Comparative study of biological activities of different extracts of root and whole plant materials of Ajuga bracteosa from Bhimber Azad Kashmir Pakistan

Ajuga bracteosa is native to Himalayan region, Afghanistan, China and Malaysia. People of northern areas of Pakistan call it ‘korri booti’ as it has bitter taste. Ajuga bracteosa is ethnically used against malarial fever, diabetes, cancer, and sore throat. The theme of present work was to appraise the biological significance of Ajuga bracteosa through in vitro studies. n-Hexane, chloroform, and methanol extracts of root and whole plant materials were obtained via maceration. Qualitative and quantitative analysis of phytochemical constituents was done. The antioxidant potency was judged by DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2,2azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) free radical scavenging assays. Antimicrobial efficacy was assessed by agar disc diffusion method. Phytochemical review confirmed the existence of flavonoids, saponins, phenols, tannins, terpenoids, xanthoproteins, carbohydrates, and glycosides. Total flavonoid and total phenolic contents were shown as rutin and gallic acid equivalents respectively. Root extracts gave more significant results of DPPH and ABTS potential (100 % and 92 % respectively) as compared to whole plant extracts (90 % and 75 % respectively). A. bracteosa is a plentiful source of polyphenolic compounds. Polyphenolic compounds are thought as natural antioxidants because of their ability of hydrogen atom donation and presence as stable radical intermediates preventing further creation of free radicals in the body. Root extracts showed greater resistance against pathogens as compared to extracts of whole plant material.


Introduction
Side effects of synthetic drugs have forced the medicinal world to return to nature i.e to use medicinal plants for healthcare. Humans have been cultivating plants since ancient times because of presence of natural compounds which have been used against various diseases [1,2]. World health organization had stated that only 250 out of 20,000 types of medicinal plants were inspected for their potential against diseases. Only 25% of drugs is obtained using plants and above 80 % of population of entire world is still dependent on indigenous medicinal plants to cure diseases [3,4]. Distinctive biodiversity of Pakistan includes more than 600 medicinal plants and people use these plants for medication purposes against fever, cough, and cold and to heal wounds. Ajuga bracteosa is native to Himalayan region, Afghanistan, China and Malaysia [5]. A. bracteosa being a promising herb has greater significance in rural areas of Pakistan and Azad Kashmir. People of northern areas of Pakistan call it 'korri booti' as it has bitter taste [6]. It belongs to family Labiatae of 266 genera and 301 species [7]. It had shown potential against worm infection, diabetes, cancer and fungal inflammation. Extracts of its leaves are used as blood purifier, against skin infections and malarial fever. Roots are used as an antidote to snake bite [8,9]. Different compounds of medical significance have also been isolated from A. bracteosa [10]. It is to our finest knowledge that no previous study has been done to compare its different parts for medicinal purposes. Thus present work was designed to compare presence of phytochemicals, antioxidant and antimicrobial potential of its different parts.

Plant collection
Plants were collected from district Bhimber, Azad Jammu and Kahmir, Pakistan and identified by ethno-botanists of department of Botany, Mirpur University of Science and Technology, Mirpur, Pakistan. Plants were rinsed with water to remove soil contents and shade dried at room temperature.

