six times, and outcomes were reported in order of the % recovery 99.05±0.89 and % RSD 0.85.

3.5.2 Analysis of herbal formulations:

A quantity of AEM was determined for the given formulation. For this, twenty tablets were weighted accurately (label claim: 500mg) and then powdered it properly. Weighted the powder equivalent to 100mg of AEM in the formulation and transferred it to a 100-ml volumetric flask. Mix it properly with 100mL of solvent. Before analysis, the solution was filtered with 0.45µm membrane filter paper. Aliquot 1ml of the filtered sample and transferred it into a 10ml volumetric flask. Make up the volume with solvent and mix it properly. The final solution was injected six times. From the standard calibration curve, the concentration of the solution was determined. the % recovery 94.53±1.29 and %RSD 1.68.

3.6 In vitro studies.

3.6.1 Antimicrobial activity:

The antimicrobial activities of purified AV extract (AV100, 200 and 300µg/ml) were tested using the agar-well diffusion method. The inhibition zone (in milimeters) was considered control (Rifamycin) for the microorganism S. aureus (Gram-positive bacteria) E. coli (Gram-negative bacteria). For, E.coli the minimum inhibitory concentration (MICs) value was below the 100 µg/ml for AV extract. The minimum bactericidal concentration (MBCs) of AV extract from experiment was observed it would be considered above 100µg/ml. At 300µg/ml, 200µg/ml and 100µg/ml the zone of inhibition was 86.71 %, 46.85% and 13.2% in order with respect to control Rifamycin. The % zone of inhibition for S. aureus at 300 µg/ml, 200µg/ml and 100µg/ml of AV extract 79.52%, 49.60% and 16.53% respectively with respect to control Rifamycin.

Control (Omadacycline) for microorganism E. cloacae (Gram-negative bacteria) C. albicanas (Fungi). The MICs and MBCs values for the microorganism E. cloacae of AV extract would be below 100 µg/ml, and above 100µg/ml. The zone of inhibition of AV extract 300µg/ml, 200µg/ml, and 100µg/ml were 76.25%, 35.25% and 10.79% respectively and for C. albicans 80.51%, 40.25% and 17.53% in order of AV extract concentration. The % inhibitions were calculated against the control Omadacycline. The results of the studies are presented in Figure 2 and 3.

Figure 2: Antimicrobial activity of partially purified Aloe vera extract against standard Rifamycin.

Figure 3: Antimicrobial activity of partially purified Aloe vera extract against standard Omadacycline.

3.6.2 Antioxidant activity:

By DPPH assay method, comparative % antioxidant activity of AEM, herbal formulation and aloe vera extract has been reported in figure 4. Experimental observation. The IC50 value of AEM (28.35 µg/ml), herbal formulation (39.84µg/ml) and aloe vera extract (31.53µg/ml) were reported. The IC50 value indicates that the extract antioxidant effect is more as compare to herbal formulation.

Figure 4: Comparison of % antioxidant activity of all three samples (Aloe emodin, Herbal formulation and Aloe vera Extract)

3.6.3 Anti-helminthic activity:

The 'time for paralysis' occurred if no motion of any kind had been seen, when the worms were aggressively shook in both the test solution and standard medication solution. The 'time for death' of the worms was determined after it had been found that they did not move while shaken vigorously or being submerged in warm water at 50°C. The paralysis time in control, Albendazole (20mg/kg), AEM (200µg/ml), Herbal formulation (200µg/ml), AV extract (200 µg/ml) have been founded 0±0, 15.06±0.32, 29.09±0.26, 41.13±0.24, and 36.17±0.34 minutes respectively, This shows that the paralysis times needed for herbal formulations are longer than those needed for AEM and AEM extract when compared to albendazole standard. Death time for all five groups are 0±0,21. 69±0.43, 38.23±0.22, 53.11±0.43, and 45.09±0.54 minutes in order. In comparison to AEM and AEM extract, the death time needed in herbal formulations is higher than the albendazole norm. This finding showed that herbal formulations contain less AEM than herbal extracts, which has also been supported by quantitative research. Figure 5.

Figure 5: Paralysis time and death time of worms in different samples (Aloe emodin, Aloe vera extract and Herbal formulation).

4. DISCUSSION:

Globally, majority of population rely on herbal remedies⁵². Bacterial and fungal strains are treated with ethanol extract of AV leaves and roots at various doses (15, 20, 25, 30µl). Escherichia coli and Agrobacterium tumefaciens exhibit an 18mm zone of inhibition, which is regarded as a successful result. At 16 mm, Bacillus subtitis and Bacillus megaterium also exhibit good results. Pseudomonas aeruginosa shows minimum zone of inhibition which is around 11mm. At 19 mm, Aspergillus niger has good performance against both root extract and leaf extract⁵³. AV leaf gel was Sextracted using four different solvents, including aloe vera leaf and root ethanol extracts. Gram-negative and Gram-positive isolates were shown to be most susceptible to these extracts⁵⁴. Aloe vera's ability to fight off microbes is assessed, as is how well it works to clean up gutta percha cones. Zones of diffusion for the three prevalent gutta percha pollutants E. coli, E. faecalis, and Staph. aureus was found. Using the agar plate, the zones of inhibition were measured as 24mm, 21mm, and 24 mm, correspondingly⁵⁵. The present finding of AV extract against E. coli, S. aureus, E. cloacae and C. albicans in 300µg/ml is good.

The antioxidant activity by DPPH method was performed and analysed that in quantitative analysis the aloe emodin content in high in AV extract as compare to herbal formulation. A comparative result of antioxidant property of aloe emodin, aloe vera extract and herbal formulation has shown effect of aloe vera extract is more compare to herbal formulation.

Again, the result of anthelmintic that has performed against aloe emodin, aloe vera extract and herbal formulation of same concentration (200µg/ml). The outcome indicates that the paralysis time and death time of aloe vera extract is less as compare to herbal formulation. This finding supports the quantitative analysis of herbal formulation and aloe vera extract.

5. CONCLUSION:

In various medicinal plants, the anthraquinone derivative AEM is present, which is an active ingredient in several formulations. A new RP-HPLC method was developed with BBD approach. This method is simple, accurate, and robust. In this research, chromatographic conditions were optimised for BBD surface response. Two dependable variables, retention time and tailing factors, were optimised by considering three independent variables: mobile phase composition, flow rate, and maximum. The developed method was validated according to ICH guidelines. The application of this method in the routine analysis of herbal extracts and formulations containing AEM.

Antimicrobial activity of AV extract of different concentration against the E. coli, S. aureus, E. cloacae and C. albicans have shown concentration dependent effects. Antioxidant property of the pure AEM, AV extract, and herbal formulation had shown the effective concentration of active bioactive compound (aloe emodin) by DPPH method. Anthelmintic activity also has shown the concentration of AEM in the samples.

6. CONFLICTS OF INTEREST:

The authors declare that they have no conflicts of interest.

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Received on 07.11.2023 Revised on 24.06.2024

Accepted on 30.10.2024 Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):165-172.

DOI: 10.52711/0974-360X.2025.00025

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