Development and Validation of UV-Visible Method to Determine Gallic Acid in Hydroalcoholic Extract of Erythrina fusca Leaves

Shivkanya Fuloria1, Lau TiewWei1, Sundram Karupiah1, Vetriselvan Subramaniyan2, Christina Gellknight3, Yuan Seng Wu2, Saminathan Kayarohanam4, Neeraj Kumar Fuloria*1 1Faculty of Pharmacy, AIMST University, Kedah 08100, Malaysia 2Faculty of Medicine, MAHSA University, Kuala Lumpur 42610, Malaysia 3Faculty of Medicine, AIMST University, Kedah 08100, Malaysia 4Faculty of Bio-economy and Health Sciences, Geomatika University College, Malaysia


INTRODUCTION
The expense of health care is rising at an alarming rate across the globe. The world market for phytopharmaceutical is also growing steadily. An estimation of the World Bank shows that trade in medicinal plants, raw materials and botanical drug products are growing at an annual rate of 5-15% (Patwardhan et al., 2005). Around 75-80% of the world population use herbal medicines (Pan et al., 2014). Standardization is considered as the critical factor to assure herbal drug quality. Access to herbal drug quality is attributed to active constituent concentration. Phytochemical constituents are crucial for the pharmacological action of herbal formulation. Thus, it is essential to set up a system of standardization for every plant medicine in the market (Kumar et al., 2011;Sitapara et al., 2011). It is very challenging for investigators to develop various authentic and accurate analytical protocols that could screen the phyto composition, including quantitative analysis of marker/bio active compounds is a signi icant challenge for scientists (Rasheed et al., 2012). Studies revealed that Erythrina fusca contains gallic acid (GA) that possess signi icant antioxidant, antidiabetic, antimicrobial property and high ef icacy against periodontitis (Fuloria et al., 2019).
Facts suggest many methods for the determination of GA as total phenolic content (TPC) individually and in combination with other drugs (Fernandes and Salgado, 2016). Various studies reported quanti ication and validation of GA in different plant extracts using Folin-Ciocalteu reagent based UV spectrophotometric method. This is because GA shows maximum absorption near 272 nm (Blainski et al., 2013;Kamboj et al., 2015). The study suggests Erythrina fusca plant possess high antioxidant potential (Subal et al., 2010;Innok et al., 2009). But till date, none of the studies performed the validation and development of estimation of GA in Erythrina fusca hydro-alcoholic extract (EFLHE). Hence, the present study was intended to quantify the amount of gallic acid (GA) in EFLHE using UV-Visible spectrophotometric method (Folin-Ciocalteu reagent method).

MATERIALS AND METHODS
The solvents, reagents and consumable were procured from Sigma Aldrich, SD Fine, Merck, and R&M chemicals. The glasswares were cleaned using deionised H 2 O and dried at 160 • C for 2 hours before the experiment. The spectrophotometric analysis was done using Shimadzu double beam UV-Visible spectrophotometer, model U-2800 (200-800 nm).

Preparation of EFLHE
The E. fusca leaves were collected (from the campus of AIMST University, Malaysia), washed (with H 2 O to remove dust), air-dried (until crispy) and powdered (coarse). The preparation of EFLHE was based on established maceration protocol with slight modi ication (Anjum and Chandra, 2015). Brie ly, in a 500 ml conical lask 100 g of EFL and mixture of ethanol and distilled H2O (1:1) were added with 1:10 w/v sample to solvent ratio. The obtained mixture was swirled for seven days on mechanical shaker maintained at 100 rpm. After seven days, the supernatant liquid was iltered, and the iltrate was dried using rotavapor at 70 • C. Next, the EFLHE was air-dried (for completely drying), kept in a desiccator (to remove moisture) and inally stored in a refrigerator.

Solvent Selection
The procedure for the selection of solvents for UV-Visible analytical method development was based on standard protocol with slight modi ication (Bhardwaj et al., 2017). In the protocol, various solvents were tested for the UV-Visible analytical method development, out of which Methanol and H2O (1:9) was selected based on solubility, peak quality, and non-interference at a speci ied wavelength.

Wavelength Selection
A representative spectrum of GA solution (10 µg/ml) in Methanol and deionised H 2 O (1:9) was obtained by scanning them in the UV range (200-400 nm) in 10 mm cell against blank solvent. Current protocol was founded on the established procedure with small variation (Bhardwaj et al., 2017).

