The Journal of Animal & Plant Sciences, 30(1): 2020, Page: 219-228
ISSN: 1018-7081

 PHYTOCHEMICAL ANALYSIS AND IN VITRO INVESTIGATION OF ANTI-INFLAMMATORY AND XANTHINE OXIDASE INHIBITION POTENTIAL OF ROOT EXTRACTS OF BRYOPHYLLUM PINNATUM

A. Latif1*, K. Ashiq1, 2, S. Ashiq3, E. Ali1, I. Anwer1and S. Qamar4

1University College of Pharmacy, University of the Punjab, 54000, Lahore, Pakistan.
2Department of Pharmacy Superior College, 17-km Raiwind Road Lahore, Pakistan.
3Centre for Applied Molecular Biology, University of the Punjab Lahore, Pakistan.
4Institute of Pharmaceutical Sciences, University of Veterinary and Animal Sciences Lahore, Pakistan.
Corresponding Author E-mail: abidalatif43@yahoo.com

ABSTRACT

The aim of present study is to emphasize the phytochemical and biological studies of root extracts of Bryophyllum pinnatum, as little work was documented on the roots. Phytochemical analysis of powder and extracts were evaluated by the standard methods. UV/ Visible scanning, FTIR and atomic absorption spectroscopy were also performed for the purposes of standardization. In vitro anti-inflammatory investigation was done by protein denaturation method and xanthine oxidase inhibition assay was performed to confirm anti-gout effect. Phytochemical analysis results revealed the presence of primary metabolites (%w/w); carbohydrates (48.54+0.6) > proteins (21.06+0.6) > lipids (1.32+0.001). Secondary metabolites (mg/g) were also isolated; methanol extract has higher concentrations of flavonoids (95.81+0.026) and glycosaponins (39.9+0.103) while in water extract polysaccharide (139.4 + 0.026) and polyphenols (114.93+0.0234) were found to be in maximum quantities as compared to the other extracts. UV/Visible and FTIR scanning results have shown the presence of various significant functional groups. In vitro anti-inflammatory evaluation showed that aqueous extract (87.46%) was found to be the most active with IC50 (570.24ug/ml) as compared to the reference drug diclofenac sodium. In vitro xanthine oxidase inhibition activity was performed and methanol extract (88.24%) found to be the most effective. The findings confirm the plant nutritional value and rationale behind its use in traditional medicine for the treatment of inflammation and gout. 
Key words: Bryophyllum pinnatum, anti-inflammatory, gout, xanthine oxidase, phytochemical analysis.

INTRODUCTION

                Globally, medicinal plants are extensively used for the cure of various disorders. Drugs obtained from medicinal herbs are low-priced, conveniently accessible and have lesser side effects as compared to the synthetic drugs (Mekuria et al., 2017). Bryophyllum pinnatum (family: Crassulaceae) is also known as Bryophyllum calycinum or Kalanchoe pinnatum (Sadhana et al., 2017). The word “Bryophyllum” is derived from the Greek word which means “sprout leaf” (leaf cuttings can grow) and “pinnatum” is coined from the Latin word which means “quill” (Nagaratna and Hegde, 2015). It is a perennial herb about 3 to 5 meters high and having divergent glabrous leaves (Afzal et al., 2012). It has a sour taste, sugary post digestive effect and hot strength. Herb contains a wide range of valuable chemicals that are responsible for various beneficial therapeutic effects (Jaiswal and Sawhney, 2006; Latif et al., 2019). Bryophyllum pinnatum grows naturally and is native to Madagascar. It is found in temperate regions, including: Asia, Galapagos, Polynesia, New Zealand, Melanesia,West Indies, the Pacific and Caribbean, Hawaii and Australia (Zamora et al., 1998; Naz et al., 2009).
                Several significant therapeutic active constituents and secondary metabolites of plant have been isolated and documented. Among these the most remarkable are flavonoids and bufadienolides (Fürer et al., 2016). The major bufadienolides isolated from the plant leaves are Bryophyllin B and A (Potterat et al., 2013). The herb is an outstanding reserve of vitamins such as niacin, thiamine and ascorbic acid. Minerals are also found in plant which includes: calcium, magnesium, sodium, phosphate, zinc and potassium (Milad et al., 2014).
                Worldwide, Bryophyllum pinnatum is used for the treatment of conjunctivitis, edema, cholera, cuts, eczema, piles, chest colds, epilepsy, constipation, asthma, chicken pox, menstrual disorders, and fever (Majaz et al., 2011a). It is frequently used for the cure and management of blisters, burns, cough suppression, rheumatoid arthritis, psychiatric disorders, insect bites and abdominal discomforts. Moreover, it acts as a tocolytic agent to prevent premature labor (Gupta et al., 2016; Sadhana et al., 2017) and also employed for the treatment of leg edema, gout, fever, otitis, abscesses and palpitations (Afzal et al., 2012).
                This study is aimed to investigate the phytochemical composition of the plant root extracts and in vitro anti-inflammatory and xanthine oxidase inhibition effects. The roots were selected because there is little scientific documentation on them and there is also need to explore the therapeutic value of the plant (Majaz et al., 2011).

