Pharmacognostic Specifications , Quercetin and Quercitrin Quantification in Bauhinia malabarica Leaf

Introduction: Bauhinia malabarica Roxb. is a tropical tree that found throughout Thailand. Leaves have a sour taste and have been used in Thai remedies for wound healing, diuretic, dysentery and emmenagogue. Objective: This study aimed to focus on pharmacognostic specification and quantitative analysis of quercetin and quercitrin in B. malabarica leaves. Methods: Various methods such as macroscopic and microscopic evaluations of B. malabarica leaf were studied along with physico-chemical parameters and quantitated quercetin and quercitrin using RP-HPLC. Results and Conclusion: Whole plant, structures of dried powder crude drug, cross section of midrib and leaf measurement were established. Paracytic stomata and multicellular trichome were found on lower epidermis. B. malabarica leaves from 15 sources throughout Thailand were examined the pharmacognostic specification according to WHO guideline. Physico-chemical parameters showed that loss on drying, total ash, acid insoluble ash and water content should not be more than 8.00, 7.08, 1.79 and 8.28 % of dry weight while ethanol and water soluble extractive values should not be less than 13.78 and 16.47 % of dry weight respectively. Quercetin and quercitrin were the markers for quantitative analysis using RP-HPLC with diode array detector in B. malabarica ethanolic extract. Quercetin and quercitrin contents were found to be 0.18 g and 0.38 g in 100 g of dried crude drug. Method validation was determined according to ICH guideline. All results were in the acceptable range and could be used for identity, safety, efficacy and quality of B. malabarica leaves in Thailand.


INTRODUCTION
Bauhinia is a large genus of the family Leguminosae that approximately 300 species of trees, shrubs and climbers.This genus is native in tropical zones such as Africa, Asia and South America.They are commonly known as cow's paw or cow's hoof because of leaf shape. 1 Mostly Bauhinia species are planted for showy flowers and ornamental shrubs.Bauhinia malabarica is a tropical tree that found throughout Thailand.Young leaves and flowers are edible.Leaves have a sour taste and have been used for wound healing, diuretic, dysentery and emmenagogue.Previous study, seven flavonol derivatives have been purified from B. malabarica leaves using chromatographic techniques such as 6,8-di-C-methylkaempferol 3 methyl ether, kaempferol, afzelin, quercetin, isoquercitrin, quercitrin and hyperoside. 2 Quercetin is a flavonoid normally found in many fruits, vegetables, leaves and grains.The primary benefits of quercetin are possesses strongly antioxidant and anticancer. 3Quercitrin is a glycoside formed from quercetin and deoxy sugar rhamnose.Quercitrin has been demonstrated biological activities such as anti-inflammatory effect, preventing diarrhea Pharmacognosy Journal, Vol 11, Issue 1, Jan-Feb, 2019

Sample extraction
Five grams of B. malabarica fine powdered were exhaustively extracted with 95% ethanol using Soxhlet apparatus, filtered, the filtrate were evaporated to dryness and stored in refrigerator for quantitative quercetin and quercitrin using reverse phase high performance liquid chromatography (HPLC).

Morphological examination
B. malabarica fresh mature leaves were macroscopically and microscopically observed.Fine powder of B. malabarica leaves was mixed with a few drops of water on the slide and observed under microscope with the magnification of 10x to 40x.The middle part of fresh mature leaf was cut into 0.5x0.5 cm, soaked in 50% sodium hypochlorite in water until clear, washed and boiled in chloral hydrate solution (4:1, chloral hydrate: water) until translucent, examined under microscope for palisade, trichome and epidermal cell numbers.Palisade cells in four epidermal cells were counted and divided by 4 for calculation palisade ratio.Trichome index and stomatal index were obtained from this formula (S or T×100)/(S+E+T) whereas S=stomatal number, E=number of epidermal cells and T=trichome number.The midrib of B. malabarica fresh mature leaf was thinly cross-section with blade by hand and observed the anatomy under microscope with the magnification of 10x to 40x.

Physico-chemical characteristics
Fine powder of B. malabarica dried leaves was examined for water content, loss on drying, ashes, extractive matters and volatile oil content according to "Quality control methods for medicinal plants materials" that established by World Health Organization (WHO). 5All samples were analyzed in triplicate.Grand mean and pooled standard deviation were calculated.

Thin layer chromatographic fingerprint
Three microliters of crude ethanolic extract solution were spotted onto the silica gel GF 254 TLC plate.TLC plate was developed in saturated TLC chamber with toluene: acetone: chloroform: formic acid (2: 8: 8: 2).After development, the plate was observed under day light, UV 254 nm, UV 365 nm, stained with p-anisaldehyde and 1% aluminium chloride in ethanol and detected under UV 365 nm.

