Investigation on Photodecomposition of Standardised Ethyl Acetate Fraction of Katha

The use of herbal medicines has become a global subject with medical and economic ramifications over the past few decades.1 Acacia catechu (L.f.) Willd. known by the name ‘khadira’ in Ayurveda belongs to Leguminosae family.2 The plant is a small moderate sized tree about 10-13 meter height extensively scattered throughout India.3 The medicinal significance of this plant is reflected by the use of leaves, bark and heartwood in several Ayurvedic formulations for thousand years.4 Heartwood of A. catechu has more potent medicinal activity in comparison to its leaves and bark.5 The heartwood extract of A. catechu has been reported to have various pharmacological effects like antioxidant,6 anti-inflammatory,7 antimicrobial,8 immunomodulatory,9 antipyretic, hypoglycaemic,10 anti-diarrhoeal and hepatoprotective11 etc. The plant contains polyphenols like tannins, flavonoids alongwith carbohydrates and proteins.12 The major constituents of the plant includes catechin (-), epicatechin, epigallocatechin, epicatechingallate and epigallocatechin gallate. Other constituents include rocatechin, phloroglucin, protocatechuic acid, quercetin, poriferasterol glucosides, lupenone, lupeol, procyanidin AC, kaempferol, dihydrokaemferol, toxifolin, (+)-afzelchin gum and minerals.13 The heartwood of this plant is known by the name ‘Katha’ which is well known for its diverse pharmacological properties.14,15 In the present study the stability of standardised ethyl acetate extract and changes in antioxidant potential was investigated through in-vitro studies as illustrated in Figure 1. Storage conditions were already reported to exert marked influence over chemical composition of crude drugs.16,17 The aim of this study was to determine antioxidant capacity of ethyl acetate fraction of Katha obtained from the heart wood of plant A. catechu and to access its photodecomposition based on temperature and storage conditions.


INTRODUCTION
The use of herbal medicines has become a global subject with medical and economic ramifications over the past few decades. 1 Acacia catechu (L.f.) Willd. known by the name 'khadira' in Ayurveda belongs to Leguminosae family. 2 The plant is a small moderate sized tree about 10-13 meter height extensively scattered throughout India. 3 The medicinal significance of this plant is reflected by the use of leaves, bark and heartwood in several Ayurvedic formulations for thousand years. 4 Heartwood of A. catechu has more potent medicinal activity in comparison to its leaves and bark. 5 The heartwood extract of A. catechu has been reported to have various pharmacological effects like antioxidant, 6 anti-inflammatory, 7 antimicrobial, 8 immunomodulatory, 9 antipyretic, hypoglycaemic, 10 anti-diarrhoeal and hepatoprotective 11 etc. The plant contains polyphenols like tannins, flavonoids alongwith carbohydrates and proteins. 12 The major constituents of the plant includes catechin (-), epicatechin, epigallocatechin, epicatechingallate and epigallocatechin gallate. Other constituents include rocatechin, phloroglucin, protocatechuic acid, quercetin, poriferasterol glucosides, lupenone, lupeol, procyanidin AC, kaempferol, dihydrokaemferol, toxifolin, (+)-afzelchin gum and minerals. 13 The heartwood of this plant is known by the name 'Katha' which is well known for its diverse pharmacological properties. 14,15 In the present study the stability of standardised ethyl acetate extract and changes in antioxidant potential was investigated through in-vitro studies as illustrated in Figure 1. Storage conditions were already reported to exert marked influence over chemical composition of crude drugs. 16,17 The aim of this study was to determine antioxidant capacity of ethyl acetate fraction of Katha obtained from the heart wood of plant A. catechu and to access its photodecomposition based on temperature and storage conditions.

Plant material
The heartwood of the plant was collected in the month of November 2019 from Hamirpur district of Himachal Pradesh, India which further was authenticated by Raw material herbarium and museum, NISCAIR, New Delhi, India. A voucher specimen of the plant was preserved in the herbarium for reference (NISCAIR/RHHD/ Consult/2019/3465-66).