MATERIALS AND METHODS

Plant material and extraction: Bryophyllum pinnatum whole plants were collected in July, 2017 (Figure 1). The plants were obtained from the botanical garden of University of the Punjab, Lahore, Pakistan and identified by taxonomist Prof. Dr. Zaheer-ud-Din Khan, Department of Botany, Government College University (GCU) Lahore, Pakistan. A specimen was deposited in a GCU herbarium (after authentication, voucher no. 3369 was issued). The roots were separated from the plant (Figure 2), cleaned and then dried at the room temperature under the shade for 20-25 days. The dried roots were then crushed into fine powder and stored in airtight plastic bag. Sequential extraction was carried out by using Soxhlet. During the extraction procedure, solvents of different polarity (n-hexane, chloroform, methanol and water) were used. Keeping the temperature less than the boiling point of the respective solvent, all extracts were dried by using a rotary evaporator except aqueous extract which was dried by freeze drying. All extracts were collected in clean, tarred and labeled bottles, and allowed to dry in oven at 40 0C.
Chemicals: Aluminum nitrate crystals, Bovine serum albumin, Chloroform, Copper sulphate, Glucose, Methanol sodium bicarbonate, Sodium hydroxide, Potassium tartrate (Merck, Germany), Gallic acid (Sinochem, China), n-Hexane (BDH, England), Quercitrin (Sigma life sciences, Germany), Anthrone reagent (Sigma life sciences, Germany), Folin and Ciocalteu’s phenol reagent (Unichem Chemicals, Ireland), Triton X (Unichem chemicals, Ireland), Allopurinol TCI (Tokyo Chemical Industry), DMSO (Reagent Densen), Xanthine oxidase (Sigma).
Proximate analysis: The total ash, acid soluble ash, water insoluble ash, sulphated ash, moisture content and alcohol and water soluble extractive values were determined according to standard procedures (USP 2015).
Phytochemical analysis: Bryophyllum pinnatum root powder and extracts were evaluated for the levels of metabolites (primary and secondary metabolites). These were estimated by the standard methods described as: total proteins (Lowry et al., 1951), total lipids (Besbes et al., 2004), carbohydrates (Al-Hooti et al., 1998), total polyphenols (Slinkard and Singleton, 1977), total flavonoids (Chang et al., 2002), total polysaccharides and total glycosaponins (Hussain et al., 2008).
Mineral content analysis: Atomic absorption spectroscopy and flame photometer were used to determine the mineral content of the plant. 20% w/v nitric acid was employed to clean volumetric flasks and then rinsed with deionized water. 1 g of root powder was taken into the beaker. The sample was treated with 15 ml of digestion mixture which comprised of concentrated nitric acid and hydrochloric acid in 1:3 (v/v). The mixture was heated in fuming hood till no more smoke appeared. At room temperature, contents were cooled and filtered. Deionized water was used to make a final volume up to 100 ml and stored in a stoppered bottle for analysis. Analysis of the digested sample was carried out by using the atomic absorption spectrophotometer and flame photometer. Working solution of standard (concentrations; 0, 1, 5, 10, 15, 20, 25 and 100 ppm) was prepared from standard stock solution (1000 ppm). The tests were performed in triplicates and concentrations of minerals in the sample were determined by the standard plot (Ahmad et al., 2014).
UV/Vis spectroscopy: Extracts stock solutions (1 mg/ml) were prepared in methanol for the UV/Vis scanning. Methanol was added in stock solution to make the volume up to 10 ml and the same solvent was used as blank. In UV/Vis region (200-800 nm) scans were taken and for each extract maximum wave length (λ max) was recorded.
FTIR spectroscopy: Root powder and extracts were subjected to FTIR analysis for the purposes of standardization.  Material (1mg) was mixed with 100 mg of potassium bromide (KBr). Then the mixture was transferred to die which was compressed in a hydraulic press to prepare the pellets. FTIR spectra were obtained in the range 4000-400cm-1(Pavia et al., 2008).
In vitro anti-inflammatory activity: In vitro anti-inflammatory potential of extracts was determined by the protein denaturation method. The reaction mixture (5 ml) comprised of 2.8 ml of phosphate buffer saline (pH 6.4), egg albumin (from fresh hen’s egg) 0.2 ml and 2 ml of the extracts (1%). Control composed of a similar volume of water (double distilled). Mixtures were incubated for 15 minutes at the temperature 37 ± 2 °C and then heated for five minutes at temperature 70 °C. After cooling the mixtures, absorbance was measured at 660 nm wave length by using the vehicle as a blank. Diclofenac sodium and aspirin were used as the standard drugs and treated likewise. Percentage inhibition of protein denaturation was calculated by the following formula:
% Inhibition = (Acontrol- Atest/Acontrol) x 100
Where, A is the absorbance.
                50% inhibition (IC50) of the most active extract and reference drug was determined through the plotting dose response curve by regression line equation where y=50 (Alhakmani et al., 2013).
In vitro xanthine oxidase inhibition activity: In vitro xanthine oxidase inhibition assay was used to evaluate anti-gout potential of the plant. To prepare plant extract solution, extract (2 mg) was dissolved in 1 ml 5% DMSO in water solution. In this mixture, 2.9 ml phosphate buffer (1.15 M, pH=7.5) and 0.1 ml of xanthine oxidase enzyme solution (purchased from Sigma) were added. After 15 minutes of mixture pre incubation at 25 0C temperature, the reaction was started by adding a substrate solution (2 ml).Then after 30 minutes, the reaction was stopped by adding 1 ml of IN HCl. Absorbance was measured at 290nm. Likewise, blank was prepared but enzyme was being added to assay mixture after addition of the HCl. Xanthine oxidase activity was calculated by following formula and expressed as the percentage inhibition of xanthine oxidase.
% Inhibition of xanthine oxidase = 1-(A extract/A no extract) x 100
Where,
                A is the absorbance (Guerrero et al., 2011).