Preparation of extracts
Roots were separated from remaining plants body in order to prepare two fractions i.e fraction of root material and fraction of whole plant material (stem and leaves) and crushed into a fine powder. Extraction was done by using three different solvents n-Hexane, chloroform, and methanol. 85 g powder of each fraction was extracted with 200 mL of one of mentioned solvents in increasing order of their polarity. Extracts were filtered and then evaporated at 40 C through rotary evaporator. Following formula was used to calculate percent recovery of extracts. % Recovery = (recovered mass of extract/ actual mass of sample powder) x 100 Qualitative analysis of phytochemical constituents For phytochemical analysis extracts of both the fractions were tested to confirm the presence of bioactive constituents using standard phytochemical protocols. Total phenolic contents Deo et al. [11] with minor modifications was adopted for estimation of total phenolic contents. They were expressed as milli gram of gallic acid equivalent per gram of extract. Total flavonoid contents Determination of total flavonoid contents was quantified by adopting Ghous et al. [12] with minor modifications. They were expressed in terms of rutin equivalent. DPPH scavenging assay DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging activity was determined by adopting Ghous et al. [12] with minor modifications. All extracts (0.5 mL and 1 mL) were mixed with 3 mL and 2.5 mL of reaction mixture (DMSO and DPPH solutions) respectively. The absorbance was measured after incubation period of 0, 15, and 30 minutes in darkness at 517 nm. Percent scavenging activity = [(Ac-Ai)/Ac] x100 formula was adopted for the calculation of percent scavenging activity; here Ac is the absorbance value of standard and Ai is the absorbance value of extract. ABTS free radical cation decolourising assay ABTS (2,2'-azinobis-(3ethylbenzothiazoline-6-sulphonic acid) free radical scavenging activity was estimated by adopting described method of Ghous et al. [12] with minor modifications. Reaction mixture was prepared by mixing potassium persulfate (2.5 mM) and ABTS (3mM) and leaving for 16 hours in darkness for the generation of ABTS free radicals. All extracts (20, 100, 200 µL) were allowed to mix with ABTS •+ (1mL) and incubated for 20 minutes. Percent radical scavenging activity (% RSA) was determined as: % RSA = [(Ac-Ai/ Ac)] x 100; here Ac is absorbance value of standard and Ai represents absorbance value of extracts. Antimicrobial activity For present research activity four bacterial pathogens (Escherichia coli, Bacillus pumilus, Bacillus subtilis, and Staphylicoccus aureus) were used. All these strains were reserved from Department of Biotechnology, Mirpur University of Science and Technology, Mirpur, Pakistan. Antimicrobial efficacy was assessed using agar disc diffusion method. Luria Bertani broth (L.B.Broth) medium and agar medium were mixed to prepare L.B Agar medium. It was transferred to sterilized plates of capacity of 20mL/ plate and left for solidification. Then fresh culture of pathogens was spread over it. Sterile discs of 6 mm diameter well soaked in extract solutions (5 %) were used. All these plates were incubated 37 C for 24 hours. Antimicrobial efficacy was assessed by measuring zone of growth inhibition in mili-meter. Efficacy of extracts was expressed as zero for no efficacy, 1-5 mm for low efficacy, 6-10 mm for moderate efficacy and above 10 mm for high efficacy.