Preparation of plant extract (Katha)
The heartwood of A. catechu was dried at room temperature (25 ± 2°C) for four consecutive weeks and pulverised. Katha was obtained from the heartwood of A. catechu by boiling the chips of heartwood with 10% hydro-alcoholic solution. 18

Standardisation of katha
The specimen was processed for pharmacognostic standardisation viz morphological studies, powder microscopy, ash value determination, florescence analysis and loss on drying. Each extract was then screened for secondary metabolites. Presence of active antioxidant compound was confirmed with the help of thin layer chromatography by using desired mobile and stationary phases. These parameters are considered essential in quality control of the crude drugs. 19

Soxhlation of katha
Katha chips obtained from heartwood of A. catechu is powdered and extracted by successive solvent extraction technique using soxhlet apparatus. 100 g of coarsely powdered drug was allowed to get extracted with different solvents in order of increasing polarity viz. ether, ethyl acetate and ethanol.

Quantitative phytochemical screening
Quantitative phytochemical screenings of ethyl acetate fraction of Katha were performed as per standard protocols to detect the amount of total phenols and total flavonoids in ethyl acetate fraction.

Determination of total phenolic content
Folin-Ciocalteu reagent was used to evaluate the total phenolic content of the extract using gallic acid as standard. 20 Standard curve of Gallic acid was prepared by taking 500, 250, 125, 62.5, 31.25 and 15.625 µg/ml concentrations. Procedure for determining absorption of various concentrations is same as follows for extract fractions. All the samples were subjected to temperature of 60°C on water bath for 1h followed by cooling to room temperature. 400 µL of this solution was transferred into the test tube containing 1.6 mL of sodium carbonate (7.5% in deionized water) and 2 mL of Folin Ciocalteu reagent (0.1% in deionized water). Further all the samples were incubated for 1 h at room temperature. Absorbance was measured at 525nm using UV Spectrophotometer. All the readings were taken in triplicate. Total phenolic content was expressed in mg Gallic acid equivalent (GAE) per gram of extracts, using calibration curve.

Estimation of flavonoid content
Most commonly used method to determine total flavonoids contents by taking Quercetin as standard. 21 Different concentration of extract and standard was prepared as above and 100, 50, 25, 12.5 µg/ml extract and standard was added to the test tube containing 75 µL of 5% NaNO2 solution. Mixture was allowed to stand for 10 minute. 150 µL of a 10% AlCl3.6H2O solution was then added to every sample and were allowed to stand for 5 minutes. Further 0.5 mL NaOH (1 M) and 2.5 mL of distilled water was added to each sample. Absorbance was measured at 510 nm using UV Spectrophotometer. All the observations were taken in triplicate. Total flavonoid content was calculated as mg Querctine equivalent (QE)/g by using the linear regression equation obtained for Quercetin.

Antioxidant activity
In the present study, two commonly used antioxidant evaluation methods such as DPPH radical scavenging activity and Nitric oxide radical scavenging assay were selected to determine the antioxidant potential of ethyl acetate fraction of Katha.

Determination of Free Radical Scavenging Activity by DPPH Method
DPPH radical scavenging activity of ethyl acetate fraction of Katha was determined according to the standard method with slight alteration. The reaction mixture containing 500, 1000 µg/ml of extract concentration and 2 ml of DPPH (0.1 Mm in methanol) was allowed to stand for 15 minutes in dark at room temperature. Absorbance was measured using double beam UV-VIS spectrophotometer (shimadzu-1601) and tested against the blank. The scavenging potential was calculated by using the following equation. 22 Where Bº is the absorbance of negative control B I is the absorbance of the reaction mixture

Determination of Free Radical Scavenging Activity by Nitric Oxide Radical Method
Griess reagent was prepared by mixing same amounts of 1% sulphanilamide in 2.5% phosphoric acid and 0.1% naphthylethylene diamine dihydrochloride in 2.5% phosphoric acid. Ethyl acetate extract

Phytochemical Studies
Preliminary phytochemical screening of ether, ethyl acetate and ethanolic extract of Katha showed the presence of tannins, flavonoids, saponins and triterpenes as shown in Table 2 but the main allure of screening was presence of polyphenols (tannins, flavonoids) in ethyl acetate extract. Tannins reported in this plant have already been documented to exhibit pharmacological as well as physiological properties (6)(7)(8)(9)(10)(11). Furthermore, ethyl acetate extract of Katha is having pink colour with no odour and obtained as fine powder as shown in Figure 2a. Ethyl acetate extract is highly soluble in methanol, ethyl acetate, slightly soluble in benzene, petroleum ether and insoluble in water. Melting point of the extract was found to be between 150-210ºC and in UV light extract showed magenta colour fluorescence.