Method validation
Calibration range, specificity, accuracy, repeatability, intermediate precision, limit of detection (LOD), limit of quantitation (LOQ) and robustness were examined following to the ICH guideline. 6

Morphological examination
B. malabarica Robx.leaves are green color on both sides.The leaf shape was is bilobed, rough surface, thick leaf, 6 -10 cm wide, 5 -7 cm long (Figure 1) and sour taste.Flowers are small white or light green.Powdered of B. malabarica leaves consist of multicellular trichome, paracytic stomata, epidermis, spiral vessel, prism of calcium oxalate and fibers (Figure 2).Microscopic leaf constant numbers including epidermal cell area, stomatal number, stomatal index, trichome number, trichome index and palisade ratio were shown in Table 1.Anatomical characteristic of B. malabarica leaf (midrib part cross section) was shown in Figure 3. Physico-chemical properties of B. malabarica dried leaves including water content, ethanol soluble extractive, water soluble extractive, loss on drying, total ash, acid insoluble ash and volatile oil content were demonstrated in Table 2. Plants extracts can provide a chromatographic "fingerprint" used for identification purposes.The colors of the separated spots and their position relative to standard substances are important characteristics for the plant extract identification. 7In this study, TLC fingerprint was the preliminary identification of B. malabarica (Figure 4).Pharmacognostic parameters were shown the specific characteristics of B. malabarica leaf constant numbers.In morphological examination part, the specific character of B. malabarica leaves was sour taste, only this species has the sour taste, so, unknown sample which looks like B. malabarica needs to be tasted in the first step.Another specific character was not only multicellular trichome and paracytic stomata on lower epidermis, but also the average of palisade ratio.Palisade ratio is the diagnostic value in differentiating the species and dose not based on geographical variations. 8

HPLC analysis
The content of quercetin and quercitrin in B. malabarica dried leaves were shown in Table 3.The content values were calculated and shown as grams of quercetin or quercitrin per 100 grams of dried crude drug.The different values of quercetin or quercitrin in B. malabarica leaf ethanolic extract were depended on the geographic variation that affected to secondary metabolite of plants. 9

Method validation
The method validation parameters of reversed phase high performance liquid chromatography were optimized by performing the effect of the mobile phase and detection wavelength on resolution and sensitivity.An isocratic elution method was shown good separation in both standard and B. malabarica ethanolic extract.In this study, the range of absorbance (200 -800 nm) was screened in both quercetin and quercitrin, the Pharmacognosy Journal, Vol 11, Issue 1, Jan-Feb, 2019      result was shown that the detection wavelength at 255 nm was presented the optimum absorbance wavelength.Linear calibration curves in the range of 20, 40, 60, 80 and 100 µg/ml were created for each compound by plotting the peak area with the concentration.The regression equation of quercetin and quercitrin were y = 18199x -31136 and y = 14702x -6863.3.The linear demonstrated excellent correlation as 0.998 in quercetin and 0.999 in quercitrin respectively (Figure 5).The correlation of method (R 2 ) value was reached 0.99 which mean that this analytical technique is acceptable. 10he peaks of quercetin and quercitrin were well separated at different retention times with resolutions of 20.324 and 11.245, respectively.
No interferences or excipient peaks co eluted with the analytes were observed, indicating the method is selective and specific in this study. 11eak purity test was based on absorbance spectrum using diode array detector which confirmed the mobile phase was suitable for analyte chromatographic peak that showed a good separation from another compound.If all the individual spectra recorded during elution of a peak are identical even detected at any periods of peak, the peak is considered as absolutely pure. 12Peak purity index of quercetin and quercitrin showed more than 0.999 (Figure 7, 8 and 9), it demonstrated that all peaks were pure.The accuracy was determined by recovery method which spiked three concentrations (20, 40 and 60 µg/ml) of standard quercetin and quercitrin into B. malabarica leaves ethanolic extract, acceptable of %recovery is between 80 -120 %, 6 the results ranged from 97.378 -99.177 and 98.181 -102.288%recovery as shown in the Table 4.The precision, both repeatability and intermediate precision were done on spiked sample with three different concentrations in the same day and three different days.The result of %RSD was determined the error of the method, acceptable of %RSD was not more than 15 %RSD. 13The result of repeatability and intermediate precision in quercetin and quercitrin were shown in Table 4.
The residual standard deviation of a regression line and the slope of calibration range in both quercetin and quercitrin were used to calculate LOD and LOQ analysis.LOD and LOQ of quercetin were 4.755 and 14.408 µg/ml and 1.941 and 5.883 µg/ml in quercitrin, respectively.
The slightly changes of column temperature, flow rate and wavelength were calculated from one sample for robustness method.Column temperature was varied from 34 -36°C, the flow rate was set in the range of 0.950 -1.050 ml/min and the detection wavelength was defined at 252nm, 255nm and 258nm in both quercetin and quercitrin.The results were shown in the Table 5.The variations of column temperature, flow rate and detection wavelength were demonstrated that there were no differences (%RSD < 8) in the retention time and the area of the curve of quercetin and quercitrin.So, the robustness was showed reliably of the method. 10

Figure 4 :
Figure 4: TLC fingerprint of B. malabarica leaf ethanolic extract; A=detection under UV 254 nm, B=detection under UV 365 nm, C=stained with p-anisaldehyde and D=stained with 1% aluminium chloride and detected under UV 365 nm.

Figure 5 :
Figure 5: The calibration curve of quercetin and quercitrin.

Figure 7 :
Figure 7: The peak purity of quercetin in B. malabarica leaf ethanolic extract.

Figure 9 :
Figure 9: The peak purity of quercitrin in B. malabarica leaf ethanolic extract.

Table 2 : Physico-chemical parameters of B. malabarica dried leaves. Parameter Content (% by weight)
*The parameters were shown as grand mean ± pooled standard deviation.Samples were from 15 different places throughout Thailand and each sample was done in triplicate.

Table 5 : Robustness of quercetin and quercitrin in B. malabarica leaves.
*Rt= Retention time