Results and Dicussion % Recovery of Extracts
The recovery of extracts is as following; Root extracts (n-Hexane= 0.63 %, chloroform= 0.87 %, methanol= 3.71 %) Whole plant extracts (n-Hexane 0.67%, chloroform= 1.64 %, methanol= 5.99 %) Phytochemical screening Scientists have been working to know chemical composition of herbal medicines. Bioactive compounds have been isolated from medicinal plants to know about their pharmacological significance. Present comparative study has uncovered different phytochemicals in both the fractions. All extracts of both the fractions showed the presence of flavonoids, carbohydrates, tannins, saponins, diterpenes, steroids, and glycosides and absence of resins and carboxylic acids. Phenols were absent only in n-Hexane extracts. Quinones and amino acids were present in methanolic extracts and completely absent in n-Hexane extracts with the exception that they were present in chloroform extracts of whole plant material but absent in extract of root material. Xanthoproteins were found only in methanolic extracts. Alkaloids were present in all extracts of whole plant material and absent in all extracts of root material (Table  1). Total phenolic contents Polyphenols, important secondary metabolites of plants, can donate hydrogen atoms of phenolic hydroxyls to reduce reactive species of oxygen. Methanolic extract of root has 2.67 mg/g presence of total phenolic contents (TPC) as compared to 2.55 mg/g of same extract of whole plant material. Comparative presence of TPC in chloroform extracts of root material and whole plant material is 1 mg/g and 0.80 mg/g respectively while n-Hexane extract of root material has 0.68 mg/g presence of TPC as compared to 0.52 mg/g in n-Hexane extract of whole plant material. Thus comparative analysis showed that TPC had greater presence in extracts of root material as compared to extracts of whole plant material. All these results were measured as gallic acid equivalent (Table 2). Total flavonoid contents Comparative estimation of total flavonoid contents (TFC) in extracts of root and whole plant material was done against rutin hydrate calibration curve. Methanolic extract of root has 7.7 mg/g TFC as compared to 7.4 mg/g in same extract of whole plant material. Comparative presence of TFC in chloroform extracts of root and whole plant materials is 5.7 mg/g and 4.97 mg/g respectively while n-Hexane extract of root material has 4.45 mg/g presence of TFC as compared to 3.9 mg/g TFC in extract of whole plant material. Thus comparative analysis exposed greater presence of TFC in root extracts as compared to whole plant extracts (Table 3).   . Extracts having ability of donation of hydrogen atom to stable free radical of DPPH are thought good antioxidants. All extracts of Ajuga bracteosa have displayed significant scavenging potential in case of DPPH assay. The present research work revealed that extracts of root material have greater scavenging potential than extracts of whole plant material and also exposed that scavenging potential exhibited decreasing trend from organic polar to organic non-polar extracts. Initially scavenging potential was compared by using 0.5 mL of all extracts of root and whole plant material. Methanolic extract of root material exhibited 89 % scavenging potential as compared to 75 % scavenging potential of methanolic extract of whole plant material. Chloroform extracts of root and whole plant material have comparative scavenging potential 71 % and 57 % respectively. 26 % scavenging potential was exhibited by n-Hexane extract of root material as compared to 15 % potential of same extract of whole plant material. It is a general observation that scavenging potential will increase by increasing volume of extacts. Thus volume of all extracts was increased to 1 mL. This change displayed incredible increase in results.
Increased Scavenging potential for methanolic, chloroform, and n-Hexane extracts of root material is 100 %, 83 %, and 39 % respectively and 90 %, 72 %, and 32 % respectively in case of whole plant material (Table 4).    (11mm against each). Chloroform extract of root material expressed his medium sensitivity against all pathogens (E. coli= 9 mm, B. subtilis= 9 mm, and B. pumilus= 7 mm) while the same extract of whole plant material showed again medium sensivity against all used pathogen (E. coli= 10 mm, B. subtilis= 7 mm, and B. pumilus= 8 mm). Sensitivity of chloroform extract of root and whole plant material was lower (4 mm) and higher (12) respectively against S.aureus. n-Hexane extract of both the fractions showed medium range sensitivity against B. subtilis, B. pumilus, and S. aureus i.e 7 mm, 10 mm, and 6 mm respectively in case of root material and 5 mm, 6 mm, and 10 mm respectively in case of whole plant material. n-Hexane extract of root displayed higher sensitivity against E. coli (11 mm) and same extract of whole plant material showed medium sensitivity against same pathogen (7 mm) ( Table 7). Their huge amount is found in more polar extracts which decreased with decrease in polarity of extracts. Chloroform and n-Hexane extracts have lesser amount of these compounds as compared to methanolic extracts. Methanolic extract of root material has more presence of these secondary metabolites as compared to the same extract of whole plant material. Metabolic and other activities in the body generate reactive oxygen and nitrogen species which are free radicals in nature. These free radicals create a chain reaction in the body producing oxidative stress which in turn causes neurodegenerative diseases, cancer, heart diseases and aging process [20][21][22]. Antioxidants are needed to outfit these reactive species. At present synthetically prepared antioxidants are available but with a disadvantage of side effects. Thus plants are considered as prospective source of natural antioxidants [16]. In our work antioxidant potential of root and whole plant materials of A. bracteosa was appraised and compared by DPPH and ABTS free radical scavenging assays. A. bracteosa is plentiful source of polyphenolic compounds. Polyphenolic compounds are thought as natural antioxidants because of their ability of hydrogen atom donation and presence as stable radical intermediates preventing further creation of free radicals in the body [23]. Our study showed more presence of these compounds in methanolic extracts of root and whole plant materials as compared to chloroform and n-Hexane extracts of the same plant materials. These compounds are significantly higher in amount in root material extracts as compared to extracts of whole plant material. Thus greater antioxidant activity was shown by extracts of root material in both the free radical scavenging assays. Decline in scavenging activity of extracts of both the root and whole plant materials was observed with decrease in polarity of the solvents used for extracts. However, all extracts of root material showed more improved and significant scavenging activity as compared to the extracts of whole plant material. Plants have been a subject of interest against various pathogenic infections [24]. While appraising the efficacy of extracts of root and whole plant materials of A. bracteosa against pathogens it was revealed that all extracts showed significant resistance against E. coli and average resistance was observed against B. subtilis. Quite interestingly methanolic extract of root material showed very low resistance against B. pumilus and S. aureus as compared to the significant resistance of methanolic extract of whole plant material against the same pathogens. Thus our study has not only revealed all possible therapeutic effects of A. bracteosa but also unveiled the supremacy of root material over whole plant material of A. bracteosa through their comparative analysis.

Conclusion
All comparative results of this research work revealed the greater importance of root over other parts of A. bracteosa in pharmacology. Keeping in view all these results it can be inferred that A. bracteosa is a prospective source of secondary metabolites playing vital role as antioxidant and antibacterial agents. It also proved that roots have greater presence of these substances as compared to other parts of the plant. This research also provides all those directions which can extend medical worth of the plant through isolation along with characterization of natural compounds.