Quantitative phytochemical screening
The standard curve of Gallic acid obtained at different concentration (µg/ml) 500, 250, 125, 62.5, 31.25 & 15.625 respectively. Total phenolic content of the extract fractions was calculated in terms of mg GAE per was added with similar volume of freshly prepared sample. Nitric oxide radical so generated was found to be inhibited by ethyl acetate extract at different concentrations. Decolouration due to reaction by polyphenols in ethyl acetate fraction with the nitric oxide free radical was then measured spectrophotometrically. The percentage inhibition of ethyl acetate extract and standard was recorded. All the experiments related to antioxidant activity were performed in triplicate. 23

Pharmacognostic data
Katha occurs in pieces of variable sizes of 4 to 4.5 cm in length and 3.5 to 4.5 cm in breath, yellowish brown in colour, fracture is hard, characteristic odour with astringent taste. Powder microscopy of Katha revealed the presence of acicular type crystals, fibers and pitted vessels as shown in Figure 1. Successive extractive values with ether, ethyl acetate and ethanol were found as 1.2 % w/w, 4.5% w/w and 3.5 % w/w respectively Table 1. Total ash and acid insoluble ash value was found to be 11.5 % w/w, 0.68 % w/w which confirms the presence of inorganic content in the drug. Moisture content of the drug was found to be between 9-13% w/w. On exposure to UV light Katha showed dark brown fluorescence.

Percentage yield of katha
Different extracts were obtained upon successively treated the sample (Katha) with different solvents as mentioned in Table 1.  Determination of free radical scavenging activity by DPPH method DPPH radical showed a strong absorption maximum at 517 nm (purple). In presence of antioxidant the colour reaction takes place. DPPH free radical scavenging activity of ethyl acetate extract of Katha at various concentrations 500 µg/ml and 1000 µg/ml was determined by taking sample with different storage conditions as shown in Figure 3 and Table 3. The first sample selected was kept at light under ordinary room temperature and the other sample was stored in dark under controlled environment.

Determination of free radical scavenging activity by nitric oxide radical method
Percentage free radical scavenging was plotted against concentration of the extracts as shown in Figure 4. The Nitric oxide free radical scavenging activity of ethyl acetate extract of Katha at various concentrations viz 500 µg/ml and 1000 µg/ml was determined by taking sample with different storage conditions Table 4. The first sample was kept at light under ordinary room temperature and the second sample was stored in dark under controlled conditions. It was observed that the sample placed in dark and in controlled conditions does not show any change in the scavenging potential whereas the sample placed in light, under ordinary temperature undergoes deterioration exponentially with time.

DISCUSSION
Katha obtained by boiling the heartwood of A. catechu with 10 % hydro-alcoholic solution resulted in increase in the percentage yield value by 10 % w/w. In chromatographic studies four active substances were identified on the basis of their respective Rf values. But the major concern of this study was to determine the photo-decomposition of ethyl acetate extract at different storage conditions. The extract is highly unstable in its solution form and its antioxidant potential degrades exponentially with time. For evaluating its antioxidant potential two methods were used (DPPH free radical scavenging and nitric oxide free radical scavenging

CONCLUSION
Standardised ethyl acetate extract of Katha was found to be highly unstable in its solution form and showed exponential decline in antioxidant potential with time. Therefore, for the experimental purpose, it should be preserved properly to avoid photodecomposition and to alleviate chances of deviation. The overall outcome of this study can be considered as very promising in establishing stability profile of plant extract with respect to storage conditions and time. Hence, from this study it might be postulated that plant extracts containing polyphenols are highly sensitive and undergoes photo-degradation exponentially with time so special care should be given while handling these plant extracts which otherwise might causes deviation in the therapeutic profile of the drugs like in our case antioxidant activity.