Preparation of Stock Solution and Standard Curve
Accurately weighed quantity of GA (10 mg) was transferred into the 10 ml of volumetric lask and dissolved by diluting up to mark using Methanol and deionised H 2 O (1:9) to get a concentration of 1000 µg/ml. From this stock solution, the aliquots of working standard solution of GA were formulated using methanol and deionised H 2 O (1:9) solution to get a concentration in a range of 0.5-10 µg/ml for GA (Bueno et al., 2012).
The absorbance value for each concentration of the standard solution of GA was measured at λ max of 212 nm. A calibration curve between concentrations versus absorbance was built to study the Beer-Lambert's Law and regression equation.

Total Phenolic Content
Accurately 10 mg of EFLHE was dissolved ad diluted with Methanol and deionised H 2 O (1:9) to get a concentration of 1000 µg/ml. From this stock solution, aliquots of EFLHE solution were prepared with Methanol and deionised H 2 O (1:9) to get a concentration in the range of 0.5-10 µg/m (Guideline, 2005). The reaction mixtures were prepared by mixing 0.5 ml of each EFLHE solution with 2.5 ml of 1% Folin-Ciocalteu reagent dissolved in distilled H 2 O and 2.5 ml of sodium bicarbonate. Next, the reaction mixtures were incubated at 45 • C for 15 minutes. The prepared solutions absorbances were measured at 212 nm against a blank. The quanti ication of TPC of EFLHE was founded on a standard curve of GA that acts as a standard phenolic compound and expressed as mg of GA equivalent (GAE) per gram of EFLHE. The TPC was estimated using the following expression: Where: T = Total phenolic content in mg/g as GAE of EFLHE, C = Concentration of GA in mg/ml (established from calibration curve), V = Volume in ml of EFLHE solution, and M = Weight of EFLHE in gram.

Linearity
The 0.5 ml of each GA standard solution (0.5-10 µg/mL) was mixed with 2.5 ml of 1% Folin-Ciocalteu reagent (dissolved in distilled H 2 O) and 2.5 ml of sodium bicarbonate. Each reaction mixture was incubated at 45 • C for 15 minutes and scanned at 212 nm against Methanol and H 2 O (1:9) as blank in triplicate. A calibration curve was constructed by plotting concentration against absorbance (Figure 1). The regression equation and correlation coef icient were determined using GA standard concentrations (0.5-10 µg/ml). The relation between drug and absorbance is expressed as y = MX + c. Where, m = slope, c = intercept and x = concentration. Based on GA standard curve, TPC of EFLHE was estimated and expressed as mg of GAE per gram of plant extracts.

Accuracy
Analytical method accuracy was estimated by carrying out a recovery analysis of 80%, 100% and 120% of EFLHE concentration as per ICH guidelines in triplicate. Percentage recovery was estimated using the following expression: Where, ' A' is EFLHE absorbance after addition of standard, ' A T ' is theoretical absorbance (sum of

Precision
The developed analytical protocol was tested for precision founded on intraday and interday variations. Intraday precision was established by analyzing the 10, 15 and 20 ug/ml of EFLHE for three times on the same day. Interday precision was calculated by analyzing the 10, 15, and 20 ug/ml of EFLHE daily for three days.

Repeatability
The proposed protocol was validated for repeatability by analyzing 10 µg/ml of sample extract solution for six times.

Ruggedness
The analytical protocol ruggedness was determined by spiking the standard six times with different analyst using the same instrument.

RESULTS AND DISCUSSION
Facts suggest GA possess a signi icant antioxidant potential and protects the human body from free radicals harmful actions (Rasool et al., 2010).  Investigation suggests GA extracted from grape seeds induced the programmed death of prostate cancer cells (Kaur et al., 2009). Besides, GA is bene icial for diabetes patients as they can trigger the release of insulin by the pancreatic cells (Sameermahmood et al., 2010). These biological activities indicate the potential use of GA (Masoud et al., 2012;Phiriyawirut and Phachamud, 2011). Based on the facts over GA to exhibit its absorption in UV region, various researchers performed GA estimation and validation studies over plant extracts involving Folin-Ciocalteu reagent based spectrophotometric analysis (Singh and Avupati, 2017;Purohit et al., 2014).
However, to date, none of the studies suggested GA estimation and validation in Erythrina fusca ethanolic extract (EFLHE). Based on these facts present study was intended to quantify the amount of GA in EFLHE using UV-Visible spectrophotometry (Folin-Ciocalteu reagent method). Application of given formula estimated associated with E. fusca leaves was found to be 32%. The per cent yield of EFLHE was estimated based on the dry weight of EFLHE (X) and EFLHE soaked (Y) using the given formula:

Development of UV-Visible spectrometric method
The earlier study suggests that solvents substantially affects the quality of the spectrophotometric signals (Bhardwaj et al., 2017). Hence, in the present study, the selection of solvents for UV-Visible method development was made by testing different solvents based on solubility, peak quality, and non-interference at a speci ied wavelength. Solvent optimization study revealed Methanol and H 2 O (1:9) as the most suitable solvent for the current protocol. For wavelength optimisation, a representative spectrum of GA solution (10 µg/ml) in Methanol and deionised H 2 O (1:9) was scanned from 200 to 400 nm. The UV-Visible spectrum revealed well-de ined λ max at 212 nm for GA. The analysis of EFLHE for GA as TPC was carried out as per the protocol given in the experimental part of the present study. The resultant data for the same is given in Table 1.

Linearity
The analytical protocol for linearity is protocol ability to deliver results in a speci ied range directly or through mathematical expression, proportional to analyze concentration (Jain et al., 2011). The linearity results for EFLHE were derived from the calibration curve of GA (0.5-10 µg/ml). The correlation coef icient (r 2 ) from the calibration curve was found to be 0.997 (Figure 1) and expressed in GAE per gram dry EFLHE weight. The content of phenolic compounds in EFLHE extracts ranged from 98 to 295.2 mg GAE/g, representing an approximate four-fold variation (  (Kaur et al., 2009).

Accuracy
Generally, the accuracy of the analytical protocol is the closeness of practical result to theoretical value (Bhardwaj et al., 2017). Accuracy study was conducted as per the experimental protocol and resulted in data for the same is given in Table 3.
The results of the accuracy study revealed percentage recovery of 86.80±0.49, 87.60±0.36 and 89.07±0.24 respectively, for the 80 %, 100 % and 120 % of the test concentration. As per the report of Andressa Blainski et al. these percentages were within the range of 85%-115% which indicate that the method has good accuracy for quanti ication of GA from EFLHE (Blainski et al., 2013).

Precision
The precision of an analytical protocol is a degree of repeatability under the normal operation conditions. Precision studies of the developed method were conducted as per the intraday and interday experimental protocol of the present study. The results for precision study are reported in Tables 4  and 5. Both intra and the inter-day precision study revealed quiet low % RSD with the highest value of 0.43%. A study claimed that %RSD less than 2% indicates good precision (Pawar and Salunkhe, 2013).

Repeatability
Repeatability study of the developed method was conducted as per the experimental protocol of the present study and resulted in data given in Table 6. The 10 µg/ml of EFLHE were analyzed for six times, and amount of GA as TPC found was of little difference with a standard deviation of 1.6, which indicates good repeatability. Amount of GA as TPC found in 10 µg/ml of sample extract (n=6) found was 295±1.6 GAE (mg/g).

Ruggedness
Ruggedness study over-developed analytical method was performed according to the present study experimental protocol, and results are given in data given in Table 7. The ruggedness of the method was assessed by spiking the standard six times with different analyst by using the same equipment. The results showed that for 20 µg/ml of sample extract, both analyst 1 and analyst 2 obtain an amount of Gallic Acid of 327.1±0.74 GAE (mg/g) and 321.2±2.48 GAE (mg/g) respectively. Both analysts had %RSD less than 2 %, which are 0.22 % and 0.77 % respectively. As the percentage RSD value is less than 2 %, so the variation of analysts will not affect the UV method in the quanti ication of GA in EFLHE (Pawar and Salunkhe, 2013). Based on resultant data, the present study reveals that quanti ication of GA in EFLHE through UV-Spectrophotometer is an economical and straight forward method. The validation of the analytical protocol developed in this study has been proven to be linear, speci ic, precise, accurate, reproducible, rugged, and easy.

CONCLUSIONS
This is the irst-time study to develop and validate the method for quanti ication the amount of gallic acid in Erythrina fusca hydro-alcoholic extract using UV-Visible spectrophotometric method (Folin-Ciocalteu reagent method). Based on the experimental results of the present study, it can be concluded that quanti ication of gallic acid in a hydro-alcoholic extract of Erythrina fusca leaves can be carried out by UV-spectro photometric technique. The developed method is quite simple and less time-consuming. Besides, this method requires less labor cost and less sophisticated and less expensive equipment. Apart from it, this method has been validated as required by ICH guidelines.
The current study recommends that in the future estimation of gallic acid in other parts of Erythrina fusca plant such as bark or stem can also be done. Moreover, variant extraction methods may also be used to replace simple maceration to compare the amount of gallic acid.