RESULTS AND DISCUSSION

Proximate analysis: Proximate analysis was done for standardization of the root powder. The findings of the analysis are summarized in Table 1.

Table 1. Proximate analysis of Bryophyllum pinnatum root powder.

                The moisture content was determined according to the USP 2015 prescribed method for botanical origin. The testing of moisture content is an important physicochemical parameter. Moisture content should be less so that the powder may be stable for a longer period of the time. The moisture content of the plant under investigation was found to be within specified range which is not greater than 14% w/w (Munir et al., 2014). Ash value testing is of great significance to identify the presence of any foreign matter like silica, oxalate etc. To evaluate the quality, purity and polarity of the root powder different ash values and extractive values were estimated. The total ash value of the plant under test was found to be 13% which indicates it is within official range (less than 20%) (Kadam et al., 2012). High alcohol and water soluble values have suggested the presence of polar substances like tannins, phenols, glycosides etc. (Baravalia et al., 2011).
Phytochemical analysis: Phytochemical analysis comprised of primary and secondary metabolites estimation. The primary metabolites evaluation included: total proteins, total lipids and total carbohydrates and their content are shown in Table 2. Assessment of the secondary metabolites was included: total polyphenols, total polysaccharides, total flavonoids and total glycosaponins and the outcomes of which is summarized in Table 3.
                Evaluation of the primary metabolites has revealed that the plant root powder contained varied amount of carbohydrates, lipids and proteins. Current findings have suggested that the carbohydrates were present in larger amount than lipids and proteins. Occurrence of the primary metabolites has indicated substantial nutritional value of the plant (Odhav et al., 2007).

Table 2. Estimation of primary metabolites.